Immunogenic constructs, compositions, and methods for inducing an immune response

Abstract

The immunogenic construct disclosed herein comprises a nanoparticle, a cationic polymer electrostatically bound to the outer surface of the nanoparticle, a stabilizer bound to the cationic polymer or a stabilizer bound to the outer surface of the nanoparticle, and an antigen or an antigen-producing factor.Optionally, the construct can comprise an adjuvant and / or one or more functional oligonucleotides (e.g., siRNA or pDNA).The immunogenic construct provided herein is also disclosed as a method for simultaneously delivering an adjuvant, an antigen, and optionally siRNA to cells, inducing an immune response in a subject, and treating or preventing infectious diseases in a subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority and benefit of the earlier filing date of U.S. Provisional Application No. 63 / 051,351, filed July 13, 2020, which is incorporated herein by reference in its entirety.

[0002] Statement of Federally Funded Research This invention was made with government support under Grant No. R44 CA217534 from the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] background During the first week of 2020, the world witnessed the emergence of a new human pathogen that achieved sufficient zoonotic spread to cause a pandemic, replacing highly pathogenic beta-coronaviruses. The 2019 novel coronavirus (SARS-CoV-2), the cause of the highly infectious disease known as COVID-19, is a new member of the beta-coronavirus family, which includes severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), which caused an epidemic in China in 2002–2003, and Middle East respiratory syndrome coronavirus 2 (MERS-CoV), which affected Saudi Arabia and neighboring countries in 2012–2013.

[0004] After first emerging in China, the rapid spread of COVID-19 has resulted in over 13 million confirmed cases and over 500,000 deaths (as of July 2020). Safe and effective treatments and prophylaxis against this novel virus are urgently needed.

[0005] Therefore, new immunogenic strategies applicable to this virus and emerging infectious diseases are needed. Summary of the Invention

[0006] Summary of the Disclosure The present disclosure provides immunogenic constructs, compositions, and methods for inducing an immune response against infectious agents. For example, the compositions of the present disclosure can be used to induce an immune response against β-coronavirus infections, such as SARS-CoV-2, SARS-CoV-1, MERS-CoV, and related viruses. The disclosed technology is also applicable to infectious diseases of animals (such as pets and livestock), as well as other current infectious diseases such as dengue fever and malaria.

[0007] In one aspect, the disclosure features an immunogenic construct containing a nanoparticle, a cross-linked cationic polymer bound to the outer surface of the nanoparticle, a stabilizer bound to the cross-linked cationic polymer or to the outer surface of the nanoparticle, and an antigen (e.g., a full-length protein, a protein subunit, a polypeptide, a peptide, or a mixture thereof) or an antigen-producing agent (such as an antigen-producing nucleic acid, e.g., mRNA or pDNA) against an infectious agent.

[0008] In some embodiments, the immunogenic construct further comprises an adjuvant. In some embodiments, the adjuvant comprises one or more of a CpG oligonucleotide, a DNA TLR agonist containing a CpG sequence, a non-CpG DNA TLR agonist, an RNA TLR agonist, an aluminum salt, an anti-CD40 antibody, a fusion protein, a cytokine, a small molecule TLR agonist, an oil-based or surfactant-based adjuvant, a lipopolysaccharide, a plant extract, or a derivative thereof. In some embodiments, the adjuvant comprises a CpG oligonucleotide (e.g., CpG ODN 1826 or CpG ODN 7909 / 2006). In some embodiments, the adjuvant comprises poly I:C. In some embodiments, the adjuvant is present at 1-20% by weight (e.g., 1-10%, 2-7%, 2-4%, 2-10%, 5-10%, 10-20%; or about 4%, about 5%, about 6%, about 7%, about 10%, or about 20% by weight) of the nanoparticle platform (NP or polymer / stabilizer-coated nanoparticle). In some embodiments, the adjuvant is present at 2-10% by weight of the NP.

[0009] In some embodiments, the nanoparticles are silica nanoparticles (e.g., mesoporous silica nanoparticles), silicon nanoparticles, iron oxide nanoparticles, gold nanoparticles, silver nanoparticles, carbon nanoparticles, or carbon nanotubes. In various embodiments, the pore size of the mesoporous nanoparticles is 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-5, 2-7, 6-10, 11-15, 16-20, 21-30, or 31-50 nm.

[0010] In embodiments, the nanoparticles are adjuvant nanoparticles or immunostimulatory nanoparticles (e.g., liposomes, lipoplex particles, lipid-based particles, polyplex particles, polymer-based particles, inorganic particles (e.g., calcium phosphate or calcium carbonate nanoparticles, aluminum salt particles, silica particles), virosomes or virus-like particles, or nanoparticles containing 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), cholesterol, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, phosphatidylcholine / cholesterol, chitosan, poly-γ-glutamic acid (γ-PGA), hyaluronic acid, polyethyleneimine (PEI), poly(propylacrylic acid), polypropylene sulfide, polylactic-co-glycolic acid (PLGA), amylopectin, maltodextrin, polystyrene, gold, cobalt oxide, alum, tri-palmitoyl-S-glycerylcysteine ​​(PAM3Cys), squalene, Montanide ISA, or other nanoparticles. 50V, Montanide ISA 51, Montanide ISA 201, Montanide ISA 206 and Montanide ISA 720).

[0011] In some embodiments, the cationic polymer is selected from the group consisting of polyethyleneimine (PEI), chitosan, polypropyleneimine, polylysine, polyamidoamine, poly(allylamine), poly(diallyldimethylammonium chloride), poly(N-isopropylacrylamide-co-acrylamide), poly(N-isopropylacrylamide-co-acrylic acid), diethylaminoethyl-dextran, poly(N-ethyl-vinylpyridinium bromide), poly(dimethylamino)ethyl methacrylate, and poly(ethylene glycol)-co-poly(trimethylaminoethyl methacrylate chloride). In some embodiments, the cationic polymer is PEI. In some embodiments, the cationic polymer has a molecular weight of about 0.8 kDa to about 25 kDa (e.g., about 0.8 kDa to about 10 kDa, about 0.8 kDa to about 5 kDa, about 0.8 kDa to about 2.5 kDa, about 2.5 kDa to about 10 kDa, or about 5 kDa to about 10 kDa). In some embodiments, the cationic polymer is present at about 1 to 50% by weight of the NP (e.g., 5 to 40%, 10 to 30%, 20 to 30%, 5 to 10%, 5 to 15%, 5 to 20%, 5 to 25%, 5 to 30%, 10 to 20%, 10 to 25%, or 25 to 40% by weight; or about 5, about 10, about 15, about 20, about 25, about 30, or about 35% by weight). In some embodiments, the cationic polymer is present at 10-20% by weight of the NP.

[0012] In some embodiments, the stabilizer is selected from the group consisting of polyethylene glycol (PEG), dextran, polysialic acid, hyaluronic acid, polyvinylpyrrolidone, polyvinyl alcohol, and polyacrylamide. In some embodiments, the stabilizer is PEG. In some embodiments, the stabilizer has a molecular weight of about 1 kDa to about 20 kDa (e.g., about 0.8 kDa to about 10 kDa, about 0.8 kDa to about 5 kDa, about 2 kDa to about 10 kDa, about 0.8 kDa to about 2.5 kDa, about 2.5 kDa to about 10 kDa, or about 5 kDa to about 10 kDa). In some embodiments, the stabilizer is present at 1-50% by weight of the NP (e.g., 5-30%, 10-20%, 10-25%, 5-15%, 5-20%, 5-25%, or 1-10%, or about 5, about 10, about 15, about 20, about 25, about 35, about 40, or about 45% by weight). The stabilizer can be introduced before or after cargo loading, or both.

[0013] In some embodiments, the infectious agent is a virus, e.g., a β-coronavirus (e.g., SARS-CoV-2, SARS-CoV-1, or MERS-CoV). In some embodiments, the antigen is a recombinant full-length protein, e.g., the full-length SARS-CoV-2 spike glycoprotein, SARS-CoV-2 nucleocapsid protein, or SARS-CoV-2 membrane protein. In some embodiments, the antigen is a protein subunit, e.g., a protein subunit corresponding to the S1 region, S2 region, or receptor binding domain (RBD) region of the SARS-CoV-2 spike glycoprotein. In some embodiments, the antigen is a peptide or mixture of peptides corresponding to an immunogenic sequence of the infectious agent. For example, the infectious agent is SARS-CoV-2 and the antigen has the peptide sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and / or 8. In some embodiments, the antigen-producing agent is mRNA or pDNA, e.g., mRNA or pDNA that is expressed or translated into an antigen in vitro (e.g., DCs) or in vivo (e.g., DCs, muscle cells). In some embodiments, the antigen or antigen-producing agent is present at 0.5-20% by weight (e.g., 0.5-10%, 1-6%, 1-15%, 1.5-10%, or 2-5% by weight) of the NP. In some embodiments, the antigen may comprise a mixture of protein subunits and peptides.

[0014] In some embodiments, the immunogenic construct comprises at least one oligonucleotide selected from siRNA, miRNA, miRNA mimic, or antisense oligonucleotide. In some embodiments, the at least one oligonucleotide is electrostatically bound to a cationic polymer. In some embodiments, the at least one oligonucleotide comprises an siRNA, e.g., one that inhibits or downregulates a gene associated with immunosuppression of cells, such as antigen-presenting cells (e.g., dendritic cells or macrophages). In some embodiments, the gene is STAT3, IDO-1, IL-6, or PD-L1. In some embodiments, the at least one oligonucleotide is present in about 1-50% by weight (e.g., 2%, 3%, 4%, 5%, 2-5%, 2-8%, 2-10%, 2-25%, or 2-50% by weight) of the NP.

[0015] In some embodiments, the immunogenic construct further comprises a targeting agent for cells such as antigen-presenting cells (e.g., dendritic cells or macrophages). In some embodiments, the targeting agent is mannose, a monoclonal or polyclonal antibody or fragment thereof that recognizes and binds to an epitope displayed on the surface of an antigen-presenting cell, an aptamer, or a ligand that binds to a surface receptor on an antigen-presenting cell. In some embodiments, the targeting agent is present at 0.1-20% by weight of the NP (e.g., 0.1-1%, 0.2-2%, 1-5%, or 1-10% by weight; or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9% by weight).

[0016] In some embodiments, the immunogenic construct further comprises a labeling agent, which is a fluorescent dye and / or a metal probe (e.g., a lanthanide probe, a quantum dot, a gold nanoparticle, or a gadolinium chelate).

[0017] In some embodiments, the immunogenic construct has a hydrodynamic diameter of about 10 nm to about 999 nm (e.g., about 80 nm to about 200 nm or about 90 nm to about 130 nm) as measured in aqueous solution (e.g., PBS, Tris buffer, or water) by dynamic light scattering techniques or a Zetasizer (Malvern Panalytical) or similar device. In some embodiments, the immunogenic construct has a hydrodynamic diameter of about 1 micron to about 10 microns (e.g., about 1 micron to about 2 microns) as measured in aqueous solution (e.g., PBS, Tris buffer, or water). In some embodiments, the nanoparticles have a diameter of about 5 nm to 999 nm (e.g., about 20 nm to about 200 nm, about 30 nm to about 60 nm, about 10 nm, about 20 nm, about 30 nm, about 50 nm, about 60 nm, about 200 to about 750 nm, or about 500 to 999 nm) as measured, for example, by transmission electron microscopy.

[0018] The present disclosure further features immunogenic constructs containing nanoparticles, a lipid layer, and an antigen (e.g., a full-length protein, protein subunit, polypeptide, or peptide) or antigen-producing agent (antigen-producing nucleic acid, such as, e.g., mRNA or pDNA) against an infectious agent.

[0019] In some embodiments, the immunogenic construct further comprises an adjuvant. In some embodiments, the adjuvant comprises one or more of a CpG oligonucleotide, a DNA TLR agonist containing a CpG sequence, a non-CpG DNA TLR agonist, an RNA TLR agonist, an aluminum salt, an anti-CD40 antibody, a fusion protein, a cytokine, a small molecule TLR agonist, an oil-based or surfactant-based adjuvant, a lipopolysaccharide, a plant extract, or a derivative thereof. In some embodiments, the adjuvant comprises a CpG oligonucleotide (e.g., CpG ODN 1826 or CpG ODN 7909 / 2006). In some embodiments, the adjuvant is loaded into the NP. In some embodiments, the adjuvant is loaded onto or within the lipid layer. In some embodiments, the adjuvant is present at 1-20% by weight of the NP (e.g., 1-10%, 2-7%, 2-4%, 2-10%, 5-10%, 10-20%; or about 4%, about 5%, about 6%, about 7%, about 10%, or about 20% by weight). In some embodiments, the adjuvant is present at 2-10% by weight of the NP.

[0020] In some embodiments, the nanoparticles are silica nanoparticles (eg, mesoporous silica nanoparticles), silicon nanoparticles, iron oxide nanoparticles, gold nanoparticles, silver nanoparticles, or carbon nanotubes.

[0021] In some embodiments, the nanoparticles are adjuvant nanoparticles or immunostimulatory nanoparticles (e.g., liposomes, lipoplex particles, lipid-based particles, polyplex particles, polymer-based particles, inorganic particles (e.g., calcium phosphate nanoparticles, aluminum salt particles, silica particles), virus-like particles, or nanoparticles containing 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), cholesterol, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, phosphatidylcholine / cholesterol, chitosan, poly-γ-glutamic acid (γ-PGA), hyaluronic acid, polyethyleneimine (PEI), poly(propylacrylic acid), polypropylene sulfide (PPS), polylactic-co-glycolic acid (PLGA), amylopectin, maltodextrin, polystyrene, gold, cobalt oxide, alum, tri-palmitoyl-S-glycerylcysteine ​​(PAM3Cys), squalene, Montanide ISA, or other nanoparticles. 50V, Montanide ISA 51, Montanide ISA 201, Montanide ISA 206 and Montanide ISA 720).

[0022] In some embodiments, the lipid layer comprises a neutral lipid (e.g., a prostaglandin, an eicosanoid, or a glyceride), a fatty acid-modified lipid (e.g., 2-diphytanoyl-sn-glycero-3-phosphocholine or 1-(12-biotinyl(aminododecanoyl))-2-oleoyl-sn-glycero-3-phosphoethanolamine), a phospholipid (e.g., phosphatidylcholine, phosphatidylethanolamine, 1,2-distearoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), or a phospholipid (e.g., phosphatidylcholine, phosphatidylethanolamine, 1,2-distearoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). The NPs are monolayer or multilayer membranes comprising one or more of the following lipids: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, dimethyldioctadecylammonium bromide, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, and distearoyl-rac-glycerol-PEG2K. In some embodiments, the lipid layer comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, dimethyldioctadecylammonium bromide, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine, and distearoyl-rac-glycerol-PEG2K. In some embodiments, the lipid layer is present at 0.1-99.9% by weight of the NPs.

[0023] In some embodiments, the infectious agent is a virus, e.g., a β-coronavirus (e.g., SARS-CoV-2, SARS-CoV-1, or MERS-CoV). In some embodiments, the antigen is a recombinant full-length protein, e.g., the full-length SARS-CoV-2 spike glycoprotein, SARS-CoV-2 nucleocapsid protein, or SARS-CoV-2 membrane protein. In some embodiments, a combination of antigens (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different antigens) is used in the formulation. In some embodiments, the antigen is a protein subunit, e.g., a protein subunit corresponding to the S1 region, S2 region, or RBD region of the SARS-CoV-2 spike glycoprotein. In some embodiments, the antigen is a peptide or mixture of peptides corresponding to an immunogenic sequence of the infectious agent. For example, the infectious agent is SARS-CoV-2 and the antigen has the peptide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and / or 8. In some embodiments, the antigen-producing agent is mRNA or pDNA, e.g., mRNA or pDNA that is expressed or translated into an antigen in vitro (e.g., DCs) or in vivo (e.g., DCs, muscle cells). In some embodiments, the antigen or antigen-producing agent is present at 0.5-20% by weight (e.g., 1-15%, 1.5-10%, or 2-5% by weight) of the NP. In some embodiments, the antigen may comprise a mixture of protein subunits and peptides.

[0024] In some embodiments, the infectious agent is a bacterium. In some embodiments, the antigen is a toxoid, e.g., an inactivated toxin intended to immunize against a particular bacterial toxin. In some embodiments, the antigen is a bacterial polysaccharide intended to generate immunity against the bacterial sugar coating. In some embodiments, the antigen consists of one or more recombinant proteins derived from bacteria.

[0025] In some embodiments, the immunogenic construct comprises at least one oligonucleotide selected from siRNA, miRNA, miRNA mimic, or antisense oligonucleotide. In some embodiments, the at least one oligonucleotide comprises an siRNA, e.g., one that inhibits or downregulates a gene associated with immunosuppression of cells, such as antigen-presenting cells (e.g., dendritic cells or macrophages). In some embodiments, the gene is STAT3, IDO-1, IL-6, or PD-L1. In some embodiments, the at least one oligonucleotide is loaded onto NPs. In some embodiments, the at least one oligonucleotide is loaded onto or within the lipid layer. In some embodiments, the at least one oligonucleotide is present in an amount of 0.01 to 10% by weight of the NPs.

[0026] In some embodiments, the immunogenic construct further comprises a targeting agent for a cell, such as an antigen-presenting cell (e.g., a dendritic cell or macrophage). In some embodiments, the targeting agent is mannose, a monoclonal or polyclonal antibody or fragment thereof that recognizes and binds to an epitope displayed on the surface of the antigen-presenting cell, an aptamer, or a ligand that binds to a surface receptor on the antigen-presenting cell.

[0027] In some embodiments, the immunogenic construct further comprises a labeling agent, which is a fluorescent dye and / or a metal probe (e.g., a lanthanide probe, a quantum dot, a gold nanoparticle, or a gadolinium chelate).

[0028] In some embodiments, the immunogenic construct has a hydrodynamic diameter of 10 nm to 10 microns. In some embodiments, the immunogenic construct has a hydrodynamic diameter of about 10 nm to about 999 nm (e.g., about 80 nm to about 200 nm or about 90 nm to about 150 nm) when measured in an aqueous solution (such as PBS, Tris buffer, or water). In some embodiments, the immunogenic construct has a hydrodynamic diameter of about 1 micron to about 10 microns (e.g., about 1 micron to about 2 microns) when measured in an aqueous solution (such as PBS, Tris buffer, or water). In some embodiments, the nanoparticles have a diameter of about 5 nm to about 999 nm (e.g., about 20 nm to about 200 nm, about 30 nm to about 60 nm, about 200 to about 750 nm, or about 500 to 999 nm) when measured, for example, by transmission electron microscopy.

[0029] In some embodiments, the nanoparticles are antioxidant nanoparticles.

[0030] In another aspect, the disclosure features a pharmaceutical composition including an immunogenic construct of the disclosure and a pharmaceutically acceptable excipient. In some aspects, the pharmaceutical composition further includes a non-conjugated adjuvant as described.

[0031] In another aspect, the disclosure features a vaccine that includes an immunogenic construct of the disclosure and a pharmaceutically acceptable excipient.

[0032] In another aspect, the disclosure features a method for co-delivering an oligonucleotide (e.g., siRNA), an antigen or antigen-producing agent (e.g., mRNA or pDNA), and / or an adjuvant to a cell (e.g., a muscle cell or an antigen-presenting cell, e.g., a dendritic cell or macrophage). The method includes contacting the cell with an immunogenic construct of the disclosure. In some embodiments, the immunogenic construct includes at least one antigen-producing agent (e.g., mRNA or pDNA) and is administered intramuscularly to a subject and taken up by muscle cells, where the immunogenic construct induces the muscle cells to produce at least one antigen for subsequent immune cell activation.

[0033] In another aspect, the disclosure features a method of inducing an immune response to an infectious agent in a subject. The method includes administering to the subject an immunogenic amount of an immunogenic construct of the disclosure. In some embodiments, the subject is human. In some embodiments, the subject is immunocompromised (e.g., an elderly or geriatric subject, e.g., over 50, 55, 60, 65, 70, 75, or 80 years of age, or a subject who is immunocompromised and has underlying medical conditions such as diabetes and cancer that are known to predispose to infection). In some embodiments, the immunogenic construct is administered by intramuscular injection.

[0034] In another aspect, the disclosure features a method of increasing an immune response to an infectious agent in a subject. The method includes administering to the subject an effective amount of an immunogenic construct of the disclosure. In some embodiments, the subject is human. In some embodiments, the subject is immunocompromised (e.g., an elderly or geriatric subject, e.g., over 50, 55, 60, 65, 70, 75, or 80 years of age, or a subject who is immunocompromised and has an underlying medical condition known to predispose to infection). In some embodiments, the immunogenic construct is administered by intramuscular injection. In some embodiments, the immunogenic construct is administered by inhalation.

[0035] In another aspect, the disclosure features a method of vaccinating a subject against an infectious agent. The method includes administering to the subject an effective amount of an immunogenic construct of the disclosure. In some embodiments, the subject is human. In some embodiments, the subject is immunocompromised (e.g., an elderly or geriatric subject, e.g., over 50, 55, 60, 65, 70, 75, or 80 years of age, or a subject who is immunocompromised and has an underlying medical condition known to predispose to infection). In some embodiments, the immunogenic construct is administered by intramuscular injection. In some embodiments, the immunogenic construct is administered by inhalation. [Brief explanation of the drawings]

[0036] [Figure 1]Figure 1 is a schematic illustrating the use of an immunogenic construct according to one embodiment of the present disclosure to induce an immune response against infection (a viral infection with SARS-CoV-2, also known as CoV, is shown). Upon intramuscular or subcutaneous injection, the immunogenic construct (AIRISE-CoV) is taken up by antigen-presenting cells (APCs, e.g., dendritic cells and macrophages). Immune activation by CpG and inhibition of immunosuppressive genes by siRNA within the APCs enhances their activity in processing the delivered antigen for presentation (A). Activated, antigen-loaded APCs migrate from the injection site to lymph nodes (B), where they subsequently activate antigen-specific CD8+ T cells (C), which subsequently proliferate into effector and memory T cells against the virus. Activated APCs also activate B cells and CD4+ T cells, which can further activate CD8+ T cells and B cells, resulting in a humoral immune response (antibodies, D) against current and future viral infections throughout the body, including the lungs (E). If the construct delivers antigen-producing factors (such as mRNA and pDNA), muscle cells also take up the injected construct and produce antigens that are processed by APCs, followed by the same processes A to E. [Figure 2] Figure 2 is a graph showing the hydrodynamic size (diameter or Z-average diameter) of mesoporous silica nanoparticles coated with PEI and PEG (nanoparticle platform; NP) loaded with SIINFEKL peptide (SEQ ID NO: 90; SF, Anaspec) and CpG 1826 (Invivogen). [Figure 3] Figure 3 shows the hydrodynamic size of NPs (MSNP-PEI-PEG) loaded with approximately 2% and approximately 9% Poly I:C by weight of the NPs, measured in PBS. [Figure 4A]Figures 4A-4D show STAT3 knockdown in multiple cell lines from multiple species at 48 hours using (Figure 4A) NPs loaded with siRNA against STAT3 (siSTAT3) and (Figure 4B) NPs loaded with siSTAT3 and CpG. qRT-PCR analysis of STAT3 and HPRT mRNA was performed using corresponding species primers in D-17 (canine osteosarcoma), BMDC (bone marrow-derived dendritic cells from mice), J774 (mouse macrophages), B16F10 (mouse melanoma), and HCC1954 (human breast cancer). A single siSTAT3 sequence was used throughout. siSCR = scrambled siRNA control. ***p<0.001; ****p<0.0001. The siSTAT3 dose was 50 nM, and the CpG dose was 220 nM. Figure 4C is a graph showing the co-delivery of non-targeting scrambled siRNA (siSCR) and CpG by NP or Dharmafect into dendritic cells harvested from C3H / HEJ mice. The dose of each siRNA was 50 nM and 2.0% by weight of NP, and the dose of CpG was 4% by weight of NP. The siRNA-Dharmafect formulation was prepared according to the manufacturer's protocol. Forty-eight hours after treatment, mRNA was analyzed using qRT-PCR. Figure 4D is a graph showing that, in addition to siSTAT3, NP can also deliver siRNA against PD-L1 (siPDL1), resulting in effective knockdown of PD-L1 protein expression (as measured by flow cytometry) in LLC-JSP cells. Cells were treated with NPs containing 30 nM siRNA against PD-L1 (siPDL1) or 30 nM scrambled siRNA (siSCR) at 2% by weight of siRNA. After 72 hours of treatment, cells were harvested and assessed for PD-L1 protein expression by flow cytometry. RFU = relative fluorescence units. "NP" refers to mesoporous silica nanoparticles coated with cross-linked PEI and PEG, as described in Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015 and U.S. Patent Application Publication No. 2017 / 0173169. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 5A] Figures 5A-5C show that siSTAT3-CpG-NPs induce greater immunogenic effects than NPs delivering siSTAT3 or CpG alone. Mice bearing bilateral B16F10 melanoma tumors were intratumorally injected with a total of three doses at 3-day intervals into one tumor. (Figure 5A) Tumor growth curves for the locally treated tumor and (Figure 5B) the distant untreated tumor are plotted as mean ± SEM. (Figure 5C) Mouse survival curves. Dose (per injection): CpG 20 μg; siSTAT3 4 μg; NP 0.22 mg. *p<0.05, ****p<0.0001 for CpG-NPs vs. siSTAT3-CpG-NPs. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6A] Figures 6A-6C show that siSTAT3-CpG-NPs enhance CD8+ T cell proliferation within tumors and draining lymph nodes (DLNs) better than NPs delivering siSTAT3 or CpG alone. The model, treatment dose, and schedule were as shown in Figures 5A-5C. Seven days after the first treatment, cells harvested from tumors and DLNs from both local (treated) and distant (untreated) tumors were analyzed to determine the ratio of CD8+ T cells to CD4+FoxP3+ regulatory T cells in the live CD45+CD3+ T cell populations in the tumor (Figure 6A) and DLN (Figure 6B), along with the proliferation status (Ki-67) of effector (CD44+) CD8+ T cells within the lymph nodes (Figure 6C). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 for siSTAT3-CpG-NP vs. saline (n=3 / group) unless brackets specify otherwise. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7]Figure 7 is a graph showing the percentage of IFNγ-activated CD8+ T cells after incubation in the presence of SF (SEQ ID NO: 90). Cells were obtained from untreated mice, mice treated with SF and CpG-loaded NPs (CpG-SF-NPs), mice treated with SF-loaded NPs (SF-NPs), mice treated with CpG-loaded NPs (CpG-NPs), and mice treated with SF formulated with incomplete Freund's adjuvant (IFA / SF). *p<0.05. Doses used: 16 μg CpG and 40 μg SF. [Figure 8A] Figures 8A and 8B show (Figure 8A) 1) the hydrodynamic size of mesoporous silica nanoparticles coated with PEI and PEG (NPs) and loaded with approximately 3 wt% SARS-CoV-2 spike protein, 2 wt% siRNA, and 4 wt% CpG, and (Figure 8B) the successful silencing of luciferase in LM2-4luc / H2N upon treatment with 30 or 60 nM siLUC (an siRNA against luciferase) delivered by spike protein-conjugated NPs (spike-NPs) containing 2 wt% of siRNA. siSCR = scrambled siRNA control. [Figure 8B] See legend to Figure 8A. [Figure 9] Figures 9A and 9B show (Figure 9A) luciferase gene knockdown in the H2N (breast) cell line by luciferase siRNA delivered with CaP-L as in Table 5. Figure 9B shows that the treatment was not toxic to the cells, as indicated by unchanged total protein levels in treated cells compared to untreated cells. The siRNA dose was 50 nM. Protein analysis was after 2 days of treatment. [Figure 10]Figure 10 shows the population of activated dendritic cells (MHCII+CD80+CD11c+ cells) after treatment with CpG, siSTAT3-NP, CpG-NP, siSTAT3-CpG-NP (AIRISE-02), or AIRISE-CoV (an immunogenic construct containing spike protein, siSTAT3, and CpG). Mice were administered various treatments via footpad injection. The dose was 0.5 mg of NP (2% by weight siSTAT3, 4% by weight CpG, and / or 3% by weight SARS-CoV-2 spike protein antigen). Two days after treatment, draining lymph nodes (DLN) and non-draining lymph nodes (NDLN) were harvested and processed into single cells for flow cytometry analysis. *p<0.05 (n=3 / group) vs. saline. [Figure 11] Figure 11 shows humoral responses to SARS-CoV-2 spike (S) antigen in BALB / c mice vaccinated with AIRISE-CoV. Eight-week-old BALB / c mice were vaccinated by footpad injection of 80 μl of AIRISE-CoV (0.5 mg NP, 2% by weight siSTAT3, 4% by weight CpG, 3% by weight SARS-CoV-2 spike protein antigen) (Dose 1: Day 0; Dose 2: Day 17). Serum was collected on Day 16 (effect of Dose 1) and Day 38 (effect of Dose 2), and SARS-CoV-2 S IgG antibody levels were assessed by ELISA after serial dilution of serum. Data represent the mean OD450nm values ​​(mean ± SD) for n=2 (naive) and n=3 (AIRISE-CoV). [Figure 12A]Figures 12A and 12B show humoral responses to SARS-CoV-2 spike (S) antigen in BALB / c mice vaccinated with AIRISE-CoV 12 weeks later. Eight-week-old BALB / c mice (M1–M3 represent mice 1, 2, and 3) were vaccinated with two SARS-CoV-2 spike peptides (0.5 mg NP, 2% siSTAT3 by weight, 4% CpG by weight, and 3% SARS-CoV-2 spike peptide antigen) via footpad injection (Figure 12A) of 80 μl of AIRISE-CoV (0.5 mg NP, 2% siSTAT3 by weight, 4% CpG by weight, and 3% SARS-CoV-2 spike protein antigen) or (Figure 12B) of 80 μl of AIRISE-CoV (Dose 1: day 0; Dose 2: day 17). Serum was collected on day 80 to assess SARS-CoV-2 S IgG antibody levels by ELISA. Data represent the mean OD450nm values ​​(mean ± SD) of two experimental replicates for each immunized or naive mouse. [Figure 12B] See legend to Figure 12A. [Figure 13] Figure 13 shows humoral responses to SARS-CoV-2 spike (S) antigen in BALB / c mice vaccinated with two doses of AIRISE-CoV. Eight-week-old BALB / c mice were vaccinated by footpad (fp) injection with AIRISE-CoV (0.5 mg NP, 2% by weight siSTAT3, 4% by weight CpG, 3% by weight SARS-CoV-2 spike protein antigen) (Dose 1: day 0; Dose 2: day 17). Serum was collected at different weeks post-vaccination (weeks 3 to 54) and SARS-CoV-2 S IgG antibody levels were assessed by ELISA. Data represent the mean OD450nm values ​​(mean ± SD) of five immunized and one naive mouse. [Figure 14A]Figures 14A-C show humoral responses to SARS-CoV-2 spike (S) antigen in BALB / c mice vaccinated with a single dose of AIRISE-CoV, siSTAT3-spike-NP, or CpG-spike-NP, respectively. Eight-week-old BALB / c mice were vaccinated by intramuscular (i.m.) injection with a single dose of (A) AIRISE-CoV (0.4 mg NP, 2% by weight siSTAT3, 4% by weight CpG, 3% by weight SARS-CoV-2 spike protein antigen; n = 4), (B) siSTAT3-spike-NP (0.4 mg NP, 2% by weight siSTAT3, 3% by weight spike protein antigen; n = 3), or (C) CpG-spike-NP (0.4 mg NP, 4% by weight CpG, 3% by weight spike protein antigen; n = 3). Serum was collected at different weeks after vaccination (weeks 6–36) to assess SARS-CoV-2 S IgG antibody levels by ELISA. Data represent the mean OD450nm values ​​(mean ± SD) of 3–4 immunized mice and 1 naive mouse. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 15] Figure 15 shows the inhibition of SARS-CoV-2 pseudovirus infection of HEK293-hACE2 cells by immune serum (from Figure 11). Graph showing % GFP+ cells at various dilutions of serum from mice immunized with AIRISE-CoV versus naive mice. Calculated neutralization titer (dilution required to neutralize 50% of virus; NT50) values ​​are shown in Table 6. [Figure 16] Figures 16A and 16B are graphs showing the cell viability of BMDCs (Figure 16A) and J774 (Figure 16B) after treatment with siSTAT3-NP or siSTAT3-CpG-NP. The NP dose is 35 μg / ml (2% siRNA by weight; 7% CpG by weight) after 2 days of treatment.

[0037] Reference to sequence listing The nucleic acid and / or amino acid sequences described herein are shown using standard abbreviations as defined in 37 CFR § 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included in the embodiments, where appropriate. A computer-readable text file entitled "2IS9696.txt (Sequence Listing.txt)," created on or about July 12, 2021, with a file size of 16 KB, contains the sequence listing for the present application and is incorporated herein by reference in its entirety.

[0038] SEQ ID NOs: 1-8 are the amino acid sequences of representative SARS-CoV-2 T cell and / or B cell epitopes derived from the spike protein (SEQ ID NOs: 1-5), nucleocapsid protein (SEQ ID NO: 6), membrane protein (SEQ ID NO: 7) and envelope protein (SEQ ID NO: 8).

[0039] SEQ ID NOs: 8-89 and 91 are nucleic acid sequences corresponding to exemplary siRNAs as set forth in Table 2 (below). Although not included in each sequence in the sequence listing, each siRNA may optionally include a deoxythymidine dinucleotide (dTdT) overhang or other deoxydinucleotide (e.g., dTdG) overhang at the 3' end.

[0040] SEQ ID NO:90 is an ovalbumin peptide used to stimulate T cells. DETAILED DESCRIPTION OF THE INVENTION

[0041] Detailed Description Described herein are immunogenic constructs for inducing an immune response, such as for the treatment or prevention of an infectious agent, e.g., a virus, e.g., a β-coronavirus infection, e.g., SARS-CoV-2 infection, SARS-CoV-1 infection, MERS-CoV infection, or other viruses and pathogens. These novel immunogenic constructs for infectious diseases utilize a single delivery vehicle to deliver an antigen, an adjuvant, and in some embodiments, an siRNA to modulate immunosuppressive genes and stimulate immunity.

[0042] The immunogenic construct contains nanoparticles (e.g., mesoporous silica nanoparticles (MSNPs)), cationic polymers (e.g., PEI), stabilizers (e.g., PEG), and antigens, and in some embodiments, at least one adjuvant (e.g., CpG) and / or oligonucleotide (e.g., siRNA). Combinations of various additional agents are also contemplated. The immunogenic constructs of the present disclosure may also contain multiple types of cationic polymers, stabilizers, antigens, adjuvants, and / or oligonucleotides. For example, the immunogenic construct may contain multiple different oligonucleotides and / or antigens that act on the same or different target infectious agents. The use of such additional agents may provide additive or synergistic effects.

[0043] The immunogenic constructs of the present disclosure can be used to co-deliver adjuvants (e.g., CpG oligonucleotides), viral antigens (e.g., proteins or peptides) or antigen-producing agents (e.g., mRNA or pDNA), and optionally siRNA to induce potent, long-lasting immunity against novel infectious diseases (Figure 1). The immunogenic constructs utilize antigens to prime the body's antigen-presenting cells (e.g., dendritic cells, B cells, and macrophages) to activate effector and memory T lymphocytes that recognize infectious agent proteins and humoral immune responses. Such immunogenic constructs can prevent future infections or reduce disease severity.

[0044] Aspects of the present disclosure will now be described with the following additional details and options: (I) Definitions; (II) Nanoparticles; (III) Cationic Polymers; (IV) Stabilizers; (V) Adjuvants; (VI) Antigens and Antigen-Generating Factors; (VII) Oligonucleotides; (VIII) Targeting Agents; (IX) Labeling Agents; (X) Synthesis of Immunogenic Constructs; (XI) Immunogenic Constructs Comprising Lipid-Coated Nanoparticles; (XII) Immunogenic Construct Formulations and Methods of Use; (XIII) Exemplary Embodiments; (XIV) Experimental Examples (including Examples 1-8); and (XV) Final Paragraph. These headings are not intended to limit the interpretation of this disclosure and are provided for organizational purposes only.

[0045] I. Definition To facilitate understanding of this disclosure, several terms are defined below. Terms defined herein have meanings that are commonly understood by those skilled in the art to which this disclosure pertains. Terms used herein are used to describe certain aspects of this disclosure, but their use does not limit this disclosure, except as outlined in the claims.

[0046] The term "CpG motif" refers to a 5' C nucleotide linked to a 3' G nucleotide via a phosphodiester or phosphodiester derivative internucleotide linkage. In some embodiments, the CpG motif comprises a phosphodiester internucleotide linkage. In some embodiments, the CpG motif comprises a phosphodiester derivative internucleotide linkage.

[0047] As used herein, the term "class A CpG ODN" (also referred to as "A-class CpG ODN," "D-type CpG ODN," or "class A CpG DNA sequence") is used according to its general meaning in biological and chemical sciences and refers to a CpG motif comprising a poly-G sequence at the 5', 3', or both ends; an internal palindromic sequence containing a CpG motif; or an oligodeoxynucleotide comprising one or more of one or more phosphodiester derivatives binding deoxynucleotides. In some embodiments, a class A CpG ODN comprises a poly-G sequence at the 5', 3', or both ends, an internal palindromic sequence containing a CpG motif, and one or more phosphodiester derivatives binding deoxynucleotides. In some embodiments, the phosphodiester derivative is phosphorothioate. Examples of class A CpG ODNs include ODN D19, ODN 1585, ODN 2216, and ODN 2336.

[0048] The terms "class B CpG ODN" or "B class CpG ODN" or "K-type CpG ODN" or "class B CpG DNA sequence" are used according to their common meaning in biological and chemical sciences and refer to a hexameric motif containing a CpG motif; a CpG motif containing an oligodeoxynucleotide containing one or more phosphodiester derivatives that bind to every deoxynucleotide. In some embodiments, a class B CpG ODN contains a hexameric motif containing a CpG motif and one or more copies of a phosphodiester derivative that binds every deoxynucleotide. In some embodiments, the phosphodiester derivative is phosphorothioate. In some embodiments, a class B CpG ODN contains one hexameric motif containing a CpG motif. In some embodiments, a class B CpG ODN contains two copies of a hexameric motif containing a CpG motif. In some embodiments, a class B CpG ODN contains three copies of a hexameric motif containing a CpG motif. In some embodiments, the class B CpG ODN contains four copies of a hexameric motif containing a CpG motif. Examples of class B CpG ODNs include ODN 1668, ODN 1826, ODN 2006, and ODN 2007.

[0049] The term "class C CpG ODN" or "C-class CpG ODN" or "C-type CpG DNA sequence" is used according to its general meaning in biological and chemical sciences, and refers to an oligodeoxynucleotide that contains a palindromic sequence containing a CpG motif and a phosphodiester derivative (phosphorothioate) that binds every deoxynucleotide. Examples of class C CpG ODN include ODN 2395 and ODN M362.

[0050] As used herein, "immunogenicity" refers to the ability of an agent (e.g., an immunogenic construct, components thereof, or a composition containing an immunogenic construct) to elicit an immune response as measured, for example, by in vitro assays (e.g., mixed lymphocyte reaction; killing of cytotoxic T cells, upregulation of cytokines upon stimulation of immune cells with antigen, etc.), ex vivo assays (e.g., antibody neutralizing titers by microneutralization test assays; antigen-specific antibodies and antibody-secreting B cells by ELISA assays), and in vivo assays that confirm successful induction of cellular and humoral immunity (e.g., ability of immunization to protect living specimens from viral challenge).

[0051] As used herein, the term "immunogenic amount" refers to an amount of an immunogenic construct or composition that induces an immune response in a subject (e.g., as reflected by an increase in antibody titers in the subject, as determined by conventional techniques such as ELISA).

[0052] As used herein, the term "infectious agent" refers to an agent that causes infection and / or disease. Infectious agents include viruses, bacteria, fungi, and parasites, or combinations thereof. In some embodiments, the infectious agent is a virus. Additional infectious agents are described herein and / or known to those skilled in the art. In an example, an infectious agent can be referred to as a "target" of the immunogenic constructs described herein. For example, the viral target can be a coronavirus, corynebacterium, ebolavirus, orthomyxovirus, hepatovirus, Haemophilus bacteria, HIV, HPV, morbillivirus, mycobacterium, meningococcus bacteria, orthorubulavirus, norovirus, streptococcus, enterovirus, orthopneumovirus, rotavirus, rubivirus, herpesvirus, clostridium bacteria, bordatella bacteria, or flavivirus. A pathogen is also called an infectious agent.

[0053] As used herein, the term "infectious disease" refers to a disease caused by an infectious agent such as a bacterium, a virus, a parasite, or a fungus. In some embodiments, the infectious disease is a viral infection. Examples of infectious diseases include coronavirus-based infections (such as Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), and coronavirus diseases (e.g., COVID-19)); Corynebacterium-based infections (such as diphtheria); Ebola virus-based infections (such as Ebola); orthomyxoviridae virus-based infections (such as influenza A, B, or C); hepatovirus A, B, C, D, or E-based infections (such as hepatitis); Haemophilus-based infections (such as Hib disease); human immunodeficiency virus (HIV)-based infections (such as acquired immunodeficiency syndrome (AIDS)); human papillomavirus (human papillomavirus (HPV)-based infections; Morbillivirus-based infections (e.g., measles); Mycobacterium-based infections (e.g., tuberculosis); Neisseria-based infections (e.g., meningitis); Orthorbulavirus-based infections (e.g., mumps); Norovirus-based infections; Streptococcus-based infections; Enterovirus-based infections (e.g., polio); Orthopneumovirus-based infections; Rotavirus-based infections; Rubivirus-based infections (e.g., rubella); Herpesvirus-based infections (e.g., herpes, chickenpox, and shingles); Clostridium-based infections (e.g., tetanus and botulism); Bordatella-based infections (e.g., whooping cough); Flavivirus-based infections (e.g., Zika); and the like. Additional infectious diseases are described herein and / or known to those skilled in the art (e.g., Pati et al., Front Immunol. 9:2224, 2018 (page 16) and references cited therein).

[0054] As used herein, the terms "biologically acceptable excipient" and "pharmaceutically acceptable excipient" refer to any inert ingredient (e.g., a vehicle capable of suspending an immunogenic construct) that has the properties of being non-toxic and non-inflammatory in a subject. Typical excipients include, for example, carriers, binders, fillers, lubricants, emulsifiers, suspending agents, sweeteners, flavorings, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other conventional excipients and additives, and / or other additives that may improve stability, delivery, absorption, half-life, efficacy, pharmacokinetics, and / or pharmacodynamics, reduce adverse side effects, or provide other benefits for biological and / or pharmaceutical and / or dietary supplement use. In some embodiments, acceptable excipients include adjuvants that are not bound to the immunogenic construct.

[0055] As used herein, "pDNA" refers to plasmid DNA, eg, a plasmid that encodes at least one antigen of an infectious agent.

[0056] As used herein, the term "preventing" refers to reducing (e.g., by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or about 100%) the risk of contracting an infectious disease, such as a viral infection (such as an infection with a β-coronavirus, e.g., SARS-CoV-2, SARS-CoV-1, MERS-CoV, or related viruses), bacterial infection, fungal infection, or parasitic infection. To determine whether prevention is effective, a comparison can be made between subjects administered a composition of the present disclosure and similarly situated subjects (e.g., subjects at risk for a viral infection, such as SARS-CoV-2, SARS-CoV-1, or MERS-CoV, or infection with a related virus) who did not receive the composition. Comparisons can also be made between subjects administered the composition and a control, baseline, or known measured level.

[0057] As used herein, the term "subject" can be a human, a non-human primate, or a non-primate mammal, such as a dog, cat, horse, cow, pig, horse, goat, monkey, rat, mouse, and / or sheep. In some embodiments, the subject is a human.

[0058] As used herein, the term "TLR-binding DNA substituent" refers to a substituent or moiety capable of binding to a Toll-like receptor ("TLR"), comprising at least one deoxyribonucleic acid. In some embodiments, the TLR-binding DNA substituent is a nucleic acid. In some embodiments, the TLR-binding DNA substituent comprises at least one nucleic acid analog. In some embodiments, the TLR-binding DNA substituent comprises at least one nucleic acid analog with an alternative backbone (e.g., a phosphodiester derivative (e.g., phosphoramidate, phosphorodiamidate, phosphorothioate, phosphorodithioate, phosphonocarboxylic acid, phosphonocarboxylate, phosphonoacetic acid, phosphonoformic acid, methylphosphonate, boronphosphonate, or O-methylphosphoramidite), a peptide nucleic acid backbone, LNA, or linkage). In some embodiments, the TLR-binding DNA substituent comprises DNA. In some embodiments, all of the nucleotides in the TLR-binding DNA substituent are deoxyribonucleotides. In some embodiments, the TLR-binding DNA substituent comprises or is DNA having internucleotide linkages selected from phosphodiesters and phosphodiester derivatives (e.g., phosphoramidates, phosphorodiamidates, phosphorothioates, phosphorodithioates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acids, phosphonoformic acids, methylphosphonates, boron phosphonates, O-methyl phosphoramidites, or combinations thereof). In some embodiments, the TLR-binding DNA substituent comprises DNA having internucleotide linkages selected from phosphodiesters and phosphorothioates. In some embodiments, the TLR-binding DNA substituent comprises or is DNA having backbone linkages selected from phosphodiesters, phosphorothioates, and phosphorodithioates. In some embodiments, the TLR-binding DNA substituent comprises or is DNA having phosphodiester backbone linkages. In some embodiments, the TLR-binding DNA substituent comprises or is DNA having phosphorothioate backbone linkages.In some embodiments, the TLR-binding DNA substituent comprises or is DNA comprising phosphorodithioate backbone linkages. In some embodiments, the TLR-binding DNA substituent preferentially binds to TLR9 over other TLRs. In some embodiments, the TLR-binding DNA substituent specifically binds to TLR9. In some embodiments, the TLR-binding DNA substituent specifically binds to TLR3. In some embodiments, the TLR-binding DNA substituent specifically binds to TLR7. In some embodiments, the TLR-binding DNA substituent specifically binds to TLR8. In some embodiments, the TLR-binding DNA substituent specifically binds to a cell subcompartment (e.g., endosome)-associated TLR (e.g., TLR3, TLR7, TLR8, or TLR9). In some embodiments, the TLR-binding DNA substituent comprises or is a G-rich oligonucleotide. In some embodiments, the TLR-binding DNA substituent comprises a CpG motif (i.e., is a CpG oligodeoxynucleotide (ODN)). In some embodiments, the CpG motif is unmethylated. In some embodiments, the TLR-binding DNA substituent is a class A CpG oligodeoxynucleotide (ODN). In some embodiments, the TLR-binding DNA substituent is a class B CpG oligodeoxynucleotide (ODN). In some embodiments, the TLR-binding DNA substituent is a class C CpG oligodeoxynucleotide (ODN). In some embodiments, the TLR-binding DNA substituent (e.g., a TLR9-binding DNA substituent) comprises a deoxyribonucleic acid having an A, G, C, or T base and a phosphodiester and / or phosphodiester derivative linkage (e.g., a phosphorothioate linkage).

[0059] As used herein, the term "treatment" or "treating" refers to reducing, diminishing, reducing the progression of, or reducing the side effects of an infectious disease, such as a viral infection, such as a β-coronavirus (e.g., SARS-CoV-2 infection, SARS-CoV-1 infection, or MERS-CoV infection, or related viruses) infection (e.g., by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or about 100%). To determine whether a treatment is effective, a comparison can be made between treated subjects and similarly situated subjects who did not receive treatment (e.g., subjects with or at risk of a viral infection, such as an infection with SARS-CoV-2 infection, SARS-CoV-1 infection, or MERS-CoV infection, or a related viral infection). Comparisons can also be made between treated subjects and a control, baseline, or known level or measurement. Treating a viral infection (e.g., a β-coronavirus infection, e.g., a SARS-CoV-2 infection, a SARS-CoV-1 infection, or a MERS-CoV infection, or an infection by a related virus) can be achieved by reducing the viral load (e.g., by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or about 1%). 00%), reducing the number of days the subject is hospitalized (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more days), reducing the number of days the subject requires antiviral therapy (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more days), and / or reducing the dose of antiviral therapy required by the subject.

[0060] As used herein, the term "vaccine" refers to an agent (e.g., an immunogenic construct, a component thereof, or a composition containing an immunogenic construct) that can induce an immune response in a subject against an infectious agent and / or treat and / or prevent infection and / or disease associated with the infectious agent.

[0061] II. Nanoparticles Nanoparticles useful with the compositions and methods of the present disclosure include, but are not limited to, mesoporous silica nanoparticles (e.g., MSNPs), iron oxide nanoparticles, silver nanoparticles, gold nanoparticles, calcium phosphate, inorganic nanoparticles, carbon nanotubes, liposomes, lipid nanoparticles, or cationic polymer particles. Nanoparticles may or may not be porous. Exemplary sizes of the nanoparticle core are about 5 nm to about 999 nm, about 5 nm to about 90 nm, about 5 nm to about 20 nm, about 20 nm to about 400 nm, about 20 nm to about 500 nm, about 20 nm to about 100 nm, about 20 nm to 200 nm, about 30 nm to about 100 nm, about 30 nm to about 80 nm, about 30 nm to about 60 nm, about 40 nm to about 80 nm, about 50 nm to 400 nm, about 50 to 500 nm, about 70 nm to about 90 nm, about 100 nm to about 200 nm, about 200 nm to about 500 nm, about 500 nm to about 999 nm, or about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm. Generally, the nanoparticle core is spherical, although other shapes such as rods and discs can also be used. In some embodiments, the nanoparticles are mesoporous silica nanoparticles (MSNPs).

[0062] In some aspects, the nanoparticles have adjuvant or immunostimulatory properties. Exemplary nanoparticles with adjuvant or immunostimulatory properties include liposomes, lipoplexes, lipid-based particles, polyplexes, polymeric particles, inorganic particles (e.g., aluminum salt particles and calcium phosphate nanoparticles), virus-like particles, or nanoparticles such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), cholesterol, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, phosphatidylcholine / cholesterol, chitosan, poly-γ-glutamic acid (γ-PGA), hyaluronic acid, polyethyleneimine (PEI), poly(propylacrylic acid), polypropylene sulfide (PPS), polylactic-co-glycolic acid (PLGA), amylopectin, maltodextrin, polystyrene, gold, cobalt oxide, alum, tri-palmitoyl-S-glycerylcysteine ​​(PAM3Cys), squalene, Montanide ISA 50V, Montanide ISA 51, Montanide ISA 201, Montanide ISA 206 and Montanide ISA 720.

[0063] To create nanoparticle platforms (NPs), additional components are covalently or noncovalently attached to nanoparticles by various mechanisms. For example, cationic polymers can be attached to nanoparticles by charge, e.g., in the case of silica or iron oxide nanoparticles. Alternatively, the surface of the nanoparticle can be modified to contain reactive moieties for conjugation to the cationic polymer and / or other components, or the cationic polymer or other components can contain moieties that bind to the nanoparticle. For example, nanoparticle cores, such as silica, silicon, gold, iron oxide, and silver nanoparticles, as well as carbon nanotubes, can be modified with reactive moieties such as thiols, phosphonates, carboxylates, and amines prior to attachment to the cationic polymer and other components. The cationic polymer and other components can be modified to contain these or other moieties, including maleimides, N-hydroxysuccinimidyl (NHS) esters, or azides, prior to attachment to the nanoparticle core. Components can be attached directly to the nanoparticles, either on the surface or within the pores (if present). Large molecular cargo such as proteins, mRNA, or plasmid DNA (pDNA) are attached to the outer surface of the nanoparticle (or optionally on a coating applied to the nanoparticle core, i.e., loaded onto the NP), while smaller molecules such as dyes can be attached inside the nanoparticle or pores of the NP, as well as to the outer surface. The immunogenic constructs of the present disclosure advantageously maintain their submicron size after being loaded with additional components.

[0064] III. Cationic Polymers In some embodiments, nanoparticles such as MSNPs are coated with cationic polymers or other compounds. The cationic polymers can be attached to the surface of the nanoparticles using any suitable means. In some embodiments, the cationic polymers are attached to the nanoparticles via electrostatic interactions. The cationic polymer can be any polymer with a positive charge, such as, but not limited to, PEI, polyamidoamine, poly(allylamine), poly(diallyldimethylammonium chloride), chitosan, poly(N-isopropylacrylamide-co-acrylamide), poly(N-isopropylacrylamide-co-acrylic acid), poly(L-lysine), diethylaminoethyl-dextran, poly(N-ethyl-vinylpyridinium bromide), poly(dimethylamino)ethyl methacrylate), or poly(ethylene glycol)-co-poly(trimethylamine-ethyl methacrylate chloride). Other cationic polymers will be apparent to those skilled in the art and can be found, for example, in Polymer Handbook, 4th Edition, Edited by: Brandrup, EH Immergut, and EA Grukle; John Wiley & Sons, 2003).

[0065] The cationic polymer can be linear or branched. In some embodiments, the cationic polymer can range in size from about 500 Da to about 25 kDa and can be branched or linear. For example, branched PEI with an average size of 1.8 kDa to 10 kDa can be loaded onto nanoparticles (yielding a nanoparticle platform; NP). The ratio of cationic polymer to nanoparticles can vary depending on the desired results. The cationic polymer can be present at 1-50% by weight of the NP, e.g., 5-40%, 10-30%, 20-30%, 5-15%, 5-20%, 5-25%, 5-30%, 10-20%, 10-25%, or 25-40% by weight, e.g., about 5, about 10, about 15, about 20, about 25, about 30, or about 35% by weight. In some embodiments, the cationic polymer is present at 10-20% by weight of the NP.

[0066] In some embodiments, the cationic polymer is crosslinked, e.g., by a cleavable disulfide bond, before or after coating onto the nanoparticles. In some embodiments, the attached cationic polymer is crosslinked after binding to the nanoparticles, e.g., MSNP, using, e.g., DSP (dithiobis[succinimidylpropionate]), DTSSP (3,3'-dithiobis(sulfosuccinimidylpropionate) and DTBP (dimethyl 3,3'-dithiobispropionimidate). Crosslinking can occur in the absence or presence of free cationic polymer in solution. In other embodiments, the cationic polymer is not crosslinked.

[0067] IV. Stabilizers The stabilizer can be conjugated to the nanoparticles and / or cationic polymers, for example, by any suitable means. In some embodiments, the stabilizer is conjugated to the amine group or other reactive group of the cross-linked cationic polymer coated on the nanoparticles (e.g., MSNPs). Exemplary stabilizers include PEG, dextran, polysialic acid, hyaluronic acid, polyvinylpyrrolidone, polyvinyl alcohol, and polyacrylamide, or a combination thereof.

[0068] The stabilizer may have multiple chemically reactive groups for attachment to, for example, nanoparticles, cationic polymers, and / or other components. For example, a reactive stabilizer, such as a PEG derivative, may have two functional moieties, such as maleimide-PEG-N-hydroxysuccinimidyl ester (Mal-PEG-NHS), which contains both a maleimide and an activated ester at each end. Stabilizers, such as PEG, used in conjunction with the compositions and methods of the present disclosure generally have molecular weights ranging from 500 Da to 40 kDa, e.g., 2 to 10 kDa. The stabilizer may be present at 1 to 50% by weight of the NP, e.g., 5 to 30%, 10 to 20%, 10 to 25%, 5 to 15%, 5 to 20%, 5 to 25%, or 1 to 10% by weight, e.g., about 5, about 10, about 15, about 20, about 25, about 35, about 40, or about 45% by weight.

[0069] In some embodiments, stabilizers (e.g., PEG) are introduced to enhance NP stability (e.g., reduce aggregation and precipitation) and / or to protect cargo molecules such as siRNA, miRNA, mRNA, and pDNA from enzymatic degradation.

[0070] Stabilizers (e.g., PEG) used in conjunction with the compositions and methods of the present disclosure generally have a molecular weight ranging from 500 Da to 40 kDa, e.g., 5 kDa, 2-5 kDa, 2-10 kDa, or 5-10 kDa. In various embodiments, the stabilizer is present at 1-50% by weight of the nanoparticle platform (NP), e.g., 5-30%, 10-20%, 10-25%, 5-15%, 5-20%, 5-25%, or 1-10%, e.g., about 5, about 10, about 15, about 20, about 25, about 35, about 40, or about 45% by weight. The size and density of the stabilizer can be optimized to accommodate large cargoes such as proteins and mRNA.

[0071] In some embodiments, the stabilizer is introduced before cargo loading. In some embodiments, the stabilizer is introduced after cargo loading. In some embodiments, the stabilizer is introduced both before and after cargo loading and / or simultaneously with loading of at least one cargo molecule.

[0072] Some nanoparticles (such as mesoporous silica nanoparticles and iron oxide nanoparticles coated with PEI and PEG) can protect antigens and antigen-producing factors during long-term storage and transportation, eliminating the need to store the materials at very low temperatures (e.g., -80 °C).

[0073] V. Adjuvants The constructs provided herein may include at least one adjuvant. The adjuvant may be contained within the nanoparticle, or may be associated with the nanoparticle, cationic polymer, or stabilizer through non-covalent or covalent interactions, including hydrogen bonding with moieties on the nanoparticle, van der Waals interactions, electrostatic interactions, hydrophobic interactions, and chemical conjugation. Chemical conjugation includes thiol-maleimide, NHS ester-amine, azide-alkyne, and other click chemistry. In some embodiments, the adjuvant is thiolated and conjugated to a stabilizer containing a maleimide group via a thiol-maleimide reaction (see International Application No. PCT / US2016 / 022655, the entire contents of which are incorporated herein by reference). In some embodiments, the adjuvant is electrostatically loaded onto a cationic polymer coated on the nanoparticle. The adjuvant may be present at 1-20% by weight of the NP, e.g., 1-10%, 2-7%, 2-4%, 2-10%, 5-10%, 10-20% by weight, e.g., about 4%, about 5%, about 6%, about 7%, about 10% or about 20% by weight.

[0074] Adjuvants may also be part of or conjugated to a therapeutic agent; for example, oligonucleotides such as siRNAs that knock down target genes may be designed to contain immunostimulatory sequences.

[0075] Generally, an adjuvant is any substance that, when mixed into a vaccine composition, enhances or otherwise modifies the immune response to an antigen. The ability of an adjuvant to enhance the immune response to an antigen is typically manifested by a significant increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies raised against the antigen, and an increase in T cell activity is typically manifested by increased antigen-specific T cell proliferation, target cell death, or cytokine secretion. Adjuvants can also alter the immune response, for example, by shifting a predominantly humoral or Th2 response to a predominantly cellular or Th1 response.

[0076] Suitable adjuvants include TLR-binding DNA substituents, such as CpG oligonucleotides (e.g., ISS1018; Amplivax; CpG ODN 7909, CpG ODN 1826, CpG ODN D19, CpG ODN 1585, CpG ODN 2216, CpG ODN 2336, ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN 2395, ODN M362, and SD-101), DNA TLR agonists containing CpG sequences (e.g., dSLIM), non-CpG DNA TLR agonists (e.g., EnanDIM), and cationic peptide-conjugated CpG oligonucleotides (e.g., IC30, IC31); RNA TLR agonists (e.g., poly I:C and poly-ICLC); aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, aluminum chloride, and aluminum potassium sulfate); anti-CD40 antibodies (e.g., CP-870, 893); cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF); small molecule TLR agonists (e.g., imiquimod, resiquimod, gardikimod, and 3M-052); fusion proteins (e.g., ImuFact IMP321, CyaA, and ONTAK); oil-based or surfactant-based adjuvants, such as MF59, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, and Montanide ISA-51; plant extracts, such as QS21 stimulon derived from saponin (Aquila Biotech, Worcester, Mass., USA); mycobacterial extracts and synthetic bacterial cell wall mimics, such as lipopolysaccharides (e.g., monophosphoryl lipid A, OM-174, OM-197-MP-EC and Pam3Cys); xanthenone derivatives (e.g., vadimezan); mixtures thereof (e.g., AS-15); and other proprietary adjuvants, such as Ribi's Detox, Quil or Superfos.Several immunological adjuvants (e.g., dendritic cell-specific MF59) and their preparations have been previously described (Dupuis et al., Cell Immunol. 186(1):18-27, 1998; Allison, Dev Biol Stand.; 92:3-11, 1998). Cytokines may also be used. Several cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), promoting dendritic cell maturation into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589), and functioning as immune adjuvants (e.g., IL-12) (Gabrilovich et al., J Immunother Emphasis Tumor Immunol. (6):414-418, 1996). Toll-like receptors (TLRs), or agents that activate TLRs, may also be used as adjuvants and are important members of a family of pattern recognition receptors (PRRs) that recognize conserved motifs shared by many microorganisms, called "pathogen-associated molecular patterns" (PAMPs).

[0077] In some embodiments, the adjuvant comprises a CpG oligonucleotide. CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in vaccine settings. Without being bound by any specific mechanistic theory, CpG oligonucleotides function at least in part by activating the innate (non-adaptive) immune system via Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates, in both prophylactic and therapeutic vaccines. More importantly, CpG-induced TLR9 activation promotes the maturation and differentiation of dendritic cells, even in the absence of CD4 helper T cells, leading to the activation of T cells. HTLR9 stimulates T cells and enhances their activation, generating potent cytotoxic T lymphocytes (CTLs). H 1 bias is usually T H This effect is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which promote a 2-bias. CpG oligonucleotides exhibit even greater adjuvant activity when formulated or coadministered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, which are particularly necessary to induce strong responses when the antigen is relatively weak. They also accelerate immune responses, allowing in some experiments to reduce antigen doses by two orders of magnitude while obtaining antibody responses comparable to full-dose vaccines without CpG (Krieg, Nature Reviews, Drug Discovery, 5:471-484, 2006). U.S. Patent No. 6,406,705 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. A commercially available CpG TLR9 antagonist is dSLIM (double stem loop immunomodulator) manufactured by Mologen (Berlin, Germany). Other TLR binding molecules may also be used, for example, RNA-binding TLR 7, RNA-binding TLR 8 and / or RNA-binding TLR 9.

[0078] For example, xanthenone derivatives such as vadimezan or AsA404 (also known as 5,6-dimethylxanthenone-4-acetic acid (DMXAA)) can also be used as adjuvants according to embodiments of the present disclosure. Alternatively, such derivatives can also be administered in parallel with the vaccines of the present disclosure, e.g., via systemic or intratumoral delivery, to stimulate immunity at the tumor site. Without being bound by theory, such xanthenone derivatives are thought to function by stimulating interferon (IFN) production through stimulators of the IFN gene (IFN-ISTING) receptor (see, e.g., Conlon et al., J Immunology, 190:5216-5225, 2013; and Kim et al., ACS Chem Biol, 8:1396-1401, 2013). Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), poly I:C (e.g., poly i:CI2U), non-CpG bacterial DNA or non-CpG bacterial RNA, and immunologically active small molecules and antibodies, such as cyclophosphamide, sunitinib, bevacizumab, Celebrex™, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which can function therapeutically and / or as adjuvants. In the context of the present disclosure, the amounts and concentrations of useful adjuvants and additives can be easily determined by those skilled in the art without undue experimentation. Additional adjuvants include colony stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim).

[0079] In some embodiments, the adjuvant comprises poly-ICLC. Poly-ICLC is a synthetically prepared double-stranded RNA containing polyI and polyC strands with an average length of 5000 nucleotides, stabilized against heat denaturation and hydrolysis by serum nucleases by the addition of polylysine and carboxymethylcellulose. This compound activates the RNA helicase domains of TLR3 and MDA5, both members of the PAMP family, leading to the activation of DCs and natural killer (NK) cells and the production of a "natural mixture" of type I interferons, cytokines, and chemokines. Furthermore, poly-ICLC exerts more direct, broad-spectrum host-targeting anti-infective and potentially anti-tumor effects mediated by two IFN-inducible nuclear enzyme systems: 2'5'-OAS and P1 / eIF2a kinase, also known as PKR(4-6), as well as RIG-I helicase and MDA5.

[0080] Examples of immunological adjuvants that can be combined with the immunogenic construct include TLR ligands, C-type lectin receptor ligands, NOD-like receptor ligands, RLR ligands, and RAGE ligands. TLR ligands can include lipopolysaccharide (LPS) and its derivatives, as well as lipid A and its derivatives, including monophosphoryl lipid A (MPL), glycopyranosyl lipid A, PET-lipid A, and 3-O-desacyl-4'-monophosphoryl lipid A. In a specific embodiment, the immunological adjuvant is MPL. In another embodiment, the immunological adjuvant is LPS. TLR ligands can also include TLR3 ligands (e.g., polyinosinic-polycytidylic acid (poly I:C)), TLR7 ligands (e.g., imiquimod and resiquimod), and TLR9 ligands.

[0081] VI. Antigens and Antigen-producing Factors The immunogenic constructs provided herein comprise at least one antigen or antigen-producing agent. Typical constructs include both at least one antigen and at least one antigen-producing agent. In embodiments in which the immunogenic construct contains multiple antigens and / or antigen-producing agents, these antigens may correspond to / be derived from different infectious agents or may correspond to / be derived from the same infectious agent. An antigen or antigen-producing agent may be considered "of" or "from" an infectious agent if the antigen is capable of eliciting an immune response against the corresponding agent, for example, if the antigen (or antigen-producing agent) is synthesized, engineered, recombinant, and / or produced in a laboratory, or is isolated or extracted from the infectious agent itself.

[0082] The antigen or antigen-producing agent may be partially or completely contained within the nanoparticle, or may be associated with the nanoparticle, cationic polymer, and / or stabilizer through non-covalent or covalent interactions, including hydrogen bonding with moieties on the nanoparticle, van der Waals interactions, electrostatic interactions, hydrophobic interactions, and chemical conjugation. Chemical conjugation includes thiol-maleimide, NHS ester-amine, azide-alkyne, and other click chemistry. In some embodiments, the antigen or antigen-producing agent is thiolated and conjugated to a stabilizer containing a maleimide group via a thiol-maleimide reaction (see International Application No. PCT / US2016 / 022655). In some embodiments, the antigen or antigen-producing agent is loaded onto the cationic polymer through hydrophobic interactions with the nanoparticle. In some embodiments, the antigen or antigen-producing agent is electrostatically loaded onto the cationic polymer. The antigen or antigen-producing factor may be present at 2%, 3%, 4%, 5%, 0.5-20% by weight of the NP, for example 1-15%, 1.5-10%, 1-6% or 2-5% by weight.

[0083] An antigen is any substance that is recognized by the body as "foreign" and consequently induces an antigen-specific immune response by the body's immune cells. Antigens are often phagocytosed by the body's antigen-presenting cells (e.g., dendritic cells) and processed into epitopes that are presented to T cells and / or B cells via the major histocompatibility complex to induce antigen-specific immunity. Immune responses can be cellular and / or humoral. An increase in cellular immunity is typically manifested by an increase in antigen-specific T cell activity, proliferation, and an enhanced ability of T cells to recognize and eliminate antigens. An increase in humoral immunity is typically manifested by an increase in antigen-specific B cell activity and proliferation, which produce antibodies capable of recognizing and neutralizing the antigen of interest.

[0084] One category of antigens is recombinant full-length proteins or protein subunits corresponding to specific proteins associated with (or derived from) the infectious agent (target) of interest. For example, the antigen can be the full-length SARS-CoV-2 spike glycoprotein, which has been identified as immunogenic (Grifoni et al. Cell Host Microbe. 2020;27(4):671-80; Ou et al. Nat Commun. 2020,11(1):1620; Walls et al. Cell. 2020;181(2):281-92). Furthermore, antigens can correspond to SARS-CoV-2 nucleocapsid proteins, membrane proteins, etc. Antigens can also correspond to specific functional regions of proteins (i.e., protein subunits or protein domains). For example, antigens can correspond to the S1 region, S2 region, or RBD region of the SARS-CoV-2 spike glycoprotein.

[0085] An antigen can also be a peptide (or several peptides) corresponding to (or derived from) an immunogenic sequence within the infectious agent of interest (target infectious agent). Peptides act as epitopes capable of eliciting various immune responses. Antigens can be epitopes selected based on predicted immunogenicity as analyzed by bioinformatics and / or experimental data implicating them in immune cell stimulation. For example, peptides can correspond to positions 494-508 or 1056-1070 of the SARS-CoV-2 spike glycoprotein, which are predicted to have both cellular and humoral immunogenicity (Fast et al. bioRxiv.2020:2020.02.19.955484).

[0086] For antigens composed of several peptides, the antigen can be a cocktail of overlapping (or non-overlapping) peptides encompassing the entire (or nearly the entire) protein, or a mixture of peptides corresponding to immunogenic regions of a single protein or two or more different proteins (which may target one or different target infectious organisms). For example, the antigen can be a mixture of peptides including the SARS-CoV-2 spike protein, nucleocapsid protein, and membrane protein. Table 1 shows examples of SARS-CoV-2 T cell and / or B cell epitopes predicted by immunogenicity-based bioinformatic prediction methods, e.g., the Immune Epitope Database and Analysis Resource (IEDB) and Discotope 2.0 prediction algorithm, and high sequence similarity (e.g., >90%, >80%, >70%, >60%, or >50%) to SARS-CoV-1, the coronavirus best characterized in terms of epitope response (Grifoni et al. Cell Host Microbe. 2020;27(4):671-80; Fast et al. bioRxiv. 2020:2020.02.19.955484).

[0087] (Table 1) TIFF2023534232000001.tif42147

[0088] Other examples of predicted immunogenic epitopes can be found throughout the literature (Prachar et al. bioRxiv.2020:2020.03.20.000794; Chour et al. medRxiv.2020;2020.05.04.20085779) and on the websites of SARS-CoV-2 antigen vendors (e.g., Sino Biological, Creative Diagnostics, Sengenics, ABclonal Technology). Prediction tools for identifying immunogenic regions based on MHC binding capacity are also widely available.

[0089] In various embodiments, the antigen-producing agent is a nucleic acid, such as mRNA or pDNA, encoding a specific protein or peptide corresponding to or specific to the target infectious agent. When administered to a subject, the mRNA or pDNA enters the cytoplasm of cells, where it is expressed (mRNA translation or pDNA transcription / translation) into a desired protein that can ultimately activate cellular and humoral immune responses. The antigen-encoding sequence can be any sequence encoding a specific protein or protein subunit, such as the SARS-CoV-2 spike protein, spike RBD domain, spike S1 domain, etc. To increase potency, stability, and protein yield, the mRNA or pDNA can be subjected to codon optimization, use of modified nucleosides, polyadenylation, etc. For example, the design of the 5'UTR and 3'UTR is important for mRNA stability, translation, protein production, and structure. Several online tools exist to optimize the design of the 5'UTR and 3'UTR based on the mRNA of interest. For efficient antigen expression, mRNA is synthesized to contain the following: 5' cap, 5' untranslated region (UTR), antigen coding sequence, 3' untranslated region (UTR), and poly(A) tail. mRNA can also be unmodified, nucleoside-modified, or self-amplifying. For example, modified uridine or cytidine can be incorporated to avoid premature recognition by innate immune molecules and improve translation efficiency.

[0090] Suitable additional target antigens are known in the art (e.g., Pati et al., Front Immunol. 9:2224, 2018 (page 16), and references cited therein) and are available from commercial, government, and scientific sources. Additional exemplary antigens are provided below.

[0091] Exemplary Viral Antigens: Viral antigens may be isolated from any virus, including, but not limited to, viruses from any of the following viral families: Adenovirus, Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus , Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., coronaviruses, e.g., severe acute respiratory syndrome (SARS) virus, including COVID-19), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus, virus) and Ebola virus (e.g., Zaire strain, Reston strain, Ivory Coast strain, or Sudan strain)), Flaviviridae (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, and Dengue virus 4)4), Hantaviridea (e.g., hantavirus), Hepadnaviridae, Herpesviridae (e.g., human herpesvirus 1, 3, 4, 5, and 6, and cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., influenza virus A, B, and C), Papillomaviridae (human papillomavirus (HPV) and animal papillomavirus (APV)), papillomavirus), Papovaviridae, Paramyxoviridae (e.g., measles virus, mumps virus, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia virus and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentiviruses, e.g., human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae (e.g., rabies virus)virus, measles virus, respiratory syncytial virus, etc.), Rotaviridae (e.g., rotavirus A-J), Togaviridae (e.g., rubella virus, dengue virus, etc.), Spongiform viruses, and Totiviridae. Suitable viral antigens also include all or part of dengue protein M or protein E, dengue D1NS1, dengue D1NS2, and dengue D1NS3.

[0092] Viral antigens include those specific strains of papillomavirus, herpesvirus, e.g., herpes simplex 1 and 2; hepatitis virus, e.g., hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), delta hepatitis D virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV); tick-borne encephalitis virus; parainfluenza, varicella-zoster, cytomegalovirus, Epstein-Barr, rotavirus, rhinovirus, adenovirus, coxsackievirus, equine encephalitis, and the like. In an additional embodiment, the viral antigen markers include peptides expressed by CMV, common cold virus, Epstein-Barr, influenza virus, hepatitis A virus, hepatitis B virus, and hepatitis C virus, herpes simplex virus, HIV virus, influenza virus, Japanese encephalitis virus, measles virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, smallpox virus, varicella-zoster virus, or West Nile virus.

[0093] As further specific examples, cytomegalovirus antigens include envelope glycoprotein B and CMV pp65; Epstein-Barr antigens include EBV EBNAI, EBV P18, and EBV P23; hepatitis antigens include HBV S, M, and L proteins, HBV pre-S antigen, HBCAG DELTA, HBV HBE, hepatitis C virus RNA, HCV NS3, and HCV NS4; herpes simplex virus antigens include immediate early proteins and glycoprotein D; HIV antigens include gene products of the gag, pol, and env genes, e.g., HIV gp32, HIV gp41, HIV gp120, HIV gp160, HIV P17 / 24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV Antigens include GP36, Nef protein, and reverse transcriptase; influenza antigens include hemagglutinin and neuraminidase; Japanese encephalitis virus antigens include proteins E, ME, ME-NS1, NS1, NS1-NS2A, and 80%E; measles antigens include measles virus fusion protein; rabies antigens include rabies glycoprotein and rabies nucleoprotein; respiratory syncytial virus antigens include RSV fusion protein and M2 protein; rotavirus antigens include VP7sc; rubella antigens include proteins E1 and E2; and varicella-zoster virus antigens include gpI and gpII. Additional specific exemplary viral antigen sequences include Nef(66-97); Nef(116-145); Gag p17(17-35); Gag p17-p24(253-284); and Pol 325-355 (RT 158-188). For additional examples of viral antigens, see Fundamental Virology, Second Edition, eds. Fields, BN and Knipe, DM (Raven Press, New York, 1991).

[0094] Exemplary bacterial antigens: Bacterial antigens include those from Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, and the like. Rhizobium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Haemophilus influenzae type B (HIB), Hyphomicrobium, Legionella, Leptospirosis, Listeria steria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Mycobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum ), Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, as well as Treponema, Vibrio,Antigens targeting bacteria can be derived from any bacteria, including bacteria such as anthrax, gram-negative bacilli, chlamydia, diphtheria, Helicobacter pylori, Mycobacterium tuberculosis, pertussis toxin, pneumococcus, rickettsiae, staphylococcus, streptococcus, and tetanus.

[0095] Bacterial infections in which the subject immunogenic constructs and methods can be used include both gram-negative and gram-positive bacteria. Examples of gram-positive bacteria include Pasteurella species, Staphylococcus species, and Streptococcus species. Examples of gram-negative bacteria include Escherichia coli, Pseudomonas species, and Salmonella species.

[0096] Specific examples of infectious bacteria include Actinomyces israelii, Bacillus anthracis, Bacteroides species, Borrelia burgdorferi, pathogenic Campylobacter species, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium species, Enterococcus species, Enterobacter aerogenes, Erysipelothrix rhusiopathie, Escherichia coli, Fusobacterium nucleatum, and the like. nucleatum, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophilia, Leptospira, Listeria monocytogenes, Mycobacteria species (e.g., Mycobacteria tuberculosis, M. avium, M. intracellilare, M. kansaii, M. godnae),gordonae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Rickettsia, Shigella flexneri, Shigella dysenteriae, Staphylococcus aureus, Streptococcus pyogenes (Group A streptococci), Streptococcus agalactiae (Group B streptococci), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis These include Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, and Vibrio cholerae.

[0097] Specific examples of bacterial antigens include anthrax protective antigen, gram-negative bacillus antigens include lipopolysaccharide, diphtheria antigens include diphtheria toxin, Mycobacterium tuberculosis antigens include mycolic acid, heat shock protein 65 (HSP65), 30 kDa major secretory protein and antigen 85A, pertussis toxin antigens include hemagglutinin, pertactin, FIM2, FIM3 and adenylate cyclase, pneumococcal antigens include pneumolysin and pneumococcal capsular polysaccharide, rickettsial antigens include rompA, streptococcal antigens include M protein, and tetanus antigens include tetanus toxin.

[0098] Exemplary Parasite Antigens: Parasite antigens can be any parasite, for example, Babesia microti, Babesi divergans, Candida albicans, Candida tropicalis, Chlamydia psittaci, Chlamydia trachomatis, Cryptococcus neoformans, Entamoeba histolytica, Giardia lamblia, Histoplasma capsulatum, Leishmania tropica, Leishmania species, Leishmania braziliensis, Leishmania donovani, donovdni, Mycoplasma pneumoniae, Nocardia asteroides, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Rickettsia rickettsii, Rickettsia typhi, Schistosoma mansoni, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi cruzi), Gambian trypanosoma (Trypanosoma gambiense), Rhodesian trypanosoma (TrypanosomaThe parasite may be derived from antigens derived from helminth organisms or worms, or may be, but is not limited to, hookworm / hookworm, anisakiasis, Ascaris - parasitic pneumonia, Ascaris - raccoon roundworm, taeniasis - tapeworm infection, Clonorchiasis, Dioctophyme renalis infection, Diphyllobothriasis - tapeworm, Dracunculiasis - dracunculiasis, Echinococcosis - tapeworm, Pinworm - intestinal enterobiasis, Fasciola hepatica - fascioliasis, Fasciola hepatica - intestinal fluke, Gnathostomiasis, Hymenococcosis, Loa loa loa) can be an organism causing diseases including Filariasis, Calabar swelling, Mansonellosis, Filariasis, Paragonimiasis - intestinal fluke, River blindness, Chinese liver fluke, Paragonimiasis, Lung fluke, Schistosomiasis - bilharzia, Bilharzia or snail fever (all types), Intestinal schistosomiasis, Urinary schistosomiasis, Schistosomiasis due to Schistosoma japonicum, Asian intestinal schistosomiasis, Sparganosis, Strongyloidiasis - parasitic pneumonia, Taenia saginata, Taenia solium, Toxocariasis, Trichinellosis, Swamp dermatitis, Trichuris and Elephantiasis Lymphatic filariasis. The parasite may be a disease causing organism or organisms including worms, Harzan's syndrome, myiasis, sand fleas, dermatophyte and candidiasis. The parasite may be a disease causing ectoparasite or organism including bed bugs, head louse-pediculosis, body louse-pediculosis, pubic louse-pediculosis, Demodex-demodex, scabies, spiral worms and Cochliomyia.

[0099] Antigens include all or part of a Sporozoan antigen, a Plasmodium antigen, such as a circumsporozoite protein, a sporozoite surface protein, a liver stage antigen, an apical membrane associated protein, or a merozoite surface protein. Histoplasma antigens include heat shock protein 60 (HSP60); Leishmania antigens include gp63 and lipophosphoglycan; Plasmodium falciparum antigens include merozoite surface antigen, sporozoite surface antigen, peri-sporozoite antigen, gametocyte / gamete surface antigen, blood-stage antigen pf 155 / RESA, and other parasitic antigens; Schistosome antigens include glutathione-S-transferase and paramyosin; Toxoplasma antigens include SAG-1 and p30; Trypanosoma cruzi antigens include the 75-77 kDa antigen and the 56 kDa antigen; and Ringworm antigens include trichophytin.

[0100] Exemplary fungal antigens: Examples of fungal pathogens include Aspergillus species, Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, Candida albicans and other Candida species, Chlamydia trachomatis, Histoplasma capsulatum, Chlamydia trachomatis, Nocardia species, and Pneumocystis carinii. Antigens targeting fungi can be derived from, for example, Candida, Coccidioides, Cryptococcus, and Histoplasma. Specific examples of fungal antigens include coccidioides antigens and capsular polysaccharides for Cryptococcus.

[0101] As noted above, antigens derived from bacteria, viruses, fungi, and parasites may be formulated into vaccines of the present disclosure and administered according to the methods of the present disclosure. Non-limiting examples of antigens include those that form infectious agents that infect animals, such as:

[0102] Swine: Erysipelothrix rhusiopathiae, Actinobacillus pleuroneumonlae, Mycoplasma hyopneumoniae, Escherichia coli K88, K99, F41, and 987P, Clostridium perfringens type c, Salmonella choleraesuis, Pasteurella multocida, Bordetella bronchiseptica, Leptospira bratislava, Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo hardjo, Leptospira promona, Leptospira ictero, Porcine influenza virus, Circovirus, Porcine reproductive and respiratory syndrome virus (PRRSV), Swine pox, Rotavirus, Porcine respiratory coronavirus, Parvo virus, Pseudorabies, Transmissible gastroenteritis agent.

[0103] Horses: Streptococcus equi, Clostridium tetani, Equine Influenza Virus A1 and A2, Equine Rhinopneumonitis types 1, 1b, and 4, Eastern Equine Encephalomyelitis, Western Equine Encephalomyelitis, Venezuelan Equine Encephalitis, Equine Rotavirus, Equine Herpesvirus, Equine Infectious Anemia Virus, West Nile Virus, Candida albicans, Aspergillus, Coccidioides immitis, Cryptococcus neoformans, Histoplasma farciminosum.

[0104] Cattle: Escherichia coli O157:H7, Pasteurella multocida, Pasteurella haemolytica, Canine Leptospira, Leptospira grippotyphosa, Leptospira hardjo, Leptospira promona, Leptospira ictero, Clostridium perfringens type C, Clostridium perfringens type D, Clostridium chauvoel, Clostridium novyl, Clostridium septicum, Clostridium tetanus, Clostridium haemolyticum, Clostridium sodellii, Salmonella dublin and Salmonella typhimurium Typhimurium, Bovine Rotavirus, Bovine Coronavirus, Bovine Rhinotracheitis, Bovine Diarrhea Virus, Parainfluenza-3, Respiratory Syncytial Virus, Trichophyton verrucosum.

[0105] Poultry: Salmonella typhimurium, Serpulina pilosicoli, Marek's disease virus, infectious bursal disease, infectious bronchitis, Newcastle disease virus, Reo virus, turkey rhinotracheitis, coccidiosis.

[0106] Dogs: Canine Leptospira, Leptospira grippotyphosa, Leptospira hardjo, Leptospira promona, Leptospira ictero, Canine Borrelia burgdorferi, Canine Ehrlichia canis, Canine Bordetella bronchiseptica, Canine Giardia lamblia, Canine distemper, Canine adenovirus, Canine coronavirus, Canine parainfluenza, Canine parvovirus, Canine rabies, fleas, giardia, lungworm, dog hookworm (Ancylostoma caninum), dog hookworm (Uncinaria stenocephala), dog microsporum canis.

[0107] Cats: Feline infectious peritonitis virus, feline rhinotracheitis, feline panleukopenia, feline calicivirus, feline coronavirus, feline alphaherpesvirus 1, feline immunodeficiency virus, feline leukemia virus, rabies lyssavirus. Bordetella, Bacillus, Bartonella, Burkholderia, Chlamydia, Clostridium, Corynebacterium, Salmonella, Proteus, Escherichia, Proteus, Moraxella, Nocardia, Pasteurella, Haemophilus, Pasteurella, Pseudomonas, Staphylococcus, Streptococcus, Microsporum canis, Nannizzia gyps, Nannizzia fulva, Nannizzia nana, Trichophyton mentagrophytes, Trichophyton verrucosum, Hookworm, Cryptosporidium, Dirofilaria immitis, fleas, Giardia, Isospora species, lungworms, Ollanulus tricuspi, Physaloptera hispida, Sarcoptes scabiei, tapeworms, Toxascaris leonina, Toxocara cati, Toxoplasma gondii, hookworms and whipworms.

[0108] VII. Oligonucleotides In some embodiments, the immunogenic construct comprises one or more oligonucleotides, such as siRNA, miRNA, miRNA mimic, or antisense oligonucleotide. The oligonucleotides can be attached by any means. In some embodiments, negatively charged siRNA is attached to a positively charged cationic polymer on a nanoparticle, such as MSNP, using electrostatic interactions. The oligonucleotides can target one or more genes expressed in cells, such as those that inhibit or downregulate genes associated with immunosuppression of antigen-presenting cells (e.g., dendritic cells), such as STAT3, PD-L1, IDO-1, and IL-6. In some embodiments, a single oligonucleotide can target multiple genes with varying efficacy. In other embodiments, multiple oligonucleotides can target a single gene. In further embodiments, multiple oligonucleotides can target multiple genes.

[0109] The oligonucleotide may be present in an amount of about 1% to 10% by weight of the NP, for example, about 2% to about 6% by weight. In some embodiments, for example, a weight ratio of about 10:1 to about 100:1 of NP per siRNA (NP / siRNA) is used during the conjugation process to achieve complete conjugation. Complete conjugation can be achieved up to 40% by weight of siRNA / NP.

[0110] Typically, the oligonucleotide inhibits or downregulates genes whose upregulation is related to some aspect of immunosuppression of antigen-presenting cells (e.g., dendritic cells).Those skilled in the art will understand how to utilize representative sequences of these targets, which are readily available in public sequence databases.In some embodiments, the oligonucleotide is an siRNA, such as STAT3, PD-L1, IDO-1, IL-6, etc.Exemplary siRNAs are shown in Table 2.

[0111] (Table 2) TIFF2023534232000002.tif177152TIFF2023534232000003.tif92152

[0112] VIII. Targeting agents In some aspects, the immunogenic construct may further comprise a targeting agent, e.g., for specific delivery of the immunogenic construct to a target site. The targeting agent may be used to target the site and, optionally, to assist or direct internalization into the cell.

[0113] Exemplary targeting agents include monoclonal antibodies, single-chain variable fragment (scFv) antibodies, other antigen-binding fragments of antibodies, aptamers, small targeting molecules (e.g., ligands that bind to cell surface receptors, e.g., N-acetylgalactosamine, mannose, transferrin, and folate), aptamers, carbohydrates, and peptides that have binding affinity for cells or tissues, e.g., immune cells such as antigen-presenting cells (e.g., dendritic cells or macrophages).

[0114] In some embodiments, the targeting agent targets immune cells such as antigen-presenting cells (e.g., dendritic cells or macrophages). Targeting agents include monoclonal or polyclonal antibodies or fragments thereof that recognize and bind to epitopes displayed on the surface of immune cells, and ligands that bind to cell surface receptors on immune cells. One such receptor, the lectin DEC-205, has been used in vitro and in mice to enhance both humoral (antibody-based) and cellular (CD8 T cell) responses by 2 to 4 orders of magnitude (Hawiger et al., J. Exp. Med., 194(6):769-79, 2001; Bonifaz et al., J. Exp. Med., 196(12):1627-38, 2002; Bonifaz et al., J. Exp. Med., 199(6):815-24, 2004). In these reports, antigens were fused to anti-DEC205 heavy chains and recombinant antibody molecules were used for immunization.

[0115] Various other endocytic receptors, including mannose-specific lectins (mannose receptors) and IgG Fc receptors, have also been targeted in this way, similarly improving antigen presentation efficiency. Other suitable receptors and surface proteins that can be targeted include DC-SIGN, 33D1, SIGLEC-H, DCIR, CD11c, CD40, DEC-205, heat shock protein receptors, and scavenger receptors. Targeting moieties for these receptors can be attached to immunogenic constructs for preferential uptake into immune cells that express these receptors. An example is mannose attached to the immunogenic construct for targeted delivery to macrophages and DCs, which have high levels of mannose receptors.

[0116] Other receptors that can be targeted include toll-like receptors (TLRs). TLRs recognize and bind to pathogen-associated molecular patterns (PAMPs). PAMPs target TLRs on the surface of dendritic cells and signal internally, potentially increasing DC antigen uptake, maturation, and T cell stimulatory capacity. PAMPs that can be conjugated or co-encapsulated on particle surfaces include unmethylated CpG DNA (bacterial), double-stranded RNA (viral), lipopolysaccharide (bacterial), peptidoglycan (bacterial), lipoarabinomannin (bacterial), zymosan (yeast), mycoplasmal lipoproteins such as MALP-2 (bacterial), flagellin (bacterial), poly(inosinic-cytidylic) acid (bacterial), lipoteichoic acid (bacterial), or imidazoquinoline (synthetic).

[0117] The targeting agent may be attached to the immunogenic construct by any means, and suitable conjugation chemistries are known in the art and described herein. In some embodiments, the targeting agent is thiolated and subsequently conjugated to Mal-PEG-PEI-MSNP via a thiol-maleimide reaction. In some embodiments, the targeting agent is attached to a PEG stabilizer prior to conjugation to the NP by reaction of an NHS ester with an amine. The targeting agent may be present at 0.1-10% by weight of the NP, e.g., 0.1-1% or 1-5% by weight, e.g., 1-10% by weight for antibodies, or 0.1-2% by weight for scFVs, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9% by weight.

[0118] IX. Labeling Agents In some embodiments, the immunogenic construct can be labeled with, for example, a lanthanide, a fluorescent dye, a quantum dot, a radioactive tracer, or a gold nanoparticle. The label can be any substance that can help a machine, a detector, a sensor, a device, a column, or the human eye, enhanced or unenhanced, distinguish a labeled composition from an unlabeled composition. Examples of labels include radioisotopes (e.g., PET tracers), dyes, stains, quantum dots, gold nanoparticles, enzymes, non-radioactive metals (e.g., MRI contrast agents), magnets, biotin, protein tags, any antibody epitope, or any combination thereof. Exemplary fluorescent dyes include FITC, RITC, Cy™ dyes, amine-reactive Dylight® dyes, and amine-reactive Alexa Fluor® dyes. In some embodiments, lanthanides can be loaded onto hydroxyl, thiol, amine, or phosphonate groups of nanoparticles, such as MSNPs, by covalent binding or adsorption. Lanthanides can facilitate sample detection with high sensitivity and resolution, for example, by mass spectrometry, while fluorescent dyes allow sample quantification by fluorescence imaging techniques. Immunogenic constructs containing lanthanides, such as gadolinium, can also serve as MRI contrast agents for imaging disease sites.

[0119] In some embodiments, a label, such as a fluorescent dye, is loaded inside the pores of a nanoparticle, e.g., an amine-MSNP, via, for example, nucleophilic acyl substitution between one or more nanoparticle-bound amines and an activated ester moiety (e.g., an NHS ester) attached to the fluorescent dye. Such a label results in an immunogenic construct that can be tracked using fluorescent imaging techniques. Such a label can be added before or after loading of the cationic polymer and / or stabilizer (i.e., the label can be applied to the nanoparticle or NP). In further embodiments, the label can be attached to the cationic polymer, stabilizer, or other component of the NP (e.g., an oligonucleotide) by any suitable means before or after their attachment to the nanoparticle.

[0120] X. Synthesis of Immunogenic Constructs The components may be attached to the nanoparticle, or other components of the NP, or to the immunogenic construct by any means, including covalent and electrostatic bonds. Various conjugation chemistries are known in the art and are described herein. In some embodiments, one or more of the components are attached to the surface of the nanoparticle or NP. In other embodiments, one or more of the components are attached within the pores of the nanoparticle (e.g., MSNP). In further embodiments, one or more of the components are attached to each other. In some embodiments, the adjuvant and / or antigen or antigen-producing agent is covalently attached to the stabilizer. The stabilizer may be covalently attached (e.g., via an amine) to the cationic polymer, which may then be electrostatically attached to the exterior of the nanoparticle. In some embodiments, the adjuvant and / or antigen or antigen-producing agent is attached to the cationic polymer via chemical conjugation, electrostatic interaction, hydrophobic interaction, hydrogen bonding, or van der Waals interaction. For example, the antigen or antigen generating agent may be covalently bound to the stabilizer, while the adjuvant is electrostatically or hydrophobically bound to the cationic polymer.

[0121] In some embodiments in which the nanoparticle has a pore, the pore has a first opening at a first location on the exterior surface of the nanoparticle (e.g., MSNP) and a second, different opening at a second location on the exterior surface of the nanoparticle. The component may bind anywhere along the interior length of the pore, although the size of the pore and the size of the component will affect binding.

[0122] Nanoparticles such as MSNPs can be commercially available or produced by any method. In some embodiments, MSNPs are formed by combining a first surfactant with a second, different surfactant to form a first mixture, heating the first mixture, adding a silica precursor to the first mixture to form a second mixture, maintaining the temperature for a period of time to produce MSNPs, and recovering the MSNPs by centrifugation. The surfactant can be removed by mixing the MSNPs in an acidic solvent under reflux conditions. In some embodiments, the first mixture can be heated before adding the silica precursor. In other embodiments, the first mixture can be at room temperature, and the second mixture can be heated. The resulting MSNPs can have a uniform or non-uniform particle size and have high porosity.

[0123] For example, to form uniform MSNPs, cetyltrimethylammonium chloride (CTAC) may be combined with triethanolamine (TEA) in water and heated to 95°C while adding tetraethyl orthosilicate. The size of the resulting MSNPs can be varied by varying the amount of TEA while keeping the amount of CTAC constant. In some embodiments, the amount of TEA is about 100 to about 600 μL, about 200 to about 450 μL, or about 200 to about 350 μL. In some embodiments, the amount of TEA is 0.1 to 1% v / v, e.g., 0.35% v / v. Heterogeneous MSNPs can be prepared using a strong base such as NaOH. For example, cetyltrimethylammonium bromide (CTAB) may be used as a surfactant, and NaOH may be used as a base catalyst.

[0124] Iron oxide nanoparticles can be purchased (e.g., Feraheme) or synthesized. Gold and silver nanoparticles can be synthesized according to various published protocols or purchased from vendors such as Sigma Aldrich, Nanocs, nanoComposix, etc. Carbon nanotubes can be synthesized according to various published protocols or purchased from vendors such as Sigma Aldrich, US Research nanomaterials, and American Elements.

[0125] In some embodiments, functional groups such as, but not limited to, thiol, amine, carboxylate, or phosphonate can be added to the exterior surface of nanoparticles, e.g., MSNPs, during synthesis using one or more reagents, e.g., organosilanes such as (3-aminopropyl)triethoxysilane and (3-aminopropyl)trimethoxysilane. The organosilanes can be added before or after the surfactant is removed from the MSNPs. Similar and other organic reagents, e.g., glutathione, mercaptopropionic acid, DMSA, PEG-thiol, oleic acid, and dextran, can also be used to modify iron oxide nanoparticles, silver nanoparticles, gold nanoparticles, and carbon nanotubes. Functionalized nanoparticles can also be purchased directly; for example, carbon nanotubes surface-modified with carboxylic acids, amides, polyaminobenzenesulfonic acid, octadecylamine, and PEG can be purchased from Sigma-Aldrich.

[0126] The resulting NPs (e.g., MSNPs after surface modification) can be any appropriate size, for example, about 20 nm to about 200 nm, about 20 nm to about 400 nm, about 20 nm to about 500 nm, about 20 nm to about 100 nm, about 30 nm to about 100 nm, about 40 nm to about 200 nm, about 50 nm to about 200 nm, about 50 nm to 400 nm, about 50 to 500 nm, about 30 nm to about 80 nm, 40 nm to about 80 nm, about 30 nm, about 40 nm, about 30 nm to about 60 nm, about 50 nm, about 60 nm, about 80 nm, about 100 nm, about 120 nm, or about 150 nm.

[0127] The resulting immunogenic construct, e.g., NP loaded with an antigen or antigen-producing factor, can be of an appropriate size, e.g., about 20 nm to about 200 nm, about 30 nm to about 100 nm, about 40 nm to about 200 nm, about 50 nm to about 200 nm, about 30 nm to about 80 nm, 40 nm to about 80 nm, about 30 nm, about 40 nm, about 30 nm to about 60 nm, about 100 nm to 200 nm, about 100 nm to about 500 nm, about 100 nm to about 999 nm, about 100 nm to about 400 nm, about 50 nm, about 60 nm, about 80 nm, about 100 nm, about 120 nm, about 150 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 999 nm.

[0128] XI. Immunogenic Constructs Comprising Lipid-Coated Nanoparticles Also disclosed herein are immunogenic constructs comprising a lipid-coated nanoparticle core (e.g., any of the nanoparticles disclosed herein). In some embodiments, the immunogenic constructs comprising lipid-coated calcium phosphate are comprised of a calcium phosphate core (CaP-L), formed, for example, by the reaction of CaCl2 with Na2HPO4 and a surrounding lipid layer. The Ca / P molar ratio can range from 10 to 200. The size of the CaP core nanoparticles can range from 5 to 999 nm (e.g., about 20 nm to about 200 nm, about 30 nm to about 100 nm, about 40 nm to about 200 nm, about 50 nm to about 200 nm, about 30 nm to about 80 nm, 40 nm to about 80 nm, about 30 nm, about 40 nm, about 30 nm to about 60 nm, about 50 nm, or about 60 nm). The thickness of the lipid layer can be in the range of 1 to 999 nm (e.g., about 20 nm to about 200 nm, about 30 nm to about 100 nm, about 40 nm to about 200 nm, about 50 nm to about 200 nm, about 30 nm to about 80 nm, 40 nm to about 80 nm, about 200 to about 750 nm, about 500 to 999 nm, about 30 nm, about 40 nm, about 30 nm to about 60 nm, about 50 nm, or about 60 nm). The lipid layer comprises one or more of a cationic lipid (e.g., DOTAP, dimethyldioctadecylammonium bromide, D-Lin-MC3-DMA), a PEGylated lipid (e.g., DMG-PEG 2000, DSG-PEG 2000), a functionalized PEGylated lipid having a functional group (e.g., -SH, -NH2, -COOH), a PEGylated lipid conjugated with a targeting agent (e.g., mannose, or any of those disclosed herein), a phospholipid (e.g., 1,2-distearoyl-sn-3-phosphocholine (DSPC), dioleoylphosphatidic acid (DOPA) or dioleoylphosphatidylethanolamine (DOPE)), and cholesterol. Each of the above lipids may account for 0 to 100% (w / w) of the lipid layer (e.g., 0 to 10%, 0 to 20%, 0 to 30%, 0 to 40%, 0 to 50%, 0 to 60%, 0 to 70%, 0 to 80%, 0 to 90%, 5 to 15%, 5 to 25%, 10 to 50%, 25 to 75%, 50 to 90%, or about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100%).PEGylated lipid / functionalized PEGylated lipid / targeted PEGylated lipid enhances stability and prolongs circulation of immunogenic constructs in blood, while phospholipid composition and cholesterol form and stabilize lipid-coated structures.Functionalized PEGylated lipid is for further conjugation with nucleic acid and / or antigen.Targeted PEGylated lipid is for enhancing uptake efficiency into targeted cells. The calcium phosphate core can account for 0.1 to 99.9% (w / w) of the CaP-L (e.g., 0 to 10%, 0 to 20%, 0 to 30%, 0 to 40%, 0 to 50%, 0 to 60%, 0 to 70%, 0 to 80%, 0 to 90%, 5 to 15%, 5 to 25%, 10 to 50%, 25 to 75%, 50 to 90%, or about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 99.9%). The lipid layer can comprise 0.1-99.9% (w / w) of the CaP-L (e.g., 0-10%, 0-20%, 0-30%, 0-40%, 0-50%, 0-60%, 0-70%, 0-80%, 0-90%, 5-15%, 5-25%, 10-50%, 25-75%, 50-90%, or about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 99.9%). One or more surfactants (e.g., Tween 80, Tween 20, Span 80, Span 20, PVP, SDS, SLS, PEG) can be included to aid in CaP-L formation. The surfactant:CaP-L weight ratio can range from 0-50%. The hydrodynamic size of CaP-L, with or without loaded cargo, can range from 10 nm to 10 microns (e.g., about 80 nm to about 200 nm, or about 90 nm to about 150 nm, about 1 micron to about 2 microns).

[0129] In some embodiments, one or more oligonucleotides (e.g., one or more of siRNA, mRNA, shRNA, miRNA, DNA, and CpG oligonucleotides) are present in the core of the nanoparticle (e.g., Ca 2+The oligonucleotides are loaded into the calcium phosphate core via ionic interactions between the cations and the phosphate groups of the nucleic acid backbone of the oligonucleotides. In some embodiments, one or more oligonucleotides (e.g., one or more of siRNA, mRNA, shRNA, miRNA, DNA, and CpG oligonucleotides) are loaded into the lipid layer via ionic interactions between the cationic lipids and the phosphate groups of the nucleic acid backbone. In some embodiments, one or more oligonucleotides (e.g., one or more of siRNA, mRNA, shRNA, miRNA, DNA, and CpG oligonucleotides) are conjugated to functionalized PEGylated lipids in the lipid layer. For example, the loading of one or more oligonucleotides into CaP-L can range from 0.01 to 10% by weight of the nanoparticles.

[0130] In some embodiments, one or more antigens or antigen-producing factors (e.g., one or more of peptides, proteins, and polysaccharides) are loaded into the core of the nanoparticle (e.g., a calcium phosphate core). In some embodiments, one or more antigens or antigen-producing factors (e.g., one or more of peptides, proteins, and polysaccharides) with an appropriate hydrophilic-lipophilic balance are inserted into the lipid layer. In some embodiments, one or more antigens or antigen-producing factors (e.g., one or more of peptides, proteins, and polysaccharides) are attached to the nanoparticle (e.g., via van der Waals interactions, and / or the Ca of the calcium phosphate core). 2+ The antigens or antigen-producing factors are adsorbed onto the surface of the nanoparticles (e.g., calcium phosphate nanoparticles) via ionic interactions with ions. In some embodiments, one or more antigens or antigen-producing factors (e.g., one or more of peptides, proteins, and polysaccharides) can be conjugated to the functionalized PEGylated lipids of the lipid layer via covalent bonds. The loading of one or more antigens or antigen-producing factors onto nanoparticles (e.g., calcium phosphate nanoparticles) can range from 0.01 to 10% by weight of the nanoparticles.

[0131] XII. Immunogenic Construct Formulations and Methods of Use The immunogenic constructs can be formulated for therapeutic, diagnostic, or research use, as known in the art. The immunogenic constructs can be used for in vivo or ex vivo applications. The effect of the agent contained in the immunogenic construct can occur intracellularly or extracellularly.

[0132] Immunogenic constructs can be used immediately after formulation or can be stored.In some embodiments, immunogenic constructs can be freeze-dried to dryness using a lyoprotectant such as sugar-like trehalose.Optimal trehalose and freeze-drying conditions can preserve the particle size and charge and effect of immunogenic constructs compared to freshly produced materials, for example, the gene knockdown effect of immunogenic constructs containing certain siRNAs.The immunogenic constructs of the present disclosure are stable for at least 6 months when freeze-dried.

[0133] The immunogenic construct can be formulated with a pharmaceutically effective excipient in a pharmaceutical composition. The pharmaceutical composition can contain active agents such as adjuvants, lyoprotectants, stabilizers, preservatives and / or solubilizers that are not bound to the immunogenic construct. The effective amount of the immunogenic construct for therapeutic administration can be easily determined by those skilled in the art, depending on, for example, clinical and patient-specific factors.

[0134] These and other effective unit dosages can be administered in the form of a single dose or multiple daily, weekly, or monthly doses, for example, twice a week for a 3-week cycle. In additional embodiments, dosages can be administered in any suitable dosage regimen in conjunction with other therapeutic regimens, depending on clinical and patient-specific factors. The amount, timing, and mode of delivery of the compositions of the present disclosure, including the immunogenic amount of the immunogenic construct, are routinely adjusted for each individual, depending on factors such as the individual's weight, age, sex, and condition, the acuity of the disease and / or associated symptoms, whether the administration is prophylactic or therapeutic, and based on drug delivery, absorption, pharmacokinetics including half-life, and other factors known to affect efficacy.

[0135] The formulations of the present disclosure are usually selected to approximate the minimum dosage regimen necessary and sufficient to substantially prevent or alleviate the disease symptoms of mammalian subjects, including human beings, including cancer, fibrosis and inflammation.Therapeutic dosage and administration protocol often include repeated administration for several days, or even for one or more weeks, or for one or more years.Effective therapeutic regimen can also involve the prophylactic dosage administered daily or multiple times per day, which lasts for several days, weeks, months or even years.

[0136] In some embodiments, the immunogenic constructs of the present disclosure are formulated for parenteral administration, e.g., intravenously, intramuscularly, intratumorally, intranasally, subcutaneously, intradermally, or intraperitoneally, including aqueous and non-aqueous sterile injection solutions, which may optionally contain antioxidants, buffers, bacteriostats, and / or solutes that render the formulation isotonic with the blood of a mammalian subject, and aqueous and non-aqueous sterile suspensions, which may include suspending agents and / or thickening agents. Formulations may be provided in unit-dose or multi-dose containers. Additional compositions and formulations of the present disclosure may include polymers for sustained release after parenteral administration. Parenteral preparations may be solutions, dispersions, or emulsions suitable for such administration. The subject agents may also be formulated in polymers for sustained release after parenteral administration. Pharmaceutically acceptable formulations and components are typically sterile or easily sterilizable, biologically inert, and easily administered. Such materials are well known to those skilled in the art of pharmaceutical formulations. Parenteral preparations typically contain buffers and preservatives, and a pharmaceutically and physiologically acceptable injectable fluid such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc. Extemporaneous injection solutions, emulsions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

[0137] In some embodiments, the immunogenic construct is formulated for oral administration and can be in any orally acceptable dosage form, including capsules, tablets, emulsions, and aqueous suspensions, dispersions, and solutions. In some embodiments, the dosage form is an oral dosage form, such as a compressed tablet, a hard or soft gel capsule, an enteric-coated tablet, an osmotic release capsule, or a unique combination of excipients. For tablets, commonly used excipients include lactose, mannitol, and cornstarch. Lubricants, such as, but not limited to, magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose, mannitol, glucose, sucrose, cornstarch, potato starch, or cellulose. In additional embodiments, the dosage form includes a capsule, which contains a mixture of materials to provide the desired sustained-release formulation. When an aqueous suspension or emulsion is administered orally, the active ingredient can be suspended or dissolved in an oily phase in combination with an emulsifier or suspending agent. If desired, certain sweeteners, flavorings, or coloring agents can be added.

[0138] In some embodiments, the immunogenic construct is formulated for intranasal administration or inhalation. Compositions for nasal administration or inhalation can be conveniently formulated as aerosols, droplets, gels, and powders. Aerosol formulations typically contain a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually provided in single or multiple doses in a sterile form in a sealed container, which can be in the form of a cartridge or refill for use with a spray device. Alternatively, the sealed container can be an integrated dispensing device, such as a single-dose nasal inhaler or an aerosol dispenser with a metered-dose valve for disposal after use. When the dosage form includes an aerosol dispenser, it contains a propellant, which can be a compressed gas such as compressed air or an organic propellant such as a fluoro-chloro-hydrocarbon. The aerosol dosage form can also be in the form of a pump-atomizer.

[0139] A topical carrier may be used to deliver the immunogenic construct. In some embodiments, the topical carrier is an emulsion, gel, or ointment. In other embodiments, the immunogenic construct may be formulated into a spray formulation. Emulsions, such as creams and lotions, are dispersion systems containing at least two immiscible phases, one of which is dispersed in the other as droplets ranging from 0.1 μm to 100 μm in diameter. To improve stability, an emulsifier is typically included. When water is the dispersed phase and oil is the dispersion medium, the emulsion is called a water-in-oil emulsion. When oil is dispersed as droplets throughout the aqueous phase, the emulsion is called an oil-in-water emulsion. Emulsions, such as creams and lotions, that can be used as topical carriers, and their preparation, are described in Remington: The Science and Practice of Pharmacy (Loyd V. Allen, 2002), incorporated herein by reference. nd This is disclosed in the Journal of Applied Physics, Vol. 1, No. 1, pp. 20

[0140] Ointments can be homogeneous, viscous, semisolid preparations, most commonly thick, greasy oils (80% oil - 20% water). Ointments can be used as emollients or to apply active ingredients to the skin for protective, therapeutic, or prophylactic purposes where some degree of occlusion is desired.

[0141] Creams are emulsions of roughly equal parts oil and water. Creams penetrate the outer layer of the skin, the stratum corneum, very well. Creams are generally thinner than ointments and retain their shape when removed from their container.

[0142] The vehicle of an ointment / cream is known as an ointment base. The choice of base depends on the clinical indication of the ointment. Various types of ointment bases include hydrocarbon bases, such as hard paraffin, soft paraffin, microcrystalline wax, and ceresin; absorbent bases, such as wool fat and beeswax; water-soluble bases, such as macrogol 200, 300, and 400; and emulsifying bases, such as emulsifying wax and vegetable oils (olive oil, coconut oil, sesame oil, almond oil, and peanut oil). The immunogenic construct is dispersed in the base and is divided after the drug penetrates the wound. Ointments / creams can be formulated to incorporate hydrophobic, hydrophilic, or water-emulsifying bases to provide preparations that are immiscible, miscible, or emulsifiable with skin secretions. They can also be derived from fatty hydrocarbons, absorbent bases, water-removable bases, or water-soluble bases. For example, a cream / ointment base can contain, among other ingredients, up to 100% of the active agent, white petrolatum, water, allantoin, EDTA, stearyl alcohol, Brij 721, Brij 72, methylcellulose, isopropyl myristate, sorbitan monooleate, polyoxyl 40 stearate, butylated hydroxytoluene, propylene glycol, methylparaben, propylparaben, deionized water, and buffered to neutral pH.

[0143] In another embodiment, the topical carrier used to deliver the immunogenic constructs of the present disclosure is a gel, e.g., a two-phase or single-phase gel. A gel is a semi-solid system consisting of a suspension of small inorganic particles or large organic molecules interpenetrated by a liquid. If the gel mass contains a network of small, discrete inorganic particles, it is classified as a two-phase gel. In some embodiments, the liquid can be water or another aqueous medium, and the gel mass is defined as a hydrogel. Hydrogels can include alginate, polyacrylate, polyalkylene oxide, and / or poly-N-vinylpyrrolidone. Hydrogels can also be amorphous, i.e., viscous gels, as opposed to solids, such as formulations of carboxymethylcellulose containing humectants such as propylene glycol or glycerin. Exemplary amorphous hydrogels include maltodextra-β-glucan, acemannan, carboxymethylcellulose, pectin, xanthan gum, collagen, keratin, and honey.

[0144] The immunogenic construct may be packaged in a biodegradable capsule for oral administration. Alternatively, a suspension of the immunogenic construct may be placed inside the bladder. This is similar to intravesical chemotherapy, in which drugs administered to the bladder come into direct contact with cancer cells lining the bladder.

[0145] The following exemplary embodiments and examples are included to demonstrate particular aspects of the present disclosure. Those skilled in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein without departing from the spirit and scope of the disclosure and still obtain like or similar results. In the following examples, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of various aspects of the present disclosure. However, those skilled in the art will understand that the present disclosure may be practiced without these specific details. In other examples, known structures and devices are shown or described more generally to avoid obscuring the invention. It should be noted that there are many different alternative configurations, devices, and techniques to which the disclosed invention may be applied. The following examples are illustrative of the disclosed methods. In light of the present disclosure, those skilled in the art will recognize that variations of these and other examples of the disclosed methods are possible without undue experimentation.

[0146] XIII. Illustrative Embodiments Exemplary Embodiment Set 1: 1. An immunogenic construct comprising a nanoparticle, a cross-linked cationic polymer bound to the outer surface of the nanoparticle, a stabilizer bound to the cross-linked cationic polymer or a stabilizer bound to the outer surface of the nanoparticle, and an antigen or antigen-producing agent for an infectious agent. 2. The immunogenic construct of embodiment 1, further comprising an adjuvant. 3. The immunogenic construct of embodiment 2, wherein the adjuvant comprises one or more of a CpG oligonucleotide, a DNA TLR agonist comprising a CpG sequence, a non-CpG DNA TLR agonist, an RNA TLR agonist, an aluminum salt, an anti-CD40 antibody, a fusion protein, a cytokine, a small molecule TLR agonist, an oil-based or surfactant-based adjuvant, lipopolysaccharide, a plant extract, or derivatives thereof. 4. The immunogenic construct of embodiment 2 or 3, wherein the adjuvant comprises a CpG oligonucleotide. 5. The immunogenic construct of any one of aspects 2 to 4, wherein the adjuvant comprises poly I:C. 6. The immunogenic construct of any one of aspects 2 to 5, wherein the adjuvant is present at 1 to 20% by weight of the nanoparticles. 7. The immunogenic construct of any one of aspects 1-6, wherein the nanoparticle is a silica nanoparticle, a silicon nanoparticle, an iron oxide nanoparticle, a gold nanoparticle, a silver nanoparticle, a carbon nanotube, or an adjuvant nanoparticle. 8. The immunogenic construct of any one of aspects 1-7, wherein the nanoparticles are mesoporous silica nanoparticles. 9. The immunogenic construct of any one of aspects 1-8, wherein the cationic polymer is selected from the group consisting of PEI, chitosan, polypropyleneimine, polylysine, polyamidoamine, poly(allylamine), poly(diallyldimethylammonium chloride), poly(N-isopropylacrylamide-co-acrylamide), poly(N-isopropylacrylamide-co-acrylic acid), diethylaminoethyl-dextran, poly(N-ethyl-vinylpyridinium bromide), poly(dimethylamino)ethyl methacrylate, and poly(ethylene glycol)-co-poly(trimethylaminoethyl methacrylate chloride). 10. The immunogenic construct of any one of aspects 1-9, wherein the cationic polymer is PEI. 11. The immunogenic construct of any one of aspects 1-10, wherein the cationic polymer has a molecular weight of from about 0.8 kDa to about 25 kDa. 12. The immunogenic construct of any one of aspects 1-11, wherein the cationic polymer is present in an amount of 1-50% by weight of the nanoparticle. 13. The immunogenic construct of any one of aspects 1-12, wherein the stabilizer is selected from the group consisting of PEG, dextran, polysialic acid, hyaluronic acid, polyvinylpyrrolidone, polyvinyl alcohol, and polyacrylamide. 14. The immunogenic construct of any one of aspects 1-13, wherein the stabilizer is PEG. 15. The immunogenic construct of any one of aspects 1-14, wherein the stabilizer has a molecular weight of from about 1 kDa to about 20 kDa. 16. The immunogenic construct of any one of aspects 1-15, wherein the stabilizer is present in 1-50% by weight of the nanoparticle. 17. The immunogenic construct of any one of aspects 1-16, wherein the infectious agent is a virus. 18. The immunogenic construct of any one of aspects 1-17, wherein the infectious agent is a β-coronavirus. 19. The immunogenic construct of any one of aspects 1-18, wherein the infectious agent is SARS-CoV-2, SARS-CoV-1, or MERS-CoV. 20. The immunogenic construct of any one of aspects 1-19, wherein the infectious agent is SARS-CoV-2. 21. The immunogenic construct of embodiment 20, wherein the antigen or antigen-producing agent is a recombinant full-length protein. 22. The immunogenic construct of embodiment 21, wherein the antigen or antigen-producing agent is a full-length SARS-CoV-2 spike glycoprotein, a SARS-CoV-2 nucleocapsid protein, or a SARS-CoV-2 membrane protein. 23. The immunogenic construct of embodiment 20, wherein the antigen or antigen-producing agent is a protein subunit. 24. The immunogenic construct of embodiment 23, wherein the antigen or antigen-producing agent is a protein subunit corresponding to the S1 region, S2 region, or RBD region of the SARS-CoV-2 spike glycoprotein. 25. The immunogenic construct of embodiment 20, wherein the antigen or antigen-producing agent is a peptide corresponding to an immunogenic sequence of the SARS-CoV-2 spike glycoprotein. 26. The immunogenic construct of embodiment 25, wherein the antigen or antigen-producing agent has the peptide sequence of any one of SEQ ID NOs: 1-8. 27. The immunogenic construct of embodiment 20, wherein the antigen or antigen-producing agent is mRNA or pDNA. 28. The immunogenic construct of any one of aspects 1 to 27, wherein the antigen or antigen-producing agent is present at 0.5 to 20% by weight of the nanoparticle. 29. The immunogenic construct of any one of aspects 1-28, further comprising at least one oligonucleotide. 30. The immunogenic construct of embodiment 29, wherein at least one type of oligonucleotide is electrostatically bound to the cationic polymer. 31. The immunogenic construct of embodiment 30, wherein at least one type of oligonucleotide comprises an siRNA, miRNA, miRNA mimic, or antisense oligonucleotide. 32. The immunogenic construct of embodiment 31, wherein at least one type of nucleotide comprises an siRNA. 33. The immunogenic construct of embodiment 32, wherein the siRNA inhibits or downregulates a gene whose upregulation is associated with cellular immunosuppression. 34. The immunogenic construct of embodiment 33, wherein the cell is an antigen-presenting cell. 35. The immunogenic construct of embodiment 34, wherein the antigen-presenting cell is a dendritic cell or a macrophage. 36. The immunogenic construct of embodiment 35, wherein the gene is STAT3, IDO-1, IL-6, or PD-L1. 37. The immunogenic construct of any one of aspects 29-35, wherein the at least one type of oligonucleotide is present in 1-10% by weight of the nanoparticles. 38. The immunogenic construct of any one of aspects 1 to 37, further comprising a targeting agent for a cell. 39. The immunogenic construct of embodiment 38, wherein the cell is an antigen-presenting cell. 40. The immunogenic construct of embodiment 39, wherein the antigen-presenting cell is a dendritic cell or a macrophage. 41. The immunogenic construct of embodiment 39 or 40, wherein the targeting agent is mannose, a monoclonal or polyclonal antibody, or a fragment thereof, that recognizes and binds to an epitope displayed on an antigen-presenting cell, or a ligand that binds to a surface receptor on an antigen-presenting cell. 42. The immunogenic construct of any one of aspects 38-41, wherein the targeting agent is present at 0.1 to 10% by weight of the nanoparticle. 43. The immunogenic construct of any one of aspects 1-42, further comprising a labeling agent. 44. The immunogenic construct of embodiment 43, wherein the labeling agent is a fluorescent dye and / or a metal probe. 45. The immunogenic construct of any one of aspects 1-44, having a hydrodynamic diameter of about 10 nm to about 10 microns. 46. ​​The immunogenic construct of embodiment 45, having a hydrodynamic diameter of about 90 nm to about 150 nm. 47. The immunogenic construct of any one of aspects 1-46, wherein the nanoparticles have a diameter of from about 5 to about 999 nm. 48. An immunogenic construct comprising a nanoparticle, a lipid layer coating the outer surface of the nanoparticle, and an antigen or antigen-producing agent for an infectious agent. 49. The immunogenic construct of embodiment 40, further comprising an adjuvant. 50. The immunogenic construct of embodiment 49, wherein the adjuvant comprises one or more of a CpG oligonucleotide, a DNA TLR agonist comprising a CpG sequence, a non-CpG DNA TLR agonist, an RNA TLR agonist, an aluminum salt, an anti-CD40 antibody, a fusion protein, a cytokine, a small molecule TLR agonist, an oil-based or surfactant-based adjuvant, lipopolysaccharide, a plant extract, or a derivative thereof. 51. The immunogenic construct of embodiment 49 or 50, wherein the adjuvant comprises a CpG oligonucleotide. 52. The immunogenic construct of any one of aspects 49-51, wherein the adjuvant is loaded onto nanoparticles. 53. The immunogenic construct of any one of aspects 49-52, wherein the adjuvant is loaded onto or within the lipid layer. 54. The immunogenic construct of any one of aspects 49-53, wherein the adjuvant is present at 1-20% by weight of the nanoparticles. 55. The immunogenic construct of any one of aspects 48 to 54, wherein the nanoparticle is a silica nanoparticle, a silicon nanoparticle, an iron oxide nanoparticle, a gold nanoparticle, a silver nanoparticle, a carbon nanotube, or an adjuvant nanoparticle. 56. The immunogenic construct of embodiment 55, wherein the nanoparticles are calcium phosphate nanoparticles. 57. The immunogenic construct of any one of aspects 48 to 56, wherein the lipid layer is a monolayer or multilayer comprising one or more of lipids selected from neutral lipids, fatty acid-modified lipids, phospholipids, fatty acids, polymerizable lipids, cationic lipids, sphingolipids, and sterols. 58. The neutral lipid is a prostaglandin, an eicosanoid, or a glyceride, the fatty acid-modified lipid is 1,2-diphytanoyl-sn-glycero-3-phosphocholine or 1-(12-biotinyl(aminododecanoyl))-2-oleoyl-sn-glycero-3-phosphoethanolamine, and the phospholipid is phosphatidylcholine, phosphatidylethanolamine, 1,2-distearoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine. 58. The immunogenic construct of embodiment 57, wherein the polymerizable lipid is ethanolamine, the fatty acid is stearic acid or lauric acid, the polymerizable lipid is cholesterol-PEG or distearoyl-rac-glycerol-PEG2K, the cationic lipid is 1,2-dioleoyl-3-trimethylammonium-propane or dimethyldioctadecylammonium bromide, the sphingolipid is sphingomyelin or ceramide, and the sterol is cholesterol or stigmasterol. 59. The immunogenic construct of embodiment 58, wherein the lipid layer comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, dimethyldioctadecylammonium bromide, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine, and distearoyl-rac-glycerol-PEG2K. 60. The immunogenic construct of any one of aspects 48 to 59, wherein the lipid layer is present in 0.1 to 99.9% by weight of the nanoparticle. 61. The immunogenic construct of any one of aspects 48-60, wherein the infectious agent is a virus. 62. The immunogenic construct of any one of aspects 48-61, wherein the infectious agent is a β-coronavirus. 63. The immunogenic construct of any one of aspects 48-62, wherein the infectious agent is SARS-CoV-2, SARS-CoV-1, or MERS-CoV. 64. The immunogenic construct of any one of aspects 48 to 63, wherein the infectious agent is SARS-CoV-2. 65. The immunogenic construct of embodiment 64, wherein the antigen or antigen-producing agent is a recombinant full-length protein. 66. The immunogenic construct of embodiment 65, wherein the antigen or antigen-producing agent is a full-length SARS-CoV-2 spike glycoprotein, a SARS-CoV-2 nucleocapsid protein, or a SARS-CoV-2 membrane protein. 67. The immunogenic construct of embodiment 64, wherein the antigen or antigen-producing agent is a protein subunit. 68. The immunogenic construct of embodiment 67, wherein the antigen or antigen-producing agent is a protein subunit corresponding to the S1 region, S2 region, or RBD region of the SARS-CoV-2 spike glycoprotein. 69. The immunogenic construct of embodiment 64, wherein the antigen or antigen-producing agent is a peptide corresponding to an immunogenic sequence of the SARS-CoV-2 spike glycoprotein. 70. The immunogenic construct of embodiment 69, wherein the antigen or antigen-producing agent has a peptide sequence of any one of SEQ ID NOs:1-8. 71. The immunogenic construct of embodiment 64, wherein the antigen or antigen-producing agent is mRNA or pDNA. 72. The immunogenic construct of any one of aspects 48 to 71, wherein the antigen or antigen-producing agent is loaded onto a nanoparticle. 73. The immunogenic construct of any one of aspects 48 to 72, wherein the antigen or antigen-producing agent is loaded onto or within the lipid layer. 74. The immunogenic construct of any one of aspects 48 to 73, wherein the antigen or antigen-producing agent is present at 0.01 to 10% by weight of the nanoparticle. 75. The immunogenic construct of any one of aspects 48 to 74, further comprising at least one oligonucleotide. 76. The immunogenic construct of embodiment 75, wherein at least one type of oligonucleotide comprises an siRNA. 77. The immunogenic construct of embodiment 76, wherein the siRNA inhibits or downregulates a gene whose upregulation is associated with cellular immunosuppression. 78. The immunogenic construct of embodiment 77, wherein the cell is an antigen-presenting cell. 79. The immunogenic construct of embodiment 78, wherein the antigen-presenting cell is a dendritic cell or a macrophage. 80. The immunogenic construct of embodiment 79, wherein the gene is STAT3, IDO-1, IL-6, or PD-L1. 81. The immunogenic construct of any one of aspects 74 to 80, wherein the at least one type of oligonucleotide is present in 0.01 to 10% by weight of the nanoparticles. 82. The immunogenic construct of any one of aspects 48 to 81, further comprising a targeting agent for a cell. 83. The immunogenic construct of embodiment 82, wherein the cell is an antigen-presenting cell. 84. The immunogenic construct of embodiment 83, wherein the antigen-presenting cell is a dendritic cell or a macrophage. 85. The immunogenic construct of embodiment 84, wherein the targeting agent is mannose, a monoclonal or polyclonal antibody, or a fragment thereof, that recognizes and binds to an epitope presented on an antigen-presenting cell, or a ligand that binds to a surface receptor on an antigen-presenting cell. 86. The immunogenic construct of any one of aspects 48 to 85, further comprising a labeling agent. 87. The immunogenic construct of embodiment 86, wherein the labeling agent is a fluorescent dye and / or a metal probe. 88. The immunogenic construct of any one of aspects 48 to 87, having a hydrodynamic diameter of about 10 nm to about 10 microns. 89. The immunogenic construct of embodiment 88, having a hydrodynamic diameter of about 90 nm to about 150 nm. 90. The immunogenic construct of any one of aspects 1-89, wherein the nanoparticles have a size of about 5 nm to 999 nm. 91. A pharmaceutical composition comprising the immunogenic construct of any one of aspects 1 to 90 and a pharmaceutically acceptable excipient. 92. A vaccine comprising the immunogenic construct of any one of aspects 1 to 90 and a pharmaceutically acceptable excipient. 93. A method for co-delivering an antigen and an adjuvant to a cell, comprising contacting the cell with the immunogenic construct of any one of aspects 1-90. 94. The method of embodiment 93, wherein the cell is an antigen-presenting cell. 95. The method of embodiment 94, wherein the cell is a dendritic cell or a macrophage. 96. The method of embodiment 93, wherein the cell is a muscle cell. 97. A method of inducing an immune response in a subject against an infectious agent, comprising administering to the subject an immunogenic amount of the immunogenic construct of any one of aspects 1-90. 98. The method of embodiment 97, wherein the subject is a human. 99. The method of embodiment 97 or 98, wherein the subject is immunocompromised. 100. The method of any one of aspects 97-99, wherein the immunogenic construct is administered by intramuscular injection. 101. A method of treating or preventing an infectious disease in a subject, comprising administering to the subject an immunogenic amount of the immunogenic construct of any one of aspects 1 to 90. 102. The method of embodiment 101, wherein the subject is a human. 103. The method of embodiment 101 or 102, wherein the subject is immunocompromised. 104. The method of any one of embodiments 101-103, wherein the immunogenic construct is administered intramuscularly, by inhalation, or intranasally.

[0147] Exemplary Embodiment Set 2: 1. Nanoparticles, an amount of cross-linked cationic polymer comprising polyethyleneimine (PEI) electrostatically bound to the outer surface of the nanoparticle, wherein the PEI content is at least 10% by weight of the nanoparticle platform (NP); and an amount of stabilizer comprising polyethylene glycol (PEG) covalently attached to cross-linked PEI NP, including Antigens or antigen-producing agents of infectious agents An immunogenic construct comprising: The immunogenic construct, wherein the hydrodynamic size of the construct is 1 micron or less. 2. The immunogenic construct of embodiment 1, wherein the nanoparticles are mesoporous silica nanoparticles (MSNPs). 3. Nanoparticles, a cross-linked cationic polymer attached to the outer surface of the nanoparticle; and Stabilizers bound to cross-linked cationic polymers or to the outer surface of nanoparticles a nanoparticle platform (NP) comprising: Antigens or antigen-producing agents of infectious agents An immunogenic construct comprising: 4. The immunogenic construct of embodiment 1 or embodiment 3, further comprising an adjuvant. 5. The immunogenic construct of embodiment 4, wherein the adjuvant comprises one or more of a CpG oligonucleotide, a DNA TLR agonist comprising a CpG sequence, a non-CpG DNA TLR agonist, an RNA TLR agonist, an aluminum salt, an anti-CD40 antibody, a fusion protein, a cytokine, a small molecule TLR agonist, an oil-based or surfactant-based adjuvant, lipopolysaccharide, a plant extract, or derivatives thereof. 6. The immunogenic construct of embodiment 5, wherein the adjuvant comprises a CpG oligonucleotide. 7. The immunogenic construct of embodiment 4, wherein the adjuvant comprises poly I:C. 8. The immunogenic construct of any one of aspects 4 to 7, wherein the adjuvant is present at 1 to 20% by weight of the NP. 9. The immunogenic construct of embodiment 1 or embodiment 3 or any other embodiment of this set, wherein the nanoparticle is a silica nanoparticle, a silicon nanoparticle, an iron oxide nanoparticle, a gold nanoparticle, a silver nanoparticle, a calcium carbonate nanoparticle, a calcium phosphate nanoparticle, a carbon nanotube, or an adjuvant nanoparticle. 10. The immunogenic construct of embodiment 9, wherein the nanoparticles are mesoporous silica nanoparticles (MSNPs). 11. The immunogenic construct of embodiment 10, wherein the MSNPs have an average pore size of 2 to 6 nm, 7 nm, or less than 7 nm. 12. The immunogenic construct of embodiment 9, wherein the nanoparticles are iron oxide nanoparticles. 13. The immunogenic construct of embodiment 1 or embodiment 3, or any other embodiment of this set, wherein the cationic polymer comprises PEI, chitosan, polypropyleneimine, polylysine, polyamidoamine, poly(allylamine), poly(diallyldimethylammonium chloride), poly(N-isopropylacrylamide-co-acrylamide), poly(N-isopropylacrylamide-co-acrylic acid), diethylaminoethyl-dextran, poly(N-ethyl-vinylpyridinium bromide), poly(dimethylamino)ethyl methacrylate, poly(ethylene glycol)-co-poly(trimethylaminoethyl methacrylate chloride), or a mixture of two or more thereof. 14. The immunogenic construct of embodiment 1 or embodiment 3, wherein the cationic polymer is or comprises PEI. 15. The immunogenic construct of embodiment 1 or embodiment 3 or any other embodiment of this set, wherein the cationic polymer has a molecular weight of from about 0.8 kDa to about 25 kDa. 16. The immunogenic construct of embodiment 1 or embodiment 3 or any other embodiment of this set, wherein the cationic polymer is present in 1-50% by weight of the NP. 17. The immunogenic construct of embodiment 1 or embodiment 3, or any other embodiment of this set, wherein the stabilizer comprises PEG, dextran, polysialic acid, hyaluronic acid, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, or a mixture of two or more thereof. 18. The immunogenic construct of embodiment 17, wherein the stabilizer is PEG. 19. The immunogenic construct of embodiment 1 or embodiment 3 or any other embodiment of this set, wherein the stabilizer has a molecular weight of about 1 kDa to about 20 kDa, or about 5 kDa. 20. The immunogenic construct of embodiment 1 or embodiment 3, or any other embodiment of this set, wherein the stabilizer is present in 1-50%, about 10-30%, about 5-20%, about 15%, or about 20% by weight of the NP. 21. The immunogenic construct of embodiment 1 or embodiment 3 or any other embodiment of this set, wherein the antigen comprises a protein, and the protein antigen is conjugated to a stabilizer. 22. The immunogenic construct of embodiment 1 or embodiment 3, wherein the antigen is a peptide, and the peptide antigen is electrostatically bound to the cross-linked cationic polymer. 23. The immunogenic construct of embodiment 1 or embodiment 3, or any other embodiment of this set, wherein the antigenic agent is mRNA or pDNA, and the antigenic agent is electrostatically bound to the cross-linked cationic polymer. 24. The immunogenic construct of embodiment 1 or embodiment 3 or any other embodiment of this set, wherein the infectious agent is a virus. 25. The immunogenic construct of embodiment 24, wherein the infectious agent is a β-coronavirus. 26. The immunogenic construct of embodiment 25, wherein the infectious agent is SARS-CoV-2, SARS-CoV-1, or MERS-CoV. 27. The immunogenic construct of embodiment 26, wherein the infectious agent is SARS-CoV-2. 28. The immunogenic construct of aspect 27, wherein the antigen is a recombinant full-length SARS-CoV-2 protein or the antigen-producing agent encodes a recombinant full-length SARS-CoV-2 protein. 29. The immunogenic construct of embodiment 21 or any other embodiment of this set, wherein the full-length SARS-CoV-2 protein is a SARS-CoV-2 spike glycoprotein, a SARS-CoV-2 nucleocapsid protein, or a SARS-CoV-2 membrane protein. 30. The immunogenic construct of embodiment 27, wherein the antigen is a protein subunit or the antigen-producing agent encodes a protein subunit. 31. The immunogenic construct of embodiment 30, wherein the protein subunit corresponds to the SARS-CoV-2 spike glycoprotein S1 region, S2 region, or receptor binding domain (RBD) region. 32. The immunogenic construct of embodiment 27 or any other embodiment of this set, wherein the antigen is a peptide corresponding to an immunogenic sequence of the SARS-CoV-2 spike glycoprotein, or the antigen producing agent encodes a peptide corresponding to an immunogenic sequence of the SARS-CoV-2 spike glycoprotein. 33. The immunogenic construct of embodiment 32, wherein the peptide comprises the sequence of any one of SEQ ID NOs: 1-8. 34. The immunogenic construct of embodiment 27, wherein the antigen producing agent is mRNA or pDNA. 35. The immunogenic construct of embodiment 1 or embodiment 3 or any other embodiment of this set, wherein the infectious agent is a bacterium, a parasite, a protozoan, or a fungus. 36. The immunogenic construct of embodiment 1 or embodiment 3, or any other embodiment of this set, wherein the antigen or antigen-producing agent is present at 0.5 to 20% by weight of the NP. 37. The immunogenic construct of embodiment 1 or embodiment 3, further comprising at least one oligonucleotide. 38. The immunogenic construct of embodiment 37, wherein at least the oligonucleotide is electrostatically bound to the cationic polymer. 39. The immunogenic construct of embodiment 38, wherein at least one oligonucleotide comprises an siRNA, miRNA, miRNA mimic, or antisense oligonucleotide. 40. The immunogenic construct of embodiment 38, wherein at least one oligonucleotide comprises an siRNA. 41. siRNA Genes whose expression or upregulation is associated with cellular immunosuppression 41. The immunogenic construct of embodiment 40, wherein the construct inhibits or downregulates 42. The immunogenic construct of embodiment 41, wherein the cell is an antigen-presenting cell. 43. The immunogenic construct of embodiment 42, wherein the antigen-presenting cell is a dendritic cell or a macrophage. 44. The immunogenic construct of embodiment 43, wherein the gene is STAT3, IDO-1, IL-6, or PD-L1. 45. The immunogenic construct of embodiment 37, wherein the oligonucleotide is present in 1 to 10% by weight of the NP. 46. ​​The immunogenic construct of embodiment 1 or embodiment 3, or any other embodiment of this set, further comprising a targeting agent for a cell. 47. The immunogenic construct of embodiment 46, wherein the cell is an antigen-presenting cell. 48. The immunogenic construct of embodiment 47, wherein the antigen-presenting cell is a dendritic cell or a macrophage. 49. The targeting agent is mannose, a monoclonal or polyclonal antibody, or a fragment thereof, that recognizes and binds to an epitope presented on an antigen-presenting cell; or Ligands that bind to surface receptors on antigen-presenting cells 49. The immunogenic construct of embodiment 48, comprising at least one of: 50. The immunogenic construct of embodiment 3, having a hydrodynamic diameter of about 10 nm to about 10 microns. 51. The immunogenic construct of embodiment 1 or embodiment 3, or any other embodiment of this set, having a hydrodynamic diameter of about 30 nm to about 200 nm. 52. The immunogenic construct of embodiment 1 or embodiment 3, or any other embodiment of this set, having a hydrodynamic diameter of about 80 nm to about 999 nm. 53. An immunogenic composition comprising a plurality of immunogenic constructs of embodiment 1 or embodiment 3 or any other embodiment of this set. 54. A composition comprising the immunogenic construct of embodiment 1 or embodiment 3, or any other embodiment of this set, and at least one biologically or pharmaceutically acceptable excipient. 55. A vaccine comprising the immunogenic construct of embodiment 1 or embodiment 3, or any other embodiment of this set, and a pharmaceutically acceptable excipient. 56. A method of co-delivering an antigen and an adjuvant to a cell, comprising contacting the cell with an immunogenic construct of embodiment 1 or embodiment 3, or any other embodiment of this set. 57. The method of embodiment 56, wherein the cell is an antigen-presenting cell. 58. The method of embodiment 57, wherein the cell is a dendritic cell or a macrophage. 59. The method of embodiment 56, wherein the cell is a muscle cell. 60. A method comprising administering to a subject an immunostimulatory amount of the immunogenic construct of embodiment 1 or embodiment 3 or any other embodiment of this set. 61. The method of embodiment 60, which results in the induction of an immune response against an infectious agent in a subject. 62. The method of embodiment 60, which results in treatment or prevention of an infectious disease in a subject. 63. The method of embodiment 62, wherein the subject is a human. 64. The method of embodiment 62, wherein the subject is immunocompromised. 65. The method of embodiment 62, wherein the immunogenic construct is administered transdermally, intramuscularly, by inhalation, or intranasally. [Example]

[0148] XIV. Experimental Examples Example 1. Preparation of immunogenic constructs Mesoporous silica-based nanoparticles coated with PEI and PEG were synthesized as previously described (see, e.g., International Application No. PCT / US2016 / 022655, incorporated herein by reference in its entirety). Briefly, mesoporous silica nanoparticles (MSNPs) were synthesized by sol-gel synthesis. The MSNP core was coated layer-by-layer with polyethyleneimine (PEI) and polyethylene glycol (PEG). Additionally, to enhance the efficacy and safety of oligonucleotide delivery, the PEI on the MSNPs was crosslinked (primarily to itself) (see Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015). Such crosslinking increases the buffering capacity and endosomal escape of the cargo and also reduces the surface charge of the nanoparticle platform (NP). Reducing the surface charge of NPs or immunogenic constructs confers safety to antigen-presenting cells, which is important for vaccination (see Figure 16). The pore size of the MSNPs used in this example was measured by TEM to be 2-3 nm and by Barrett-Joyner-Halenda (BJH) pore size analysis (e.g., via nitrogen adsorption and desorption) to be 6.6 nm. The cross-linked PEI and PEG-coated MSNPs are hereafter referred to as nanoparticle platforms or "NPs."

[0149] CpG 1826 (mouse sequence; Invivogen) was electrostatically loaded onto NPs by 10 min of mixing, although shorter times (2–5 min) were also effective. Loading was achieved in a fully bound manner, as confirmed by the absence of free cargo molecules in the supernatant upon separation of the cargo-loaded NPs by centrifugation. siRNA was conjugated with Dy677 dye (Dharmacon) and quantified by fluorescent signal. Unbound CpG and siRNA cargo content in the supernatant was measured by a Nanodrop spectrophotometer, a microplate spectrophotometer, or gel electrophoresis. SIINFEKL (SF; SEQ ID NO: 90) peptide was loaded via noncovalent interactions with the NPs (on the PEI layer) by 2 h of mixing with the NPs, although shorter times were also effective. Large proteins (antibodies, full-length proteins) are typically loaded by covalent attachment (Example 4), although noncovalent attachment is also possible. Large protein cargoes, such as spike proteins, are not encapsulated inside the small pores (e.g., 2-6 nm) of mesoporous silica, but instead are attached to the surface of the material (e.g., conjugated onto a PEG-PEI layer or electrostatically bound, or adsorbed onto the external silica surface).

[0150] The amount of unbound peptide or protein in the supernatant (upon centrifugation) was characterized either by the fluorescent signal of a fluorescent dye conjugated to the peptide / protein or by a BCA assay. Other protein assays can also be used. With multiple cargo loadings, the hydrodynamic size (diameter) of the resulting immunogenic constructs remained below 5 microns (e.g., below 1 micron), suitable for cellular uptake. As shown in Figure 2, when NPs were loaded with 5 wt% SF (via noncovalent bonds) and 2 wt% CpG, they maintained a hydrodynamic size of approximately 100 nm. As shown in Table 3, when the nanoparticle platform (MSNP-PEI-PEG [MSNPs loaded with 15 wt% crosslinked PEI and 10 wt% PEG] characterized by thermogravimetric analysis (TGA)) was loaded with approximately 3 wt% spike protein (e.g., via covalent bonds), 2 wt% siRNA, and 4 wt% CpG, it maintained a hydrodynamic size of less than 150 nm. siRNA and CpG are finally loaded onto the immunogenic construct. Approximately 2-4% siRNA loading by weight allows for approximately 4-9% CpG loading by weight while maintaining size below 150 nm (Table 4). CpG, siRNA, and peptide were loaded at the corresponding final weight percentages in a fully bound manner (as confirmed by the absence of unbound cargo in the supernatant upon centrifugation) by mixing with the NP solution as previously described. While CpG 1826 (mouse sequence) was used throughout Examples 1-4, CpG 7909 / 2006 (human sequence) was also evaluated and showed similar properties. All loading rates are by weight of the NP.

[0151] Table 3 shows the hydrodynamic size of 1) mesoporous silica nanoparticles (NPs) coated with 15 wt% cross-linked PEI and 10 wt% PEG, 2) NPs loaded with approximately 2 wt% siRNA and approximately 6 wt% CpG, 3) spike protein (3 wt%) conjugated NPs (Spike-NPs), and 4) Spike-NPs loaded with approximately 2 wt% siRNA and approximately 4 wt% CpG. The mean size (Z-average) and polydispersity index (PDI) are shown from triplicate measurements using a Malvern Zetasizer. All loadings are by weight of the nanoparticle platform (NPs or PEG-PEI-MSNPs).

[0152] (Table 3) TIFF2023534232000004.tif37128

[0153] Table 4 shows the hydrodynamic size of mesoporous silica nanoparticles coated with PEI and PEG (NP) and loaded with approximately 2-4% siRNA and approximately 4-9% CpG by weight of the NP.

[0154] (Table 4) TIFF2023534232000005.tif34128

[0155] In addition to CpG, other adjuvants, such as poly I:C (Adipogen), can be non-covalently loaded onto immunogenic constructs. Poly I:C was electrostatically and completely loaded onto NPs by mixing the poly I:C solution with the NP solution for 10 minutes. The hydrodynamic size remained small (88 nm) at approximately 2 wt% loading, but increased significantly (4.8 microns) at approximately 9 wt% loading (Figure 3).

[0156] Example 2. NP co-delivery of CpG and oligonucleotides (e.g., siRNA) to knockdown immunosuppressive genes (e.g., STAT3, PD-L1) STAT3 is considered a potent immunosuppressive gene. To determine the knockdown effect of NPs loaded with siRNA against STAT3 (siSTAT3), we treated immune cells (i.e., dendritic cells, macrophages) and various cancer cells (B16-F10, HCC1954, D17) with siSTAT3-NPs (mesoporous silica coated with PEI and PEG from Example 1) for 48 hours, followed by RNA isolation and qRT-PCR to determine the knockdown effect of STAT3 in the cell lines. As shown in Figure 4A, the immunogenic construct (siSTAT3-NPs) resulted in >70% target gene knockdown in both immune and cancer cell lines. The same siSTAT3 sequence was able to knockdown the STAT3 gene in human, canine, and mouse cells (Figure 4A). The same immunogenic construct loaded with both siRNA and CpG was still effective in knocking down STAT3 in both immune and cancer cells (Figure 4B). Interestingly, siSCR-NPs were also found to reduce STAT3 levels in DCs (see Figure 4C). This was not caused by NP toxicity, as cell viability was unchanged compared to untreated controls and STAT3 mRNA was normalized by housekeeping mRNA. Without being bound by any explanation, it is proposed that this may be due to the antioxidant properties of mesoporous silica nanoparticles, as antioxidants have previously been reported to counteract immunosuppressive pathways, including STAT3 activation (Yoon et al., Autophagy, 6(8):1125-1138, 2010). On the other hand, Dharmafect (a commercially available transfection agent based on cationic lipids (non-antioxidant) manufactured by Horizon Discovery) was found to increase STAT3 expression in DCs (Figure 4C), potentially resulting in an undesirable immunosuppressive TME. Figure 4D shows that nanoparticles can also be used to deliver PD-L1 siRNA to knockdown PD-L1 protein expression in lung cells.This suggests that the use of antioxidant mesoporous silica nanoparticle platforms, as described in Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015, and Morry et al., Biomaterials, 66:41-52, 2015, may be more advantageous than lipid nanoparticles for controlling STAT3-mediated pathways.

[0157] NPs (PEG-PEI-MSNPs) can be loaded with 2–4 wt% siRNA and 4–9 wt% NPs while maintaining the hydrodynamic size of the immunogenic constructs below 150 nm (Table 4). As shown in Figures 5 and 6, in vivo NP co-delivery of CpG and siSTAT3 elicits better adaptive immunity than NP delivery of CpG or siSTAT3 alone. C57BL / 6 mice were implanted with 250K and 100K B16F10 melanoma cells in both shoulders to model local (primary) and distant (metastatic) tumors, respectively. Eight days after tumor implantation, the local tumor was intratumorally injected with a total of three doses of treatment, each at 3-day intervals, while the distant tumor was left untreated. Tumor size and viability were monitored at least twice weekly, as shown in Figures 5A–C. Figure 5 shows that NP co-delivery of siSTAT3 and CpG (siSTAT3-CpG-NP) modulates the immunosuppressive environment (due to siSTAT3) and results in a superior systemic antitumor immune response, as evidenced by greater tumor reduction in both locally treated tumors (Figure 5A) and distant tumors (Figure 5B), as well as superior survival rates (Figure 5C), compared with NPs delivering CpG (CpG-NP) or siSTAT3 alone (siSTAT3-NP). Figure 6 shows that siSTAT3-CpG-NP treatment resulted in significantly higher CD8 / Treg ratios (p<0.05 vs. saline) for both local (treated) and distant (untreated) tumors (Figure 6A) and associated draining lymph nodes (DLNs) (Figure 6B), supporting successful in situ tumor vaccination. Within patient tumors, regulatory T cells (Tregs) are typically elevated, resulting in CD8 +The immunogenic constructs suppressed antitumor immune responses, including T cell activity. Activated T cells also expanded further in tumor-draining lymph nodes (Figure 6C). These results demonstrate the ability of the immunogenic constructs to knock down target genes in immune cells and other cells (e.g., cancer cells or muscle cells) and provide a rationale for using the immunogenic constructs of the present disclosure for co-delivery of siRNA and adjuvants (e.g., CpG). For in situ cancer vaccination in Figures 5 and 6, the tumor was relied upon as a depot for antigens (hence, no exogenous antigens were added to the constructs).

[0158] Example 3. NP co-delivery of CpG and antigen to induce adaptive (antigen-specific) immune responses A study was conducted to determine the ability of CpG-SF-NPs (NPs loaded with CpG and SF) to induce systemic antigen-specific immunity. Briefly, C57BL / 6 mice (n = 3 / group) were injected via the footpad with the immunogenic construct of CpG-SF-NPs, NPs loaded with SF (SF-NPs) or CpG alone (CpG-NPs), and SF formulated with incomplete Freund's adjuvant (IFA) (IFA / SF). Untreated mice were included as controls. One week after treatment, cells were harvested from the spleens of the mice, treated with Golgiblock, and incubated for 6 hours in the presence or absence of SF. Intracellular IFNγ, whose production corresponds to a CD8+ T cell response, was analyzed by flow cytometry. As shown in Figure 7, CpG-SF-NPs induced excellent CD8+ T cell responses after ex vivo peptide restimulation. The results from Examples 1-3 provide a strong rationale for using the immunogenic constructs of the present disclosure for the co-delivery of adjuvants, antigens and / or oligonucleotides.

[0159] Example 4. Synthesis of spike protein conjugated NP (immunogenic construct) As an example, we covalently conjugated the full-length SARS-CoV-2 spike glycoprotein (Sino Biological) to NPs (PEI-PEG-MSNPs). Briefly, the spike protein was thiolated for 1 hour, then mixed with the NPs and shaken overnight at 4 °C. After shaking, the final immunogenic construct was washed, and based on the amount of unbound protein determined by BCA assay of the supernatant, the final spike protein content was determined to be 3% by weight of the NPs. siRNA (against luciferase or non-targeting scrambled siRNA) and CpG were finally loaded onto the immunogenic construct via electrostatic interactions (shaking for 5–10 min at room temperature). The hydrodynamic size of the spike-conjugated NPs was less than 100 nm and remained less than 150 nm after spike glycoprotein loading (Table 3 and Figure 8A). The spike-NPs were able to effectively deliver siRNA to knockdown a model gene (luciferase) in human cells (Figure 8B). Briefly, cells were seeded at 3500 cells / well and incubated overnight at 37°C. The next day, cells were treated with spike-NPs at 30 or 60 nM siRNA doses. Forty-eight hours after treatment, cells were lysed and analyzed for luciferase activity using a luciferase glow assay kit (ThermoFisher Scientific) and for protein concentration using a BCA protein assay kit according to the manufacturer's protocol (ThermoFisher Scientific). Luciferase activity in the lysates was normalized by the corresponding protein concentration in the same well and reported as a percentage of the untreated control. All treatments were performed in quadruplicate.

[0160] Example 5. Synthesis of lipid-coated calcium phosphate nanoparticle platform (CaP-L) Calcium phosphate nanoparticles (CaPNPs) were synthesized in a water-in-oil microemulsion. Briefly, 60 μL of 2.5 M CaCl (Fisher Scientific, USA) was dispersed in 4 mL of cyclohexane (Sigma, USA) / Igepal CO-520 (Sigma, USA) (71:29, v / v) to form the calcium phase. 60 μL of 12.5 mM NaHPO (Fisher Scientific, USA) was dispersed in another 4 mL of cyclohexane / Igepal CO-520 (71:29, v / v) to form the phosphate phase. 40 μL of a 20 mM solution of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids, USA) in CHCl (Fisher Scientific, USA) was added to the phosphate phase. The phosphate phase was then added dropwise to the calcium phase. The mixture was stirred at room temperature for 0, 10, 15, or 20 minutes. An equal volume of ethanol (Decon Labs, USA) was added to break the microemulsion. CaPNPs were recovered by centrifugation at 21,000 g for 15 minutes and then washed three times with absolute ethanol to remove the residual oil phase. The pellet was suspended in 100 μL of CHCl3. CaP-L was formed by mixing 10 μL of CaPNP in CHCl with 1.4 μL of 20 mM dimethyldioctadecylammonium bromide (Sigma, USA), 1.4 μL of 20 mM cholesterol (Sigma, USA), 2.8 μL of 20 mM distearoyl-rac-glycerol-PEG2K (Avanti Polar Lipids, USA), and 0.7 μL of 20 mM 1,2-distearoyl-sn-glycero-3-phosphocholine (Sigma, USA) in CHCl, followed by slight bath sonication. The CHCl was then removed under reduced pressure and rehydrated with 100 μL of PBS 1X (pH = 7.2). The hydrodynamic size of CaP-L was measured using a Zetasizer (ZS-90 / Malvern, Malvern, UK) (Table 5).

[0161] Table 5 shows the hydrodynamic size of lipid-coated calcium phosphate NPs (CaP-L) and CaP-L loaded with 0.3% siRNA by weight of CaP-L.

[0162] (Table 5) TIFF2023534232000006.tif24128

[0163] Synthesis of siRNA-loaded lipid-coated CaP-L (siRNA-CaP-L) siRNA-loaded calcium phosphate nanoparticles were synthesized in a water-in-oil microemulsion. Briefly, 5 μg of siRNA was added to 60 μL of 2.5 M CaCl (Fisher Scientific, USA), and the resulting solution was then dispersed in 4 mL of cyclohexane / Igepal CO-520 (Sigma, USA) (71:29, v / v) to form the calcium phase. 60 μL of 12.5 mM NaHPO (Fisher Scientific, USA) was dispersed in another 4 mL of cyclohexane / Igepal CO-520 (71:29, v / v) to form the phosphate phase. 40 μL of a 20 mM solution of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids, USA) in CHCl (Fisher Scientific, USA) was added to the phosphate phase, and the phosphate phase was then added dropwise to the calcium phase. The mixture was stirred at room temperature for 10 minutes, and then an equal volume of ethanol (Decon Labs, USA) was added to break the microemulsion. The resulting siRNA-CaPNP was collected by centrifugation at 21,000 g for 15 minutes, and then washed three times with absolute ethanol to remove the residual oil phase. The pellet thus obtained was suspended in 100 μL of CHCl3. 10 μL of siRNA-CaPNP in CHCl3 was mixed with 1.4 μL of 20 mM dimethyldioctadecylammonium bromide (Sigma, USA), 1.4 μL of 20 mM cholesterol (Sigma, USA), 2.8 μL of 20 mM distearoyl-rac-glycerol-PEG2K (Avanti Polar Lipids, USA), and 0.7 μL of 20 mM 1,2-distearoyl-sn-glycero-3-phosphocholine (Sigma, USA) in CHCl3, followed by slight bath sonication to form siRNA-CaP-L. The CHCl3 was then removed under reduced pressure, and CaP-L was formed by rehydration with 100 μL of PBS 1X (pH = 7.2). The hydrodynamic size of siRNA-CaP-L was measured using a Zetasizer (ZS-90 / Malvern, Malvern, UK) (Table 5).The material was able to knock down luciferase (as a model protein) in human cells (Figure 9A) and was found to be safe for the cells (Figure 9B).

[0164] Synthesis of siRNA / CpG-loaded lipid-coated CaP-L (siRNA / CpG-CaP-L) siRNA-loaded calcium phosphate nanoparticles (siRNA-CaPNPs) were synthesized in a water-in-oil microemulsion. Briefly, 5 μg of siRNA (scrambled siRNA) was added to 60 μL of 2.5 M CaCl2 (Fisher Scientific, USA). The solution was then dispersed in 4 mL of cyclohexane (Sigma, USA) / Igepal CO-520 (Sigma, USA) (71:29, v / v) to form the calcium phase. 60 μL of 12.5 mM Na2HPO4 (Fisher Scientific, USA) was dispersed in another 4 mL of cyclohexane / Igepal CO-520 (71:29, v / v) to form the phosphate phase. 40 μL of 20 mM 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids, USA) in CHCl3 (Fisher Scientific, USA) was added to the phosphate phase. The phosphate phase was then added dropwise to the calcium phase, and the mixture was stirred at room temperature for 10 minutes. An equal volume of ethanol (Decon Labs, USA) was added to break the microemulsion, and the resulting siRNA-CaPNP was collected by centrifugation at 21,000 g for 15 minutes, and then washed three times with absolute ethanol to remove the residual oil phase. The resulting pellet was suspended in 100 μL of CHCl3. 10 μL of siRNA-CaPNP in CHCl3 was mixed with 1.4 μL of 20 mM dimethyldioctadecylammonium bromide (Sigma, USA), 1.4 μL of 20 mM cholesterol (Sigma, USA), 2.8 μL of 20 mM distearoyl-rac-glycerol-PEG2K (Avanti Polar Lipids, USA), and 0.7 μL of 20 mM 1,2-distearoyl-sn-glycero-3-phosphocholine (Sigma, USA) in CHCl3, followed by slight bath sonication to form siRNA / CaP-L. The CHCl3 was then removed under reduced pressure, and the siRNA / CpG-CaP-L was rehydrated with 100 μL of PBS 1X (pH = 7.2) containing 2 μg of CpG (CpG 7909 / 2006).

[0165] Synthesis of mRNA / siRNA / CpG-loaded lipid-coated CaPNPs (mRNA / siRNA / CpG-CaP-L) mRNA / siRNA-loaded calcium phosphate cores (mRNA / siRNA-CaPNPs) were synthesized in a water-in-oil microemulsion. 2.5 μg of siRNA (scrambled siRNA) and 2.5 μg of mRNA (firefly luciferase mRNA) were added to 60 μL of 2.5 M CaCl2 (Fisher Scientific, USA). The solution was then dispersed in 4 mL of cyclohexane / Igepal CO-520 (Sigma, USA) (71:29, v / v) to form the calcium phase. 60 μL of 12.5 mM Na2HPO4 (Fisher Scientific, USA) was dispersed in another 4 mL of cyclohexane / Igepal CO-520 (71:29, v / v) to form the phosphate phase. 40 μL of 20 mM 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids, USA) in CHCl3 (Fisher Scientific, USA) was added to the phosphate phase. The phosphate phase was then added dropwise to the calcium phase. The mixture was stirred at room temperature for 10 minutes. An equal volume of ethanol (Decon Labs, USA) was added to break the microemulsion. The mRNA / siRNA-CaPNPs were recovered by centrifugation at 21,000 g for 15 minutes and then washed three times with absolute ethanol to remove the residual oil phase. The pellet was suspended in 100 μL of CHCl3. Ten μL of the mRNA / siRNA-CaPNP solution in CHCl3 was mixed with 1.4 μL of 20 mM dimethyldioctadecylammonium bromide (Sigma, USA), 1.4 μL of 20 mM cholesterol (Sigma, USA), 2.8 μL of 20 mM distearoyl-rac-glycerol-PEG2K (Avanti Polar Lipids, USA), and 0.7 μL of 20 mM 1,2-distearoyl-sn-glycero-3-phosphocholine (Sigma, USA) in CHCl3, followed by slight bath sonication to form mRNA / siRNA / CaP-L.The CHCl3 was then removed under reduced pressure and rehydrated with 100 μL of PBS 1X (pH=7.2) containing 1 μg of CpG (CpG 7909 / 2006) to form mRNA / siRNA / CpG-CaP-L.

[0166] Example 6. Immunogenic constructs for COVID-19 vaccine (AIRISE-CoV) Synthesis and Characterization of AIRISE-CoV. AIRISE-CoV consists of a mesoporous silica nanoparticle (MSNP, 50 nm) core coated with a cross-linked polyethyleneimine (PEI) and polyethylene glycol (PEG) polymer layer. Similar to antibody loading in our previous work (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015, and U.S. Patent Application Publication No. 2017 / 0173169), the SARS-CoV-2 spike glycoprotein antigen (S) was loaded by external conjugation with PEG, followed by CpG oligonucleotides and siRNA loading via electrostatic interactions with PEI, protecting them from enzymatic degradation by the PEG layer. Bioreducible crosslinking allows the use of low-molecular-weight PEI (10 kDa) to achieve the effects of high-molecular-weight PEI (25 kDa) required for endosomal escape of siRNA or proteins to the cytosol without toxicity. The nanoparticle platform has been used for cancer vaccine delivery due to its versatility to load and protect multiple types of cargo (siRNA, CpG) while maintaining a small particle size (approximately 200 nm). For cancer vaccine applications, we do not load the antigen onto the construct but rely on the antigen within the tumor (via intratumoral injection) to generate a vaccine effect. The final composition of AIRISE-CoV is 15% PEI, 10% PEG (by TGA), and 3% spike protein (by bicinchoninic acid assay (BCA)) (all by weight of NPs). CpG and siRNA are loaded at 4% and 2% by weight of the NPs, respectively (full loading confirmed by nanodrop).

[0167] siSTAT3 and CpG on nanoparticles effectively promote DC activation. We show in Figure 10 that NPs loaded with both siSTAT3 and CpG can activate DCs in draining lymph nodes (DLNs) more effectively than NPs delivering a single component upon footpad injection. Compared to saline, only AIRISE-treated mice exhibit a significantly higher percentage of activated DCs in the draining lymph nodes (p<0.05). There appears to be no activity in non-draining lymph nodes (NDLNs), suggesting a localized effect of DC priming. Antibody staining methods for quantifying immune cell populations follow published reports (Ngamcherdtrakul et al., Advanced Material, 2021; doi:10.1002 / adma.202100628).

[0168] Presence of SARS-CoV-2 IgG antibodies in mice vaccinated with AIRISE-CoV. As shown in Figure 11, BALB / c mice vaccinated with AIRISE-CoV produced high levels of SARS-CoV-2-binding IgG antibodies in serum as assessed by endpoint titers in ELISA. Endpoint titers represent the serum dilution at which SARS-CoV-2-binding antibodies are detectable and are widely used to assess the induction of humoral immunogenicity (antibody production). AIRISE-CoV produced 10 IgG antibodies after one and two doses, respectively. 5 and 10 6 (Figure 11), which is comparable to or better than other leading COVID-19 vaccine candidates (Table 6).

[0169] In Figure 12A, we show that high levels of IgG antibodies were maintained in all immunized mice up to 12 weeks after the first dose. Furthermore, we demonstrate that replacing the full-length spike protein antigen with two immunogenic spike peptides (424–433 and 891–906, JPT Peptide Technologies) did not elicit significant antibody titers (Figure 12B), providing a strong rationale for the use of the full-length spike protein antigen. Both peptides were loaded onto the nanostructure via electrostatic interactions. Without being bound by any explanation, the two peptides did not elicit a strong response. However, the same NPs have previously been shown to be effective peptide delivery agents for eliciting antigen-specific immune responses (e.g., SF delivery, Figure 7).

[0170] We found that mice injected with AIRISE-CoV via the footpad (Fig. 13, 2 doses total) or intramuscularly (Fig. 14A, 1 dose only) have sustained high levels of antibodies to date (i.e., up to 54 weeks via the footpad route and 36 weeks via the intramuscular route), suggesting long-lasting antibody production and induction of humoral immunity.

[0171] Table 6. Antibody (Ab) responses to leading COVID-19 vaccines or vaccine candidates TIFF2023534232000007.tif55154

[0172] AIRISE-CoV bearing spike protein, siSTAT3, and CpG was developed for effective vaccination of immunosuppressed or immunocompromised subjects (e.g., aging subjects or subjects with diseases and conditions that impair the immune system). However, in normal immune mice tested, we found that PEI and PEG-coated mesoporous silica nanoparticles (NPs) loaded with spike protein alone, spike protein and siSTAT3 (Figure 14B), or spike protein and CpG (Figure 14C) could also produce high and sustained antibody levels in these mice, suggesting that NPs can be effectively used for antigen delivery to create better vaccines than free antigen (no NPs). As shown in Figures 14B-C, vaccination with one dose of siSTAT3-loaded spike-NPs (Figure 14B) or one dose of CpG-loaded spike-NPs (Figure 14C) could also induce high levels of SARS-CoV-2 IgG antibodies that persisted for at least 36 weeks. This further demonstrates that antibody generation and induction of humoral immunity are long-lasting, which may be due to the ability of NPs to protect and preserve cargoes and effectively deliver them to antigen-presenting cells.

[0173] Neutralization of SARS-CoV-2 pseudovirus by immune serum. To determine whether SARS-CoV-2-specific IgG antibodies are effective in neutralizing SARS-CoV-2, we constructed a pseudovirus (CoV2-S-PsV) neutralization assay, a widely used method to evaluate the ability of immune serum to inhibit SARS-CoV-2 pseudoviruses from transfection of HEK293-derived cell lines expressing human ACE2 (spike protein-binding receptor). This assay utilizes a replication-deficient GFP-encoding reporter lentivirus pseudotyped with the SARS-CoV-2 spike (S) protein. Thus, neutralization capacity is determined by the % GFP+ cells (i.e., a low % GFP+ suggests that the pseudovirus is unable to transfect HEK293-hACE2 cells due to the presence of neutralizing antibodies). Figure 15 shows how antibody titers in serum obtained from mice vaccinated with AIRISE-CoV can effectively neutralize CoV2-S-PsV infection (i.e., inhibit binding to the ACE2 receptor), whereas serum obtained from naive mice was ineffective. Furthermore, we measured the neutralization titers (dilution required to neutralize 50% of the virus; NT) after two doses. 50 ) were found to be higher than or equivalent to neutralizing titers found in convalescent sera, as shown in Table 7.

[0174] Table 7 shows the neutralization titers (dilution required to neutralize 50% of the virus; NT) obtained from the samples in Figure 15. 50 ) is shown.

[0175] (Table 7) TIFF2023534232000008.tif54128

[0176] Example 7. Nanoparticles (NPs) with cross-linked PEI and PEG are safe for antigen-presenting cells As shown in Figure 16, NPs described herein loaded with siSTAT3 or siSTAT3+CpG were safe for both bone marrow-derived dendritic cells (BMDCs, Figure 16A) and macrophages (J774, Figure 16B). The NP dose was 35 μg / ml (2% siRNA by weight; 7% CpG by weight). Viability was assessed by CellTiter-Glo assay 2 days after treatment according to the manufacturer's protocol.

[0177] Example 8. Synthesis of mRNA-loaded MSNP constructs Mesoporous silica-coated cross-linked PEI (MSNP-PEI) or mesoporous silica-coated cross-linked PEI and PEG (MSNP-PEI-PEG) can be used for mRNA delivery to generate vaccines. MSNP-PEI or MSNP-PEI-PEG was mixed with a PBS solution of firefly luciferase mRNA (1% by weight of nanoparticles) on an orbital shaker at 350 rpm for 15–60 minutes at room temperature. The mRNA was electrostatically bound to the outer surface of the particles. Using a Zetasizer, particle size was measured before and after mixing with mRNA (Table 8). After centrifugation of the mixture, the supernatant containing unbound mRNA was obtained. The mRNA concentration in the supernatant was analyzed using a NanoDrop (ND-1000, Thermo Scientific). More than 97% of the mRNA was bound to MSNP-PEI or MSNP-PEI-PEG.

[0178] Table 8 shows the hydrodynamic size of the mRNA-loaded nanoparticles.

[0179] (Table 8) TIFF2023534232000009.tif31128

[0180] XV. Concluding Paragraph As will be understood by those skilled in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its specified described elements, steps, ingredients, or components. Accordingly, the terms "include" or "including" should be interpreted to recite "comprise," "consist of," or "consist essentially of." The transitional terms "comprise" or "comprises" mean having, but not being limited to, and allowing for the inclusion of unspecified elements, steps, ingredients, or components, even in large amounts. The transitional phrase "consisting of" excludes any elements, steps, ingredients, or components not specified. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the specified elements, steps, ingredients, or components and those that do not substantially affect the embodiment. A significant effect would result in a statistically significant change in the activity (e.g., immunogenicity) of the nanoparticle construct / platform as described.

[0181] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like used in the specification and claims are to be understood as being modified in each instance by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Where further clarification is needed, the term "about," when used in conjunction with a stated numerical value or range, has the meaning reasonably given by one of ordinary skill in the art, i.e., to indicate something greater than or somewhat less than the stated value or range, within ±20% of the stated value, ±19% of the stated value, ±18% of the stated value, ±17% of the stated value, ±16% of the stated value, ±15% of the stated value, ±14% of the stated value, ±13% of the stated value, ±12% of the stated value, ±11% of the stated value, ±10% of the stated value, ±9% of the stated value, ±8% of the stated value, ±7% of the stated value, ±6% of the stated value, ±5% of the stated value, ±4% of the stated value, ±3% of the stated value, ±2% of the stated value, or ±1% of the stated value.

[0182] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported to the extent practicable, however, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0183] The terms "a," "an," "the," and similar referents as used in the context of describing the present invention (particularly in the context of the appended claims) should be construed to encompass both the singular and the plural unless otherwise specified herein or clearly contradicted by context. Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise specified herein, each separate value is incorporated herein as if set forth individually herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better describe the invention and does not pose a limitation on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0184] Groupings of alternative elements or aspects of the invention disclosed herein should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that for reasons of convenience and / or patentability, one or more members of a group may be included in or deleted from a group. When any such inclusion or deletion occurs, the specification includes the modified group and is therefore deemed to satisfy the written description of all Markush groups used in the appended claims.

[0185] Certain aspects of this invention have been described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described aspects will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such variations will be employed by those skilled in the art as appropriate, and the inventors intend that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

[0186] Additionally, throughout this specification, numerous references are made to patents, printed publications, journal articles, other writings, and website content (materials referenced herein). Each of the referenced materials is individually incorporated herein by reference in its entirety for the referenced teachings as of the filing date of the first application in the priority chain in which the particular reference is included. For example, with respect to compounds and nucleic acid or amino acid sequences referred to herein that are available in public databases, the information in the cited database entries is incorporated herein by reference as of the date the database identifier was first included in the text of an application in the priority chain.

[0187] It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, and not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the invention is not limited to the exact same that has been shown and described.

[0188] The details shown in this specification are by way of example and for the purpose of illustrative description of preferred embodiments of the present invention only, and are presented to provide what is believed to be the most useful and readily understood explanation of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no attempt has been made to show the structural details of the present invention in more detail than necessary for a fundamental understanding of the invention, and the description using figures and / or examples will make clear to those skilled in the art how several forms of the present invention may be embodied in practice.

[0189] The definitions and explanations used in this disclosure are meant and intended to control any future construction unless clearly and unambiguously changed in the examples or unless application of the meaning makes any construction meaningless or essentially meaningless. Definitions are provided in accordance with Webster's Dictionary, 11th Edition, or dictionaries known to those skilled in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology, 2000. nd Edition (Ed. Anthony Smith, Oxford University Press, Oxford, 2006) and / or A Dictionary of Chemistry, 8 th Edition (Eds. J. Law & R. Rennie, Oxford University Press, 2020).

Claims

[Claim 1] The invention described in the specification.

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