Albumin nanoparticles for efficient vaccine delivery

Abstract

The present disclosure provides a protein-based nanoparticle made of a protein and an amine for the delivery of a nucleic acid molecule, such as an mRNA vaccine, to a cell. The present disclosure further provides systems and methods for producing such protein-based nanoparticle including a nucleic acid molecule, and methods of delivering the nucleic acid molecule to a cell, and treating a subject with the compositions as disclosure herein.

Description

ALBUMIN NANOPARTICLES FOR EFFICIENT VACCINE DELIVERYRELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 635,548, filed April 17, 2024. The entire contents of the foregoing application are incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under 2015-68004-23131 awarded by the National Institute of Food and Agriculture. The government has certain rights in the invention.INCORPORATED BY REFERENCE OF SEQUENCE LISTING

[0003] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on March 4, 2025, is named “098121-00394.xml” and is 7,318 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.FIELD OF THE DISCLOSURE

[0004] Described herein are vaccines, and in particular to new platforms for delivery thereof.BACKGROUND

[0005] Vaccine technology has a very large market, especially after the COVID-19 epidemic. The ability to rapidly develop vaccines against unexpected pathogens is an important factor as it is to develop an efficient vaccine. The current technology for vaccination of chickens uses age- old method of deactivated live virus or variations thereof, which are more cumbersome to produce and likely to generate undesirable consequences.

[0006] Thus, what are needed are methods and apparatus to improve vaccine development and delivery or said vaccines.SUMMARY

[0007] Accordingly, in some aspects, disclosed herein is a composition comprising a nucleic acid molecule and a carrier, wherein the carrier comprises a protein and an amine, and wherein the carrier interacts with the nucleic acid molecule forming a complex to deliver the nucleic acid molecule to a cell.

[0008] In some embodiments, the nucleic acid molecule is selected from the group consisting mRNA, siRNA, shRNA, ssRNA, dsRNA, ssDNA, dsDNA, DNA, and combinations thereof. In some embodiments, the nucleic acid molecule is mRNA.

[0009] In some embodiments, the protein is between about 50 kDa and about 100 KDa. In some embodiments, the protein has a net negative charge at physiological pH.

[0010] In some embodiments, the protein is albumin, or a fragment thereof. In some embodiments, the albumin, or a fragment thereof, is derived from an animal or is synthetic. In some embodiments, the animal is selected from bovine or human.

[0011] In some embodiments, the amine is selected from the group consisting of pentaethylenehexamine (PEHA), ethylenediamine, triethylenetetraamine, spermine, histidine, polyethylenimine (PEI), spermidine, poly(L-lysine), poly(amido amine) (PAMAM) dendrimers, polypropyleneiminie dendrimers, poly(2-dimethylamino ethyl) -methacrylate (pDMAEMA), chitosan, tris(2-aminoethyl)amine and methylated derivatives thereof, ethylene diamine, diethylene triamine, tetra ethylene pentamine, and combinations thereof. In some embodiments, the amine is pentaethylenehexamine (PEHA).

[0012] In some embodiments, the nucleic acid molecule is between about 0.5 pg to about 20 pg, about 1 pg to about 5 pg, about 5 pg to about 7 pg, or about 10 pg to about 20 pg.

[0013] In some embodiments, the carrier is between about 10 pg / ml to about 150 pg / ml, about 20 pg / ml to about 125 pg / ml, about 30 pg / ml to about 110 pg / ml, about 50 pg / ml to about 100 pg / ml, or about 75 pg / ml to about 115 pg / ml.

[0014] In some embodiments, the mRNA encodes a polypeptide from a pathogen. In some embodiments, the pathogen is a bacteria, vims, fungus, parasite, prion, or protest.

[0015] In some embodiments, the polypeptide is naturally found on the cell surface of the pathogen.

[0016] In some embodiments, the pathogen infects an animal. In some embodiments, the animal is an avian or a mammal. In some embodiments, the avian is a chicken, a goose, or a duck. In some embodiments, the mammal is a sheep, a pig, a cow, a horse, a goat, or a human.

[0017] In some embodiments, the pathogen is a virus. In some embodiments, the virus is infectious bronchitis virus (IBV) or highly pathogenic avian influenza (HPAI).

[0018] In some embodiments, the avian is a chicken.

[0019] In some embodiments, the polypeptide is the spike protein (S protein) or IBV or HPAI.

[0020] In some embodiments, the virus is coronavirus.

[0021] In some embodiments, the mammal is a human.

[0022] In some embodiments, the polypeptide is the spike protein (S protein) or coronavirus.

[0023] In some aspects, disclosed herein is a pharmaceutical composition comprising the composition of any one disclosed herein and a pharmaceutically acceptable excipient.

[0024] In some aspects, disclosed herein is a method of producing a composition comprising an mRNA and a carrier, the method comprising: crosslinking a protein to an amine to generate the carrier; and contacting the carrier with the mRNA, wherein the carrier interacts with the mRNA forming a complex to deliver the mRNA to a cell.

[0025] In some embodiments, the protein is albumin, or a fragment thereof.

[0026] In some embodiments, the protein is albumin, or a fragment thereof, is derived from an animal or is synthetic. In some embodiments, the animal is selected from bovine or human.

[0027] In some embodiments, the amine is selected from the group consisting of pentaethylenehexamine (PEHA), ethylenediamine, triethylenetetraamine, spermine, histidine, polyethylenimine (PEI), spermidine, poly(L-lysine), poly(amido amine) (PAMAM) dendrimers, polypropyleneiminie dendrimers, poly(2-dimethylamino ethyl)-methacrylate (pDMAEMA), chitosan, tris(2-aminoethyl)amine and its methylated derivatives, and combinations thereof.

[0028] In some embodiments, the amine is pentaethylenehexamine (PEHA).

[0029] In some embodiments, the protein and amine are crosslinked with a carbodiimide. In some embodiments, the carbodiimide is l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0030] In some embodiments, the crosslinking occurs at a pH between pH 4 and pH 6, optionally the pH is 5.5.

[0031] In some embodiments, the method further comprises the crosslinked protein-amine molecules are dialyzed into a salt buffer at a pH between pH 6.5 and pH 7.5.

[0032] In some aspects, disclosed herein is a nanoparticle for delivery of a vaccine to a cell comprising a carrier and an mRNA, wherein the mRNA encodes a polypeptide derived from infectious bronchitis virus, and the carrier comprises a crosslinked protein-amine molecule; wherein the carrier interacts with the mRNA forming a complex to deliver the mRNA to the cell.

[0033] In some embodiments, the crosslinked protein-amine molecule comprises albumin, or a fragment thereof, and pentaethylenehexamine (PEHA).

[0034] In some embodiments, the polypeptide is a spike protein (S protein).

[0035] In some embodiments, the nanoparticle is formulated for subcutaneous injection or intranasal spray.

[0036] In some aspects, disclosed herein is a pharmaceutical composition comprising the nanoparticle of any one disclosed herein, and a pharmaceutically acceptable excipient.

[0037] In some aspects, disclosed herein is a method of vaccinating a subject, the method comprising administering the composition of any one disclosed herein, the composition producedby the method of any one disclosed herein, the nanoparticle of any one disclosed herein, or the pharmaceutical composition of any one disclosed herein.

[0038] In some embodiments, the subject is an animal.

[0039] In some embodiments, the animal is an avian or a human.

[0040] In some aspects, disclosed herein is a method of inducing an immune response in a subject, the method comprising administering the composition of any one disclosed herein, the composition produced by the method of any one disclosed herein, the nanoparticle of any one disclosed herein, or the pharmaceutical composition of any one disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1A depicts Dynamic Light Scattering of bovine serum albuminpentaethylenehexamine (BSA-PEHA): 10 mM phosphate buffer pH 7.0. FIG. IB depicts Zeta potential of BSA-PEHA. FIG. 1C depicts TEM image of BSA-PEHA (pH 4.0). FIG. ID depicts Circular Dichroism, which shows no loss in ellipticity for BSA-PEHA compared to BSA. All samples show a double minimum at 208 and 222 nm.

[0042] FIGs. 2A-2C depict cell viability of BSA-PEHA nanoparticles treated HD 11 cells using MTT assay. Cells were treated with 100 to 0.097 pg / ml of BSA-PEHA nanoparticles and incubated at 24 hours (FIG. 2A), 48 hours (FIG. 2B) and 72 hours (FIG. 2C). The data was obtained by using GraphPad Prism vlO software. Data points represent n = 3 experiments with triplicates, for each experiment ± standard deviation. *P<0.05 and ** P< 0.01.

[0043] FIG. 3 depicts cell viability assays for media, BSA, and PEHA controls.

[0044] FIG. 4 depicts confocal images of HD11 cells after incubation with FITC-labeled BSA- PEHA nanoparticles (NPs) (10 pg / ml: green channel) at different time intervals (0.5, 1 hour) with control without any treatment. The nucleus and cell membrane were stained with Hoechst (blue channel) and CellMask™ Deep red Plasma Membrane stain (red channel), respectively. The internalization of the NP was confirmed by using Nikon AIR Spectral Confocal microscope using x 60 oil immersion lens and analyzed using ImageJ. Scale bar: 20 pm.

[0045] FIG. 5A shows a confocal image of FITC labeled BSA-PEHA nanoparticles treated HD11 cells after 24 hours of incubation (Scale: 20pm). FIG. 5B are gel images showing the FITC labeled BSA-PEHA and FITC dye after UV exposure and Coomassie blue stain, respectively.

[0046] FIG. 6A depicts staining of HD11 cells with a Viability / Cytotoxicity Assay Kit for live and dead cells. Fluorescence microscopy image of HD11 cells stained with Calcein AM staining shows live cells in green and PI staining shows dead cells in red. FIG. 6B depicts FACS where live cells have positive signal for Calcein AM (FITC / green detection-upper left quadrant) (top panel) and dead cells can be visualized as positive for PI (PE- A / red detection) (bottom panel).

[0047] FIG. 7 depicts quantification of cell cytotoxicity to BSA-PEHA nanoparticles using Fmctry by staining cells using Viability / Cytotoxicity Assay Kit for live and dead cells.Following a 24-hour incubation period, HD11 cells were subjected to varying doses of BSA- PEHA nanoparticles (200 pg / ml-1.5 pg / ml). The Y-axis of a dot plot displays FITC-positive cells, while the X-axis displays PE-A-positive cells.

[0048] FIGs. 8A and 8B show Cell viability of chloroquine treated HD 11 cells using MTT assay. Cells were treated with 250 to 0.243 pM of chloroquine and incubated at 24 hours (FIG. 8A) and 48 hours (FIG. 8B). The data was obtained by using GraphPad Prism vlO software. Data points represent n = 3 experiments with triplicates, for each experiment ± standard deviation. *P<0.05, ** P< 0.01 and *** P<0.001.

[0049] FIGs. 9A-9C show expression of eGFP mRNA using Lipofectamine™ 2000 and BSA- PEHA nanoparticles as the delivery vehicle in HD11 cells. Expression of eGFP mRNA with Lipofectamine 2000 is shown in FIG. 9A. Expression of eGFP mRNA with BSA-PEHA (25 pg / ml) is shown in FIG. 9B. Expression of eGFP mRNA with BSA-PEHA (25 pg / ml) in presence of chloroquine (25pM) is shown in FIG. 9C.

[0050] FIGs. 10A-10D show CD spectra of BSA-PEHA and BSA (FIG. 10A), mRNA (FIG. 10B), BSA-PEHA / IVT IBV S protein mRNA (FIG. 10C), and BSA-PEHA / IVT IBV S protein mRNA at different pH (FIG. 10D).

[0051] FIG. 11A shows a denaturing gel electrophoresis of S protein of IBV mRNA. FIG. 11B shows a Western blot of eGFP mRNA (26 kDA) in HD11 cells, expressed in presence and absence of chloroquine. FIG. 11C shows a Western blot of IBV S protein mRNA (128 kDA) in HD 11 cells, expressed in presence and absence of chloroquine.

[0052] FIG. 12A shows an EMSA gel illustrating the binding dynamics between BSA-PEHA with IVT IBV S protein mRNA under constant nucleic acid concentration. FIG. 12B shows an EMSA gel illustrating the binding dynamics between BSA-PEHA with IVT IBV S protein mRNA under constant BSA-PEHA nanoparticle concentration. Both images show agar gel electrophoresis image under UV light and the same gel after staining with Coomassie blue. Lane 1: BSA-PEHA 100 pg / ml: 1 pg IBV mRNA; Lane 2: BSA-PEHA 50 pg / ml: 1 pg IBV mRNA; Lane 3: BSA-PEHA 25 pg / ml: 1 pg IBV mRNA; Lane 4: BSA-PEHA 12.5 pg / ml: 1 pg IBV mRNA; Lane 5: BSA-PEHA 100 pg / ml; and Lane 6: 1 pg IBV mRNA.

[0053] FIG. 13A shows the titer of an antibody against IBV S protein in different groups after first (right panel) and second dose (left panel). FIG. 13B shows a bar graph comparing first and second dose of BSA-PEHA / IVT S protein mRNA with chloroquine. W CQ: with chloroquine; WO CQ: without chloroquine.

[0054] FIG. 14 shows a bar graph of peripheral blood mononuclear cell (PBMC) stimulation with killed IBV vims.

[0055] FIG. 15 shows serum neutralization of chickens immunized with different combinations of BSA-PEHA / IVT IBV S protein mRNA at two and three weeks after vaccination.DETAILED DESCRIPTION

[0056] A protein-based nanoparticle as carrier for the delivery of mRNA vaccine is demonstrated here. In some embodiments, the carrier and mRNA can form a complex and can be used to vaccinate a subject. In some embodiments, the vaccine can be against infectious Bronchitis virus (IBV) in chickens. Vaccination of chickens using the protein based nanoparticle as the earner for mRNA corresponding to the S protein of the IBV virus induced the expression of antibodies against the viral protein with high efficiency. Control experiments showed no deleterious effects of the carrier without the vaccine and mRNA administration alone hadnegligible protection. Lymphocyte proliferation assays indicated the successful stimulation of the T-cells by the vaccine delivered by the albumin nanoparticles, while controls showed little or no stimulation of the T-cells. Albumin protein can be chosen to match the species to be vaccinated and can be matched with specific patients, thus making it highly compatible with the subject of interest. Another advantage of this technology is the ease of preparation, robustness, storage and handling of the protein nanoparticles. The nanoparticles showed strong binding to the mRNA as evidenced by circular dichroism and gel electrophoresis studies.

[0057] This technology leverages protein nanotechnology to create nanocarriers, e.g., a protein crosslinked to an amine, that bind mRNA and deliver the mRNA into target cells with high efficiency but with no detectable side effects. The platform demonstrated is broad and can be extended to other mRNAs, other pathogens and other species, including humans. For humans, for example, the albumin can be obtained from the specific individual for making the vaccine.

[0058] The use of nanotechnology and mRNA chemistry enables multiple mRNA vaccines to be co-delivered in a single dose or multiple variants can be administered for a broader protection.

[0059] As used herein, “nucleic acid molecule” may be any molecule in which molecules of nucleotide or molecules having functions equivalent to those of the nucleotide are polymerized, and examples thereof include RNA, which is a polymer of ribonucleotide; DNA, which is a polymer of deoxyribonucleotide; a polymer of a mixture of ribonucleotide and deoxyribonucleotide; and a nucleotide polymer including a nucleotide analog. Further, the nucleic acid molecule may be a nucleotide polymer including a nucleotide derivative. The nucleic acid molecule may be a single- stranded nucleic acid molecule or a double-stranded nucleic acid molecule. The concept of the double- stranded nucleic acid molecule encompasses a double-stranded nucleic acid molecule such that one strand hybridizes with the other strand under stringent conditions.

[0060] The nucleotide analog may be any molecule obtained by modifying ribonucleotide, deoxyribonucleotide, RNA, or DNA to improve nuclease resistance, to stabilize, to enhance affinity with a complementary- strand nucleic acid molecule or to enhance cell permeability as compared with RNA or DNA, or for visualization. The nucleotide analog may be a naturally-occurring molecule or a non-natural molecule, and examples thereof include a sugar-modified nucleotide analog and a phosphodiester bond-modified nucleotide analog.

[0061] Non-limiting examples of nucleic acid molecules include mRNA, siRNA, shRNA, ssRNA, dsRNA, ssDNA, dsDNA, and DNA, and the like.

[0062] As used herein, “mRNA” refers to an RNA including a nucleotide sequence that can be translated into a protein. In some embodiments, the mRNA is not limited to a particular mRNA and may be any mRNA capable of expressing a desired protein in cells. In some embodiments, the mRNA expresses a polypeptide.

[0063] The terms "polypeptide" and "protein" are used herein in the broadest sense to refer to a sequence of subunit amino acids, amino acid analogs, or peptidomimetics. The subunits are linked by peptide bonds, except where noted. These terms also apply to amino acid polymers in which one or more amino acid residues can be artificial chemical analogues of a corresponding naturally occurring amino acid(s), as well as to naturally occurring amino acid polymers. The terms also are inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ribosylation.

[0064] The term “carrier” as used herein describes a material that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the compound of the composition disclosed herein. Carriers must be of sufficiently high purity and of sufficiently low toxicity to render them suitable for administration to the mammal being treated. The carrier can be inert, or it can possess pharmaceutical benefits. The carrier can facilitate the delivery of a drug (e.g., a nucleic acid molecule) to a cell. For example, the carrier can bind to the nucleic acid molecule (e.g., RNA, e.g., mRNA) forming a protein nanoparticle. The term “protein nanoparticle” or “PNPs” as used herein describes microscopic particles composed on proteins that range in size from 10 nm to 100 nm in diameter. Protein nanoparticles can be used to deliver a drug or a therapeutic to a cell. In some embodiments, the drug or therapeutic can be a nucleic acid molecule (e.g., RNA, e.g., mRNA).

[0065] In some embodiments, the carrier includes a protein and an amine. In some embodiments, the protein may between about 50 kDa and about 100 KDa, e.g., approximately 66.3 kDa. In some embodiments, the protein has a net negative charge at physiological pH, e.g., pH 6.8-7.5, e.g., pH 7 . In some embodiments, the protein may be albumin, such as, but not limited to, bovine albumin, ovalbumin, and human albumin, hi some embodiments, the protein, such as, but not limited to, albumin, can be a fragment. A fragment of the protein, e.g., albumin, can be one or more globular domains, truncated N-terminus and / or C-terminus, or a fusion of domains which are not found next to each other in nature, for example, the N-terminus is fused to the C- terminus and the center of the protein has been deleted. In another embodiment, albumin can be synthetic. In another embodiment, albumin can be derived from serum of a donor, or serum of the subject. In another embodiment, the albumin may be a fragment of albumin. For example, albumin can be the middle region of the protein. In another embodiment, the albumin can be a peptide, or a mix of peptides derived from albumin.

[0066] In some embodiments, the amine can be pentaethylenehexamine (PEHA), ethylenediamine, triethylenetetraamine, spermine, histidine, polyethylenimine (PEI), spermidine, poly(L-lysine), poly(amido amine) (PAMAM) dendrimers, polypropyleneiminie dendrimers, poly(2-dimethylamino ethyl)-methacrylate (pDMAEMA), chitosan, tris(2-aminoethyl)amine and methylated derivatives thereof, ethylene diamine, diethylene triamine, tetra ethylene pentamine, and combinations thereof. In other embodiments, the amine may be PEHA.

[0067] As used herein, the term “crosslinking” or grammatical variations thereof, refers to the process of connecting one polymer chain to another polymer chain, typically by a covalent or ionic bond. Crosslinking can occur, for example, between two polypeptides or proteins via amino acids, between a chemical compound and a protein via an amino acid, between a chemical compound and a nucleotide, or between two nucleotides, hi some embodiments, the protein and the amine are crosslinked to generate the carrier.

[0068] “Initiators” can be reagents or compounds that initiate a crosslinking reaction between two molecules, e.g., an amino acid and an amine. For example, the initiators can be primary amines, which initiate the ring-opening polymerization of N-carboxyanhydrides (NCAs), the activated form of amino acids, through a nucleophilic attack on the carbonyl group within theNCA ring, allowing the chain to grow by adding more monomers. Other examples of initiators can include, but are not limited to, alkoxide anions (e.g., ethanol (CH3CH2OH)), tertiary amines (e.g., N-methylpiperidine and N-phenylpiperidine), hydroxyl initiators (e.g., methanol and ethanol), sodium thiophenolate, lithium hexamethyldisilazide (LiHMDS), carboxylates (e.g., tetraalkylammonium carboxylates), and metal complexes (e.g., zerovalent nickel and cobalt initiators).

[0069] “Crosslinking reagents” can be reagents or compounds which contain two or more reactive groups which covalently attach via a spacer to functional (e.g., primary amines, sulfhydryls, etc.) on proteins, nucleotides, or other molecules. Some common examples of heterobifunctional crosslinkers include MDS (m-Maleimidobenzoyl-N-hydroxysuccinimide ester), GMBS (N-y-Maleimidobutyryloxysuccinimide ester), EMCS (N-(s- Malcimidocaproyloxy) succinimide ester) and sulfo-EMCS (N-(s-Malcimidocaproyloxy) sulfo succinimide ester), to name a few. Some common examples of amine-to-amine crosslinkers include disuccinimidyl suberate or DSS (ideal for receptor ligand crosslinking), disuccinimidyl tartrate or DST (used for applications wherein crosslink cleavability is required while keeping the protein disulfide bonds intact) and dithiobis succinimidyl propionate, or DSP (ideally used for crosslinking intracellular proteins prior to cell lysis and immunoprecipitation as well as for fixing protein interactions prior to identification of weak or transient protein interactions). Some common examples of sulfhydryl-to-sulfhydryl crosslinkers include BMOE and DTME. NHS- ester diazirines or azipentanoates contain a photoactivatable diazirine ring and an N- hydroxysuccinimide (NHS) ester which efficiently reacts with primary amino groups in neutral to basic buffers (pH 7 to 9) to form stable amide bonds. In some embodiments, the crosslinking reagent can be a carbodiimide, which react with carboxylic acids to form an active ester intermediate, which can then react with nucleophiles such as amines to form stable amide bonds. In some embodiments, the carbodiimide can be l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0070] In some embodiments, the protein (e.g., albumin), or fragment thereof, and amine can be crosslinked. In some embodiments, one or more amine molecules can be crosslinked to the protein, or fragment thereof. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amine molecules can be crosslinked to the protein, or fragment thereof.

[0071] In some embodiments, the composition comprising a carrier comprising a protein and an amine can be between about 10 pg / ml to about 150 pg / ml, about 20 pg / ml to about 125 pg / ml, about 30 pg / ml to about 110 pg / ml, about 50 pg / ml to about 100 pg / ml, or about 75 pg / ml to about 115 pg / ml. In some embodiments, the carrier comprising a protein and an amine can be between about 10 pg / ml to about 150 pg / ml, about 10 pg / ml to about 125 pg / ml, about 10 pg / ml to about 100 pg / ml, about 10 pg / ml to about 90 pg / ml, about 10 pg / ml to about 80 pg / ml, about 10 pg / ml to about 70 pg / ml, about 10 pg / ml to about 60 pg / ml, about 10 pg / ml to about 50 pg / ml, about 10 pg / ml to about 40 pg / ml, about 10 pg / ml to about 30 pg / ml, about 10 pg / ml to about 20 pg / ml, about 20 pg / ml to about 150 pg / ml, about 20 pg / ml to about 125 pg / ml, about 20 pg / ml to about 100 pg / ml, about 20 pg / ml to about 90 pg / ml, about 20 pg / ml to about 80 pg / ml, about 20 pg / ml to about 70 pg / ml, about 20 pg / ml to about 60 pg / ml, about 20 pg / ml to about 50 pg / ml, about 20 pg / ml to about 40 pg / ml, 20 pg / ml to about 30 pg / ml, 30 pg / ml to about 150 pg / ml, 30 pg / ml to about 125 pg / ml, about 30 pg / ml to about 100 pg / ml, about 30 pg / ml to about 90 pg / ml, about 30 pg / ml to about 80 pg / ml, about 30 pg / ml to about 70 pg / ml, about 30 pg / ml to about 60 pg / ml, about 30 pg / ml to about 50 pg / ml, about 30 pg / ml to about 40 pg / ml, about 40 pg / ml to about 150 pg / ml, about 40 pg / ml to about 125 pg / ml, about 40 pg / ml to about 100 pg / ml, about 40 pg / ml to about 90 pg / ml, about 40 pg / ml to about 80 pg / ml, about 40 pg / ml to about 70 pg / ml, about 40 pg / ml to about 60 pg / ml, about 40 pg / ml to about 50 pg / ml, about 50 pg / ml to about 150 pg / ml, 50 pg / ml to about 125 pg / ml, about 50 pg / ml to about 100 pg / ml, about 50 pg / ml to about 90 pg / ml, about 50 pg / ml to about 80 pg / ml, about 50 pg / ml to about 70 pg / ml, about 50 pg / ml to about 60 pg / ml, about 60 pg / ml to about 150 pg / ml, about 60 pg / ml to about 125 pg / ml, about 60 pg / ml to about 100 pg / ml, about 60 pg / ml to about 90 pg / ml, about 60 pg / ml to about 80 pg / ml, about 60 pg / ml to about 70 pg / ml, about 70 pg / ml to about 150 pg / ml, about 70 pg / ml to about 125 pg / ml, about 70 pg / ml to about 100 pg / ml, about 70 pg / ml to about 90 pg / ml, about 70 pg / ml to about 80 pg / ml, about 80 pg / ml to about 150 pg / ml, about 80 pg / ml to about 125 pg / ml, about 80 pg / ml to about 100 pg / ml, about 80 pg / ml to about 90 pg / ml, about 90 pg / ml to about 150 pg / ml, about 90 pg / ml to about 125 pg / ml, about 90 pg / ml to about 100 pg / ml, about 100 pg / ml to about 150 pg / ml, about 100 pg / ml to about 125 pg / ml, or about 125 pg / ml to about 150 pg / ml.

[0072] In some embodiments, the nucleic acid molecule can be between about 0.5 pg to about 20 pg, about 1 pg to about 5 pg, about 5 pg to about 7 pg, or about 10 pg to about 20 pg. In otherembodiments, the nucleic acid molecule can be between about 0.5 ig to about 20 ig, about 0.5 pig to about 17 pig, about 0.5 pig to about 15 pig, about 0.5 pig to about 12 pig, about 0.5 pig to about 10 pig, about 0.5 pig to about 9 pig, about 0.5 pig to about 8 pig, about 0.5 pig to about 7 pig, about 0.5 pig to about 6 pig, about 0.5 pig to about 5 pig, about 0.5 pig to about 4 pig, about 0.5 pig to about 3 pig, about 0.5 pig to about 2 pig, about 0.5 pig to about 1 pig, about 1 pig to about 20 pig, about 1 pig to about 17 pig, about 1 pig to about 15 pig, about 1 pig to about 12 pig, about 1 pig to about 10 pig, about 1 pig to about 9 pig, about 1 pig to about 8 pig, about 1 pig to about 7 pig, about 1 pig to about 6 pig, about 1 pig to about 5 pig, about 1 pig to about 4 pig, about 1 pig to about 3 pig, about 1 pig to about 2 pig, about 2 pig to about 20 pig, about 2 pig to about 17 pig, about 2 pig to about 15 pig, about 2 pig to about 12 pig, about 2 pig to about 10 pig, about 2 pig to about 9 pig, about 2 pig to about 8 pig, about 2 pig to about 7 pig, about 2 pig to about 6 pig, about 2 pig to about5 pig, about 2 pig to about 4 pig, about 2 pig to about 3 pig, about 3 pig to about 20 pig, about 3 pig to about 17 pig, about 3 pig to about 15 pig, about 3 pig to about 12 pig, about 3 pig to about 10 pig, about 3 pig to about 9 pig, about 3 pig to about 8 pig, about 3 pig to about 7 pig, about 3 pig to about6 pig, about 3 pig to about 5 pig, about 3 pig to about 4 pig, about 4 pig to about 20 pig, about 4 pig to about 17 pig, about 4 pig to about 15 pig, about 4 pig to about 12 pig, about 4 pig to about 10 pig, about 4 pig to about 9 pig, about 4 pig to about 8 pig, about 4 pig to about 7 pig, about 4 pig to about 6 pig, about 4 pig to about 5 pig, about 5 pig to about 20 pig, about 5 pig to about 17 pig, about 5 pig to about 15 pig, about 5 pig to about 12 pig, about 5 pig to about 10 pig, about 5 pig to about 9 pig, about 5 pig to about 8 pig, about 5 pig to about 7 pig, about 5 pig to about 6 pig, about 6 pig to about 20 pig, about 6 pig to about 17 pig, about 6 pig to about 15 pig, about 6 pig to about 12 pig, about 6 pig to about 10 pig, about 6 pig to about 9 pig, about 6 pig to about 8 pig, about 6 pig to about 7 pig, about 7 pig to about 20 pig, about 7 pig to about 17 pig, about 7 pig to about 15 pig, about 7 pig to about 12 pig, about 7 pig to about 10 pig, about 7 pig to about 9 pig, about 7 pig to about 8 pig, about 8 pig to about 20 pig, about 8 pig to about 17 pig, about 8 pig to about 15 pig, about 8 pig to about 12 pig, about 8 pig to about 10 pig, about 8 pig to about 9 pig, about 9 pig to about 20 pig, about 9 pig to about 17 pig, about 9 pig to about 15 pig, about 9 pig to about 12 pig, about 9 pig to about 10 pig, about 10 pig to about 20 pig, about 10 pig to about 17 pig, about 10 pig to about 15 pig, about 10 pig to about 12 pig, about 12 pig to about 20 pig, about 12 pig to about 17 pig, about 12 pig to about 15 pig, about 15 pig to about 20 pig, about 15 pig to about 17 pig, or about 17 pig to about 20 pig.

[0073] In some embodiments, the carrier (e.g., protein (e.g., albumin) crosslinked to an amine) and the nucleic acid molecule form a “complex”. As used herein, the term “complex” refers to structures formed by the carrier binding to the nucleic acid molecule. The binding of the carrier to the nucleic acid molecules can be by non-covalent bonds, such as hydrogen bonds, ionic interactions, and van der Waals forces. In some embodiments, the carrier can transiently bind to the nucleic acid molecule, hi some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more carrier molecules (e.g., BSA-PEHA) can bind to a nucleic acid molecule. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more carrier-nucleic acid molecules (e.g., BSA-PEHA-mRNA) can be in a complex.Pathogens

[0074] In some embodiments, the mRNA encodes a polypeptide from a pathogen. A “pathogen” refers to an organism which is capable of causing diseases in a host, e.g., an animal, e.g., an avian or a human. Examples of pathogens can include, but are not limited to, a bacteria, a virus, a fungus, a parasite, a prion, or a protest.

[0075] The pathogen can be a unicellular organism (e.g., bacteria), a multi-cellular organism (e.g., parasite), an obligate, intracellular parasite (e.g., vims) or a protein (e.g., prion). The pathogen, e.g., a virus, can infect the host through the air, food, contact with an infected organism (e.g., another host of the same species, or of a different species), and the environment. Once the host is infected, the pathogen can multiple by the nature cell cycle pathways of the pathogen. An immune response can be trigger by the pathogen in the host. For example, a component (e.g., polypeptide or nucleic acid molecule) from the pathogen can trigger an immune response in the host.

[0076] In some embodiments, the polypeptide is naturally found in the pathogen. For example, the polypeptide can be on the surface of one or more cells of the pathogen. In another example, the polypeptide can be intracellular of the pathogen. In some embodiments, the cell surface polypeptide is a spike protein (S protein) of a virus, e.g., infectious bronchitis virus (IBV), highly pathogenic avian influenza (HP Al), or coronavirus.

[0077] In some embodiments, the pathogen infects one or more animal species. For example, the animal can be an avian or a mammal. In some embodiments, the avian can be, for example, a chicken, a goose, or a duck. In some embodiments, the animal can be, for example, a sheep, a pig, a cow, a horse, a goat, or a human.Pharmaceutical Composition and Uses Thereof

[0078] The term “pharmaceutical composition” is used herein to refer to a composition that is employed to prevent, reduce in intensity, cure or otherwise treat a target condition or disease. The terms "formulation" and "composition" are used interchangeably herein to refer to a product disclosed herein that includes all active and inert ingredients.

[0079] The phrase “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions, and / or dosage forms which arc, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0080] The phrase “pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0081] The composition and / or pharmaceutical composition, as disclosed herein, can be produced by methods disclosed herein. The composition can include a nucleic acid molecule and a carrier, which includes a protein and an amine. To produce the carrier, the amine can be crosslinked to the protein. In some embodiments, one or more amine molecules can be crosslinked to one protein molecule. For example, there can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amine molecules crosslinked to one protein molecule (e.g., one albumin molecule).

[0082] In some embodiments, the amine can be pentaethylenehexamine (PEHA), ethylenediamine, triethylenetetraamine, spermine, histidine, polyethylenimine (PEI), spermidine, poly(L-lysine), poly(amido amine) (PAMAM) dendrimers, polypropyleneiminie dendrimers, poly(2-dimethylamino ethyl)-methacrylate (pDMAEMA), chitosan, tris(2-aminoethyl)amine and methylated derivatives thereof, ethylene diamine, dicthylcnc triaminc, tetra ethylene pcntaminc, and combinations thereof. In other embodiments, the amine may be PEHA.

[0083] In some embodiments, the amine is crosslinked to the protein in the presence of a crosslinking reagent. In some embodiments, the crosslinking reagent can be reagent(s) or compounds which contain two or more reactive groups which covalently attach via a spacer to functional (e.g., primary amines, sulfhydryls, etc.) on proteins, nucleotides, or other molecules. Some common examples of heterobifunctional crosslinkers include MDS (m-Maleimidobenzoyl- N-hydroxysuccinimide ester), GMBS (N-Y-Maleimidobutyryloxysuccinimide ester), EMCS (N- (s-Maleimidocaproyloxy) succinimide ester) and sulfo-EMCS (N-(s-Maleimidocaproyloxy) sulfo succinimide ester), to name a few. Some common examples of amine-to-amine crosslinkers include disuccinimidyl suberate or DSS (ideal for receptor ligand crosslinking), disuccinimidyl tartrate or DST (used for applications wherein crosslink cleavability is required while keeping the protein disulfide bonds intact) and dithiobis succinimidyl propionate, or DSP (ideally used for crosslinking intracellular proteins prior to cell lysis and immunoprecipitation as well as for fixing protein interactions prior to identification of weak or transient protein interactions). Some common examples of sulfhydryl-to-sulfhydryl crosslinkers include BMOE and DTME. NHS- ester diazirines or azipentanoates contain a photoactivatable diazirine ring and an N- hydroxysuccinimide (NHS) ester which efficiently reacts with primary amino groups in neutral to basic buffers (pH 7 to 9) to form stable amide bonds. In some embodiments, the crosslinkingreagent can be a carbodiimide, which react with carboxylic acids to form an active ester intermediate, which can then react with nucleophiles such as amines to form stable amide bonds. In some embodiments, the carbodiimide can be l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0084] In some embodiments, the crosslinking of the protein (e.g., albumin) and amine (e.g., PEHA) can occur at a pH between pH 4 and pH 6. In some embodiments, the crosslinking can occur at pH 5.5. In some embodiments, after crosslinking the protein-amine molecule can be dialyzed into a salt buffer at a pH between pH 6.5 and pH 7.5. For example, the salt buffer can be mono sodium phosphate buffer, e.g., 10 mM NaH PCE buffer at pH 7.

[0085] As disclosed herein, the composition can be produced by contacting the protein crosslinked to the amine (e.g., crosslinked protein-amine molecules) with the nucleic acid molecule. In some embodiments, the protein can be albumin. In some embodiments, the amine can be pentaethylenehexamine (PEHA).

[0086] In some embodiments, the nucleic acid molecule can be an mRNA which encodes a polypeptide from a pathogen, e.g., a bacteria, a virus, a fungus, a parasite, a prion, or a protest. In some embodiments, the pathogen infects one or more animal species. For example, the animal can be an avian or a mammal. In some embodiments, the avian can be, for example, a chicken, a goose, or a duck. In some embodiments, the animal can be, for example, a sheep, a pig, a cow, a horse, a goat, or a human.

[0087] In some embodiments, the mRNA can encode a polypeptide is naturally found in the pathogen. For example, the polypeptide can be on the surface of one or more cells of the pathogen. In some embodiments, the cell surface polypeptide is a spike protein (S protein) of a vims, e.g., infectious bronchitis virus (IBV), highly pathogenic avian influenza (HPAI), or coronavirus.

[0088] As described herein, a composition, a pharmaceutical composition, and / or a nanoparticle including a nucleic acid molecule and a carrier can be adapted to deliver the nucleic acid molecule to a cell. In another embodiment, a composition, a pharmaceutical composition, and / ora nanoparticle including a nucleic acid molecule and a carrier can be adapted to deliver a vaccine to a cell.

[0089] In some embodiments, the vaccine (e.g., the composition, the pharmaceutical composition, and the nanoparticle) can be formulated for subcutaneous injection or intranasal spray. As used herein, the term “intranasal” or “intranasally” or grammatical variations thereof, refers to application of the compositions, as disclosed herein, to the surface of the skin and mucosal cells and tissues of the nasal passages, e.g., nasal mucosa, sinus cavity, nasal turbinates, or other tissues and cells which line the nasal passages.

[0090] The terms “subject” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans).

[0091] As described herein, the vaccine, composition, nanoparticle or pharmaceutical composition can be administered to a subject to trigger an immune response to a pathogen. The immune response of the subject can be measured by determining the titer and / or presence of antibodies against the immunogen after administration of the nanoemulsion vaccine to evaluate the humoral response to the immunogen. Seroconversion refers to the development of specific antibodies to an immunogen and may be used to evaluate the presence of a protective immune response. Such antibody-based detection is often measured using Western blotting or enzyme- linked immunosorbent (ELISA) assays or hemagglutination inhibition assays (HAI). Persons of skill in the ait would readily select and use appropriate detection methods.

[0092] Another method for determining the subject's immune response is to determine the cellular immune response, such as through immunogen- specific cell responses, such as cytotoxic T lymphocytes, or immunogen- specific lymphocyte proliferation assay. Additionally, challenge by the pathogen may be used to determine the immune response, either in the subject, or, more likely, in an animal model. A person of skill in the art would be well versed in the methods of determining the immune response of a subject and the compositions disclosed herein is not limited to any particular method.

[0093] The present inventions are further illustrated by the following examples, which are not intended to be limiting in any way. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the Figures, are hereby incorporated herein by reference.EXAMPLESExample 1: Nanoparticle synthesis and characterizationCationic BSA nanoparticle synthesis and characterization

[0094] A I M solution of pentaethylenehexamine (PEHA) was made by adding the appropriate amount of amine to an equal amount of water. The solution was kept in an ice bath while the pH was slowly adjusted to 5.5 using concentrated HC1. The resulting solution was brought up to volume using deionized H2O (DI H2O). In a clean glass vial, 1.5 g of bovine serum albumin (BSA) was dissolved into 4 mL of DI H2O by stirring for 1 h. 1 M aqueous amine (537 mL) was added to the solution and allowed to stir for 45 mins. Finally, 1 mL of 1 M l-ethyl-3-(3- dimethylaminopropyljcarbodiimide (EDC) was added to the vial and the reaction was allowed to stir for 4 h under nitrogen. The solution was then dialyzed into a 10 mM NaH2PO4 pH 7.0 buffer. Physicochemical properties of the nanoparticles were analyzed using transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analyzer. The hydrodynamic size was determined by using DLS (FIG. 1A) and the zeta potential using Zetasizer (FIG. IB). The morphology and size of the nanoparticle was determined by using transmission electron microscopy (TEM) (FIG. 1C). Circular Dichroism shows no loss in ellipticity for BSA-PEHA compared to BSA (FIG. ID). All samples show a double minimum at 208 and 222 nm.Nanoparticle uptake assay using HD 11 cells

[0095] The protocol from Da silva et al 2018 was modified and used for this study (Da Silva et al., 2018). HD11 (chicken; Gallus gallus) cells were grown in 35 mm No. 1.5 coverslip bottom dishes from MatTek with clear glass bottom. The cells were incubated in RPMI 1640 containing 10% FBS and 1% Antibiotic-Antimycotic at 42 °C for 24 hours. The cells were then treated with FITC labeled BSA-PEHA NPs at 10 ug / ml concentration and incubated at 0.5, 1 and 24 hours.At the end of each incubation period the NPs were rinsed out using PBS and the cells were stained with CellMaskTN Deep red Plasma Membrane stain for 10 min at 42°C. After the incubation, the staining solution was removed and rinsed with PBS three times. This was followed by addition of a sufficient volume of Hoechst 33342 working solution to completely cover the sample. An aluminum foil was placed over the sample to protect it from light and incubated at room temperature for 5-10 minutes. The image of the live cells was taken immediately using a Nikon AIR® Spectral Confocal microscope equipped with a 60X objective lens. Imaging was conducted at an excitation / emission wavelength of 460 / 490 nm for FITC and 649 / 666 nm for the CellMask™ Deep red Plasma Membrane stain.In vitro cytotoxicity of BSA-PEHA determined using an MTT assay

[0096] The in vitro cytotoxicity of the BSA-PEHA NPs were determined by using TACS® MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide)) Cell Proliferation Assay according to the manufacturer's instruction, in HD 11 cells. Briefly, the cells were seeded in 96- well tissue culture plate at a density of 5000 cells / well and maintained overnight at 42°C with 0.1 mL of feeding medium (90% RPMI and 10% fetal calf serum containing 1% antibiotic- antimycotic). After the incubation, the wells were treated with NPs of concentration ranging from 200 pg / ml - 6.25 pg / ml. The cells were then incubated for 24, 48 and 72 hours. The MTT reagent was added to all the wells after each incubation period and then kept for 4 hours at 37 °C. 100 pl of the detergent reagent was finally added and the cells were kept at RT overnight. After the incubation was over, the absorbance in each well was read at 570 nm using a microplate reader (Molecular Devices SpectraMax Plus 384). The controls were cell culture medium, BSA and PEHA.

[0097] Cell viability of BSA-PEHA nanoparticles treated HD11 cells using MTT assay are shown in FIGs. 2A-2C. Cells were treated with 0.097 to 100 pg / ml of BSA-PEHA nanoparticles and incubated at 24, 48, and 72 hours, FIGs. 2A, 2B, and 2C, respectively. Cell viability assays were performed for media, BSA and PEHA controls (FIG. 3), where none of media, BSA and PEHA altered cell viability significantly.Design of In Vitro Transcribed (IVT) S protein mRNA for IBV

[0098] Custom-made mRNA was utilized for this study to suit the experimental requirements. The mRNA was synthesized by Cellema Bioscience using a custom synthesis service. The ORF encoding the target antigen (S protein) was identified using National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™) database. The accession number AY851295 was selected. The ORF for the S protein started at the nucleotide position 20374 and ends at nucleotide position 23862, see Table 1. The stall and the end point were Hanked by a start codon ATG and the stop codon TAA, respectively, see Table 1. The mRNA sequence has a length of 3489 nucleotides, encoding for spike protein and was fully substituted with 5-methoxyuridine to enhance the stability and reduce immunogenicity. Capl analog was incorporated into the mRNA synthesis process, ensuring a cap structure with a purity of over 98%. The synthesized mRNA was polyadcnylatcd with a 110 bp poly A-tail.Table 1Transfection ofHDll cells with IVT S protein mRNA o IBV after binding with BSA-PEHA

[0099] The HD 11 cells were grown in a 6-well plate at the concentration of 0.3 x 106cells per well in RPMI 1640 containing 10% FBS and 1% Antibiotic-Antimycotic at 42 °C for 24 hours. 100 pl of 25 pg / ml BSA-PEHA and 2.5 pg of IVT S protein mRNA were mixed and incubated for 10 minutes and added to the cell monolayer drop wise. The cell culture medium was removed from the 6-well plates and the cells were washed with Dulbecco’s phosphate buffered saline (DPBS). 500pl of Opti-MEM I reduced serum media with chloroquine (25pM) was then added to the transfected wells and incubated for 4 hours at 42 °C. After the incubation, the transfection media was replaced by RPMI 1640 and then incubated for 24 hours at 42 °C. The cells were lysed and the supernatant was then used for western blotting.

[0100] Confocal images of HD11 cells after incubation with FITC-labeled BSA-PEHA NPs (10 pg / ml: green channel) at different time intervals (0.5, 1 hour) compared to the control without any treatment are shown in FIG. 4. The nucleus and cell membrane were stained with Hoechst (blue channel) and CellMask™ Deep red Plasma Membrane stain (red channel), respectively. Confocal image of FITC labeled BSA-PEHA nanoparticles treated HD 11 cells after 24 hours of incubation is shown in FIG. 5A. Gel image showing the FITC labeled BSA-PEHA and FITC dye are shown in FIG. 5B in protein gel and after UV exposure (left panel) and Coomassie blue stain (right panel).

[0101] Viability was determined by a viability / cytotoxicity assay kit which stains live and dead HD11 cells. Fluorescence microscopy image of HD11 cells stained with Calcein AM staining shows live cells in green and PI staining shows dead cells in red (FIG. 6A). The live cells have positive signal for Calcein AM (FITC / green detection- upper left quadrant) and dead cells can be visualized as positive for PI (PE- A / red detection)(FIG. 6B).

[0102] Quantification of cell cytotoxicity to BSA-PEHA nanoparticles using Fmetry by staining cells using Viability / Cytotoxicity Assay Kit for live and dead cells is shown FIG. 7. Following a 24-hour incubation period, HD11 cells were subjected to varying doses of BSA- PEHA nanoparticles (200 pg / ml-1.5 pg / ml). The Y-axis of a dot plot displays FITC-positive cells, while the X-axis displays PE-A-positive cells.

[0103] Cell viability was measured for HD11 cells treated with chloroquine using the MTT assay. Cells were treated with 250 to 0.243 pM of chloroquine and incubated at 24 and 48 hours, FIGs. 8A and 8B, respectively.

[0104] Chloroquine inhibits endosomal acidification (decreasing pH of endosomes), which may affect viral infection by interfering with viral-cell fusion. Expression of eGFP mRNA was measured using Lipofcctaminc 2000 and BSA-PEHA nanoparticlcs as the delivery vehicle in HD11 cells. Expression of eGFP mRNA with Lipofectamine 2000 is shown in FIG. 9A. Expression of eGFP mRNA with BSA-PEHA (25 pg / ml) is shown in FIG. 9B. Expression of eGFP mRNA with BSA-PEHA (25 pg / ml) in presence of chloroquine (25 pM) is shown in FIG. 9C.

[0105] Next, the circular dichroism spectra of BSA-PEHA, BSA, mRNA, and BSA- PEHA / IVT IBV S protein mRNA were measured under various conditions. FIG. 10A compares BSA-PEHA and BSA, similar to FIG. ID. FIG. 10B shows the CD spectra of mRNA alone. FIGs. 10C and 10D show measured at various pHs, including pH 4 through pH 7. The CD spectrum of the mixture of mRNA / BSA-PEHA is considerably different from a simple sum of the spectra of the two components of the mixture. This indicates interactions between the two binding partners (BSA-PEHA and mRNA).

[0106] FIG. 11A shows a denaturing gel electrophoresis of S protein of IBV mRNA. Western blot of eGFP mRNA (26 kDA) and IBV S protein mRNA (128kDA) in HD11 cells, respectively, expressed in presence and absence of chloroquine are shown in FIGs. 11B and 11C, respectively.

[0107] Binding dynamics of BSA-PEHA and IVT IBV S protein mRNA were measured at various concentrations of each by electrophoretic mobility shift assay (EMSA). Firstly, BSA-PEHA nanoparticles at different concentrations (100, 50, 25, 12.5 pg / ml) was allowed to react with a constant concentration of IVT IBV S protein mRNA (1 pg). Also, BSA-PEHA at a constant concentration of 100 pg / ml was allowed to conjugate with different quantities of mRNA (1, 2, 4 and 8 pg) at pH 7.0. Since mRNAs are negatively charged and BSA-PEHA nanoparticles are positively charged, free proteins will migrate quickly towards the cathode and the nucleic acid towards anode. In this study, in contrast to the free mRNA and the free BSA-PEHA nanoparticles, the mobility of the complex is clearly reduced when the BSA-PEHA conjugates with the mRNA. The results indicate that as the concentration of BSA-PEHA decreases the binding with the nucleic acid also decreases hence the free nucleic acid moves towards the cathode. However, when the concentration of nanoparticles was kept at constant (100 pg / ml) the binding of the nanoparticle with the nucleic acid seemed to remain constant. The agar gel electrophoresis was imaged under UV light for nucleic acids (left panel for each FIGs. 12A and 12B) and stained with Coomassic blue for BSA-PEHA (right panel for each FIGs. 12A and 12B). FIG. 12A shows binding dynamics when the nucleic acids are at a constant concentration and BSA-PEHA is between 12.5 pg / ml to 100 pg / ml. FIG. 12B shows binding dynamics when BSA-PEHA is constant and the nucleic acids are between 1 pg to 4 pg.Example 2: Protein nanoparticles in animal modelsAnimal Immunization

[0108] Four-week-old SPF white leghorn chickens at 4-weeks of age were inoculated via intramuscular route with two doses (5 pg and 25 pg) of IVT S protein IBV mRNA conjugated with the BSA-PEHA nanoparticle (FIGs. 13A and 13B). A booster was administrated after 2 weeks of the first vaccination. The sera from the chickens were collected at 14 and 28 days of inoculation.

[0109] The serum were then used for ELISA and virus neutralization assay. Peripheral blood mononuclear cells (PBMCs) were also collected to find out the stimulation of T-cells when incubated with inactivated virus (FIG. 14). Serum neutralization of chickens immunized with different combinations of BSA-PEHA / IVT IBV S protein mRNA at two and three weeks after vaccination.Summary

[0110] Time-dependent internalization of nanoparticles by cells was influenced by surface charge, shape and size of the nanoparticle. Nanoparticles were -10 nm in diameter by DLS data and TEM micrograph images with little to no effect in the protein structure and a positive surface charge (zeta potential ± 20-30mV). Dose dependent cytotoxicity, but well above the International Organization for Standardization ISO 10993-5:2009 threshold. ISO 10993- 5:2009 relates to test methods to assess the in vitro cytotoxicity of medical devices. These methods specify the incubation of cultured cells in contact with a device and / or extracts of a device either directly or through diffusion, e.g., BSA-PEHA / mRNA nanoparticles. S protein expression was enhanced in presence of chloroquine when in BSA-PEHA nanoparticles in HD11 cells. Immunogenicity study revealed significant increase in antibody titer against IBV S protein with scrum neutralization and also stimulation of cell-mediated immune response.

[0111] The end users will be farmers that are interested in vaccinating flocks, but the current technology is unlikely to be limited to chickens. Other birds, animals and even humans could be vaccinated using the albumin nanoparticles with corresponding mRNA variants. For humans and other species, the albumin can be matched exactly with the target species or even individuals, thus providing a great advantage of high compatibility with low toxicity. Globally, 19 billion chickens are vaccinated every 3-4 months, thus providing a very large market, and applications for other animals or humans should further expand the application of this technology. Even though current studies are focused on IBV, other viruses and bacteria could also be targeted by this technology and could be potentially expanded to cancer vaccines.

[0112] Points of novelty include, without limitation: (1) Albumin-based carrier developed can be matched with a species of interest is unique. (2) The carrier is non-toxic, biocompatible and stable at room temperature. (3) In some embodiments, this does not require the cold-chain, and hence, of great interest for storage, transportation and distribution. (4) low cost, easy to prepare and scale-up to gram quantities with relatively simple resources (5) completely biodegradable and bioabsorbable carrier, with no potential accumulation in the target, (6) low cost (7) very low dose of mRNA was sufficient (few pg) as opposed to hundreds of pg of the state of the art COVID-19 vaccines.

[0113] All statements herein reciting principles, aspects, and embodiments of the disclosure, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure.

[0114] Various other components may be included and called upon for providing for aspects of the teachings herein. For example, additional materials, combinations of materials and / or omission of materials may be used to provide for added embodiments that are within the scope of the teachings herein. Adequacy of any particular element for practice of the teachings herein is to be judged from the perspective of a designer, manufacturer, seller, user, system operator or other similarly interested party, and such limitations are to be perceived according to the standards of the interested party.

[0115] In the disclosure hereof any element expressed as a means for performing a specified function is intended to encompass any way of performing that function including, for example, a) a combination of circuit elements and associated hardware which perform that function or b) software in any form, including, therefore, firmware, microcode or the like as set forth herein, combined with appropriate circuitry for executing that software to perform the function. Applicants thus regard any means which can provide those functionalities as equivalent to those shown herein. No functional language used in claims appended herein is to be construed as invoking 35 U.S.C. § 112(f) interpretations as “means-plus-function” language unless specifically expressed as such by use of the words “means for” or “steps for” within the respective claim.

[0116] When introducing elements of the present inventions or the embodiment(s) thereof, the articles “a,” “an,” and “the” are intended to mean that there are one or more of the elements. Similarly, the adjective “another,” when used to introduce an element, is intended to mean one or more elements. The terms “including” and “having” are intended to be inclusive such that there may be additional elements other than the listed elements. The term “exemplary” is not intended to be construed as a superlative example but merely one of many possible examples.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a nucleic acid molecule and a carrier, wherein the carrier comprises a protein and an amine, and wherein the carrier interacts with the nucleic acid molecule forming a complex to deliver the nucleic acid molecule to a cell.

2. The composition of claim 1, wherein the nucleic acid molecule is selected from the group consisting mRNA, siRNA, shRNA, ssRNA, dsRNA, ssDNA, dsDNA, DNA, and combinations thereof.

3. The composition of claim 2, wherein the nucleic acid molecule is mRNA.

4. The composition of any one of claims 1-3, wherein the protein is between about 50 kDa and about 100 KDa.

5. The composition of any one of claims 1-4, wherein the protein has a net negative charge at physiological pH.

6. The composition of any one of claims 1-5, wherein the protein is albumin, or a fragment thereof.

7. The composition of claim 6, wherein the albumin, or a fragment thereof, is derived from an animal or is synthetic.

8. The composition of claim 6 or 7, wherein the animal is selected from bovine or human.

9. The composition of any one of claims 1-8, wherein the amine is selected from the group consisting of pentaethylenehexamine (PEHA), ethylenediamine, triethylenetetraamine, spermine, histidine, polyethylenimine (PET), spermidine, poly(L-lysine), poly(amido amine) (PAMAM)dendrimers, polypropyleneiminie dendrimers, poly(2-dimethylamino ethyl)-methacrylate (pDMAEMA), chitosan, tris(2-aminoethyl)amine and methylated derivatives thereof, ethylene diamine, diethylene triamine, tetra ethylene pentamine, and combinations thereof.

10. The composition of claim 9, wherein the amine is pentaethylenehexamine (PEHA).

11. The composition of any one of claims 1-10, wherein the nucleic acid molecule is between about 0.5 pg to about 20 pg, about 1 pg to about 5 pg, about 5 pg to about 7 pg, or about 10 pg to about 20 pg.

12. The composition of any one of claims 1-11, wherein the carrier is between about 10 pg / ml to about 150 pg / ml, about 20 pg / ml to about 125 pg / ml, about 30 pg / ml to about 110 pg / ml, about 50 pg / ml to about 100 pg / ml, or about 75 pg / ml to about 115 pg / ml.

13. The composition of any one of claims 3-12, wherein the mRNA encodes a polypeptide from a pathogen.

14. The composition of claim 13, wherein the pathogen is a bacteria, virus, fungus, parasite, prion, or protest.

15. The composition of claim 13 or 14, wherein the polypeptide is naturally found on the cell surface of the pathogen.

16. The composition of any one of claims 13-15, wherein the pathogen infects an animal.

17. The composition of claim 16, wherein the animal is an avian or a mammal.

18. The composition of claim 17, wherein the avian is a chicken, a goose, or a duck.

19. The composition of claim 17, wherein the mammal is a sheep, a pig, a cow, a horse, a goat, or a human.

20. The composition of any one of claims 13-19, wherein the pathogen is a virus.

21. The composition of claim 20, wherein the virus is infectious bronchitis virus (IBV) or highly pathogenic avian influenza (HP Al).

22. The composition of claim 21, wherein the avian is a chicken.

23. The composition of claim 21 or 22, wherein the polypeptide is the spike protein (S protein) or IBV or HPAI.

24. The composition of claim 20, wherein the virus is coronavirus.

25. The composition of claim 24, wherein the mammal is a human.

26. The composition of claim 24 or 25, wherein the polypeptide is the spike protein (S protein) or coronavirus.

27. A pharmaceutical composition comprising the composition of any one of claims 1-26 and a pharmaceutically acceptable excipient.

28. A method of producing a composition comprising an mRNA and a carrier, the method comprising: crosslinking a protein to an amine to generate the carrier; and contacting the carrier with the mRNA, wherein the carrier interacts with the mRNA forming a complex to deliver the mRNA to a cell.

29. The method of claim 28, wherein the protein is albumin, or a fragment thereof.

30. The method of claim 28 or 29, wherein the protein is albumin, or a fragment thereof, is derived from an animal or is synthetic.

31. The method of claim 30, wherein the animal is selected from bovine or human.

32. Thee method of any one of claims 28-31, wherein the amine is selected from the group consisting of pentaethylenehexamine (PEHA), ethylenediamine, triethylenetetraamine, spermine, histidine, polyethylenimine (PEI), spermidine, poly(L-lysine), poly(amido amine) (PAMAM) dendrimers, polypropyleneiminie dendrimers, poly(2-dimethylamino ethyl) -methacrylate (pDMAEMA), chitosan, tris(2-aminoethyl)amine and its methylated derivatives, and combinations thereof.

33. The method of any one of claims 28-32, wherein the amine is pcntacthylcnchcxaminc (PEHA).

34. The method of any one of claims 28-33, wherein the protein and amine are crosslinked with a carbodiimide.

35. The method of claim 34, wherein the carbodiimide is l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC).

36. The method of any one of claims 28-35, wherein the crosslinking occurs at a pH between pH 4 and pH 6, optionally the pH is 5.5.

37. The method of any one of claims 28-36, further wherein the crosslinked protein-amine molecules are dialyzed into a salt buffer at a pH between pH 6.5 and pH 7.5.

38. A nanoparticle for delivery of a vaccine to a cell comprising a carrier and an mRNA, wherein the mRNA encodes a polypeptide derived from infectious bronchitis virus, and the carrier comprises a crosslinked protein-amine molecule;wherein the carrier interacts with the mRNA forming a complex to deliver the mRNA to the cell.

39. The nanoparticle of claim 39, wherein the crosslinked protein-amine molecule comprises albumin, or a fragment thereof, and pentaethylenehexamine (PEHA).

40. The nanoparticle of claim 38 or 39, wherein the polypeptide is a spike protein (S protein).

41. The nanoparticle of any one of claims 38-40, wherein the nanoparticle is formulated for subcutaneous injection or intranasal spray.

42. A pharmaceutical composition comprising the nanoparticle of any one of claims 38-41 , and a pharmaceutically acceptable excipient.

43. A method of vaccinating a subject, the method comprising administering the composition of any one of claims 1-26, the composition produced by the method of any one of claims 28-37, the nanoparticle of any one of claims 38-41, or the pharmaceutical composition of claim 27 or 42.

44. The method of claim 43, wherein the subject is an animal.

45. The method of claim 44, wherein the animal is an avian or a human.

46. A method of inducing an immune response in a subject, the method comprising administering the composition of any one of claims 1-26, the composition produced by the method of any one of claims 28-37, the nanoparticle of any one of claims 38-41, or the pharmaceutical composition of claim 27 or 42.

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