Classifications

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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P31/12—Antivirals
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  • A61P37/00—Drugs for immunological or allergic disorders
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  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/543—Mucosal route intranasal
• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55505—Inorganic adjuvants
• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55583—Polysaccharides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
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  • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20071—Demonstrated in vivo effect

Definitions

• COVID-19 is a pandemic disease caused by a novel strain of coronavirus called SARS-CoV-2.
• SARS-CoV-2 This virus mainly causes respiratory tract disease and pneumonia in humans by infecting cells expressing a higher level of angiotensin-converting enzyme 2 (ACE2) receptor, which the virus uses to gain intracellular entry.
• ACE2 angiotensin-converting enzyme 2
• the body’s adaptive immune system can learn to recognize new invading pathogens.
• SARS-CoV-2 uses its surface spike glycoprotein (S) to lock onto ACE2 receptors on human cell surfaces.
• S surface spike glycoprotein
• RNA ribonucleic acid
• virus assembly occurs, and more viruses are released to infect other host cells.
• An infected patient can then initiate an immune response, whereby specialized “antigen presenting cells” engulf the virus and display portions of it to activate T helper cells.
• T helper cells then enable other immune responses: B cells make antibodies that can block the virus from infecting cells, and also mark the virus for destruction; and cytotoxic T cells identify and destroy virus -infected cells. Long-lived ‘memory’ B and T cells that recognize the virus can patrol the body for subsequent months or years, providing immunity. 5
• Anti-COVID-19 treatments and preventions are immediate priorities: The current pandemic SARS-CoV-2 strain is new, and limited clinical and immunological information are available to help in the development of drugs or vaccines that can treat and protect patients with COVID-19. It is vital and of immediate importance to develop safe and effective vaccines that provide protection from SARS-CoV-2.
• All vaccines aim to expose the body to an antigen that will not cause disease, but will provoke an immune response that can block or kill the virus if a person becomes infected.
• SARS-CoV-2 As of April 30, 2020, more than 90 vaccines of eight broad types were being developed against SARS-CoV-2 across the world: (1) Virus vaccines (weakened or inactivated viruses). (2) Nucleic acid vaccines (DNA or RNA based). (3) Viral vector vaccines (replicating and non-replicating viral vectors). (4) Protein based vaccines (protein subunits and virus-like particles). There are many relative merits and demerits for these strategies.
• SARS-CoV-2 is similar to SARS-CoV and MERS-CoV viruses with regard to their biological profiles and clinical presentations. 7 In all these viruses, the S protein is the major inducer of neutralizing antibodies. 8 11 Recombinant adenovirus-based vaccine expressing S protein of MERS-CoV induces systemic IgG, secretory IgA, and lung resident memory T cell responses when administered intranasally (IN) into B ALB/c mice, providing long-lasting neutralizing immunity to spike pseudotyped MERS virus, thereby suggesting that this IN vaccine may confer protection against MERS-CoV.
• a complex including: (a) a nanoparticle including a gold core; and (b) a pulmonary viral protein or fragment thereof, or a nucleic acid encoding the pulmonary viral protein or fragment thereof, wherein the pulmonary viral protein or nucleic acid is attached to the nanoparticle.
• a vaccine composition including a complex provided herein including embodiments thereof and a pharmaceutically acceptable excipient.
• a method of treating or preventing a pulmonary viral disease in a subject in need of such treatment or prevention including administering a therapeutically or prophylactically effective amount of a complex provided herein including embodiments thereof to the subject.
• a method of treating or preventing a pulmonary viral disease in a subject in need of such treatment or prevention including administering a therapeutically or prophylactically effective amount of a vaccine composition provided herein including embodiments thereof to the subject.
• a method for immunizing a subject susceptible to a pulmonary viral disease including administering a complex provided herein including embodiments thereof to the subject, under conditions such that antibodies that bind to the pulmonary viral protein or fragment thereof are produced.
• a nanoparticle including a plurality of nucleic acids attached thereto and plurality of proteins attached thereto, wherein each of the plurality of nucleic acids encode for a different SARS-CoV-2 viral protein, and each of the plurality of proteins is a different SARS-CoV-2 viral protein.
• a vaccine formulation including a nanoparticle as provided herein including embodiments thereof, and a pharmaceutically acceptable excipient.
• a method of preventing or treating COVID-19 in a subject in need thereof including administering to the subject a composition including an effective amount of a vaccine as provided herein including embodiments thereof, or a nanoparticle as provided herein including embodiments thereof, to a subject in need thereof.
• a method of preventing or treating a SARS-CoV-2 viral infection in a subject in need thereof including administering a composition including an effective amount of a vaccine as provided herein including embodiments thereof, or a nanoparticle as provided herein including embodiments thereof, to a subject in need thereof.
• FIG. 1 is a schematic illustration of SARS-CoV-2 mRNA vaccine delivery using PolyGION-CD-CS nanoparticles (NPs) for activation of pulmonary immune responses.
• NPs PolyGION-CD-CS nanoparticles
• FIGS. 2A-2F present an exemplary in vitro evaluation of PolyGION-CD-CS nanoparticles, and their efficiency in delivering FLuc-mRNA in cells by functional expression analysis.
• FIG. 2A shows a transmission electron microscopy (TEM) image of PolyGION.
• FIG. 2B shows Energy-dispersive X-ray spectroscopy (EDX) analysis of PolyGION.
• FIG. 2C shows RNA loading efficiency of PolyGION-CD-CS.
• FIGS. 2D-2E show a DLS analysis of PolyGION-CD-CS nanoparticles for size (FIG. 2D) and zeta potential (FIG. 2E).
• FIG. 2F shows intracellular delivery of PolyGION-CD-CS-mRNA in A549 cells by Prussian blue staining.
• FIG. 3 shows optical bioluminescence imaging of animals delivered with FLuc- mRNA using PolyGION-CD-CS NPs. Mice treated with a dose of 2 pg of FLuc-mRNA every day and images acquired 24 h after treatment.
• FIGS. 4A-4C present exemplary in vivo micro-computed tomography (microCT) and optical imaging, and ex vivo bioluminescence imaging (BLI) of tissues after five doses of PolyGION-CD-CS-FLuc-mRNA.
• FIG. 4A shows microCT images of control and animal treated using PolyGION-CD-CS-FLuc-mRNA;
• FIG. 4B shows BLI of animals imaged on the same day as microCT;
• FIG. 4C shows ex vivo BLI of tissues extracted from animal of treatment group.
• FIG. 5 is a schematic illustration of in vitro and in vivo experimental workflow with the assays proposed to assess transfection, immune response, and pathogenicity.
• FIG. 6 illustrates the DNA loading efficiency of Au-Chitosan Nanoparticles of various shapes and sizes.
• the left and middle panel show data for Au-Nanostar nanoparticles between 0.015 and 0.5 nm in diameter and the right panel shows data for Au-Nanosphere nanoparticles between 0.015 and 0.5 nm in diameter.
• FIGS. 7A-7B shows the transfection efficiency of Au-CH Nanoparticles of different sizes and shape into cells.
• FIG. 7A Representative images of cells transfected with Au-CH Nanoparticles loaded with DNA encoding a luciferase reporter protein.
• FIG. 7B Bar graph showing fluorescence intensity of the transfected cells.
• FIGS. 8A-8C illustrate DNA dose dependent transfection by Au-NS Nanoparticles in Mammalian Cells.
• FIG. 8A Representative images of cells transfected with Au-CH Nanoparticles loaded with various amounts of DNA encoding a luciferase reporter protein.
• FIG. 8B Number of events against Fluc-EGFP-Fluorescence intensity histogram showing the shift of stained cells dependent on the amount of DNA loaded on the nanoparticles.
• FIG. 8C Bar graph showing transfection efficiency of nanoparticles loaded with DNA, and dose dependent expression of loaded DNA as measured by fluorescence intensity.
• FIGS. 9A-9H show in vitro characterization of SC2 DNA vaccine loaded on AuNS-CS NPs.
• FIGS. 9A-9C FE-SEM micrographs indicate uniform morphology of AuNS-chitosan and SC2 DNA;
• FIG. 9D Evaluation of DNA loading efficiency of AuNS- chitosan by gel retardation assay;
• FIGS. 9E-9F DLS results measured for zeta potential and particle size (nm) of SC2 vaccine loaded AuNS at different ratios;
• FIG. 9G Transfection efficiency of AuNS-chitosan evaluated by delivery of pcDNA-FLuc-eGFP plasmid by bioluminescence imaging;
• FIG. 9G Transfection efficiency of AuNS-chitosan evaluated by delivery of pcDNA-FLuc-eGFP plasmid by bioluminescence imaging;
• FIGS. 10A-10D illustrate (FIG. 10A) a Schematic representation of the experimental design: Five-weeks-old BALB/c mice and C57BL/6J mice were immunized with AuNS-chitosan loaded with control DNA or SC2-S DNA vaccine administered via the IN route, the serum was collected every week and assessed for anti-SC2 antibody against purified proteins of CoV-1 and CoV-2; (FIG. 10B) Chemiluminescence based dot blot immunoassay for screening anti-SC2 antibody levels in serum collected from, (FIG. IOC) BALB/c and (FIG. 10D) C57BL/6J mice at different time points of treatment with their respective quantitative plots. The data are presented as mean ⁇ SEM.
• ns- represents no-significant difference, * for p ⁇ 0.05, ** for p ⁇ 0.01, *** for p ⁇ 0.001 and **** for pO.OOOl.
• FIGS. 11A-11G illustrate dot blot immunoassay for screening anti-SC2 antibody levels in (FIG. 11A) BALB/c and (FIG. 11B) C57BL/6J mice at different time points of treatment.
• the serum was probed against the cell lysate of HEK-293T cells transfected with plasmid encoding S protein of SC2-SA-mutant and SC2-Wuhan variant to determine the efficacy of vaccination strategy in mounting an immune response against different variants of SC2.
• FIGS. 12A-13B show anti-S protein IgA (FIG. 12A) and IgG (FIG. 12B) responses induced by mRNA vaccines
• FIGS. 13A-13G show evaluation of the specificity of lentivirus expressing SC2 S protein as a pseudovirus to cells expressing ACE2 receptor.
• FIG. 13A Mechanism of SC2 transduction in cells; lentivirus expressing SC2 S protein and Fluc-ZsGreen reporter gene were engineered, and these pseudoviruses were transduced in control and ACE2 receptor expressing cells, and subsequent infectivity was quantified using bioluminescence imaging.
• FIGGS. 13B-13C DNA vaccine-mediated induction of anti-SC2-S protein specific antibody evaluated for its neutralizing effect using engineered pseudovirus assessed by quantifying pseudovirus-mediated ZsGreen expression in the infected cells in the presence of neutralizing antibody from serum of mice treated with DNA vaccine; and
• FIGS. 13A-13G show evaluation of the specificity of lentivirus expressing SC2 S protein as a pseudovirus to cells expressing ACE2 receptor.
• Adjusted p- values were considered statistically significant if p- values ⁇ 0.05 and the symbols indicating statistical significance were as follows - ns represents no-significant difference, * represents p ⁇ 0.05, ** represents pO.Ol, *** represents p ⁇ 0.001 and **** represents pO.OOOl significance.
• FIGS. 14A-14D show in vitro delivery of Flue mRNA using AuNS-chitosan in (FIG 14A) HEK293 and (FIG 14B) A549 and cells imaged by optical bioluminescence (BLI).
• FIGG 14C In vivo BLI
• FIGG 14D ex vivo BLI of tissues after two doses of AuNS-chitosan-FLuc-mRNA delivery. There is significant expression of Firefly luciferase in the lungs and this is supported by the ex vivo tissue imaging findings.
• FIG. 15 illustrates the SARS-CoV-2 genome structure and locations where target proteins for a mRNA vaccine are encoded.
• FIGS. 16A-16E shows recruitment of immune cells in the lung (FIG. 16A) and resident T cell distribution in the spleen (FIG. 16B), lungs (FIG. 16C), thymus (FIG. 16D), and lymph nodes (FIG. 16E) of mice IN treated with DNA vaccine expressing S protein of SC2.
• the DNA vaccine induced CD4+ T cells expressing memory and tissue-resident markers were used for assessment.
• the number of lung resident CD4+ T cells increased in the lungs of the immunized mice compared to control DNA treated mice.
• the lungs, spleen, thymus and lymph nodes were dissected 14 weeks after treatment to evaluate for T cell subtypes.
• T cell subtypes CD3/CD4, CD3/CD8; macrophages: CD45/CDllb; dendritic cells: CD45/CDllc; B cells: CD19 and CD22).
• FIGS. 17A-17E illustrate flow cytometry analysis of IFNy expression in CD45+ T cells isolated from spleen (FIG. 17A), lungs (FIG. 17B), thymus (FIG. 17C), lymph nodes (FIG. 17D), and blood (FIG. 17E) of BALB/c mice treated with pDNA and SC2-spike DNA.
• FIG. 18 shows that vaccination with DNA encoding SARS-CoV-2 S protein results in T and B cell activation in the Draining Lymph Node.
• FIG. 19 shows the structures of polymers that form may form the outer layer of the nanoparticle.
• FIG. 19A shows the structures of chitosan and chitosan-cyclodextran and the reaction for synthesizing cyclodextrin conjugated chitosan (CD-CS).
• FIG. 19B shows the structure of Polyethylenimine (PEI) (top panel) and Polyamidoamine (PAMAM) (bottom panel) dendrimers.
• PEI Polyethylenimine
• PAMAM Polyamidoamine
• Nucleic acid refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides.
• polynucleotide oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides.
• nucleoside refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five-carbon sugar (ribose or deoxyribose).
• nucleosides include, cytidine, uridine, adenosine, guanosine, thymidine and inosine.
• nucleotide refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof.
• polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA.
• nucleic acid e.g. polynucleotides contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof.
• duplex in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched.
• nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides.
• the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.
• Nucleic acids can include one or more reactive moieties.
• the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions.
• the nucleic acid can include a reactive moiety that reacts with an outer layer of a nanoparticle through a covalent, non- covalent or other interaction.
• the terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
• Examples of such analogs include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine.; and peptide nucleic acid backbones and linkages.
• phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphothio
• nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art), including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids.
• LNA locked nucleic acids
• Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip.
• Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
• the intemucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.
• Nucleic acids can include nonspecific sequences.
• nonspecific sequence refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence.
• a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.
• a polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA).
• A adenine
• C cytosine
• G guanine
• T thymine
• U uracil
• T thymine
• polynucleotide sequence is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
• Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleo
• complement refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides.
• a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence.
• the nucleotides of a complement may partially or completely match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. Where the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence.
• Examples of complementary sequences include coding and a non-coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence.
• a further example of complementary sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence.
• sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing.
• two sequences that are complementary to each other may have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region).
• amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
• Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g- carboxyglutamate, and O-phosphoserine.
• Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i. e.. an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
• Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
• Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
• non-naturally occurring amino acid and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.
• Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
• polypeptide refers to a polymer of amino acid residues, wherein the polymer may In embodiments be conjugated to a moiety that does not consist of amino acids.
• the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
• a “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.
• amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion.
• numbered with reference to refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.
• An amino acid residue in a protein "corresponds" to a given residue when it occupies the same essential structural position within the protein as the given residue.
• residues corresponding to a specific position in a protein e.g ., Spike protein
• a protein e.g., Spike protein
• the identity and location of residues corresponding to specific positions of the protein are identified in other protein sequences aligning to the protein.
• a selected residue in a selected protein corresponds to glutamic acid at position 138 when the selected residue occupies the same essential spatial or other structural relationship as a glutamic acid at position 138.
• the position in the aligned selected protein aligning with glutamic acid 138 corresponds to glutamic acid 138.
• a three dimensional structural alignment can also be used, e.g., where the structure of the selected protein is aligned for maximum correspondence with the glutamic acid at position 138, and the overall structures compared.
• an amino acid that occupies the same essential position as glutamic acid 138 in the structural model corresponds to the glutamic acid 138 residue.
• Constantly modified variants applies to both amino acid and nucleic acid sequences.
• “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations,” which are one species of conservatively modified variations.
• Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
• each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
• TGG which is ordinarily the only codon for tryptophan
• amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure.
• nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.
• sequences are then said to be "substantially identical.”
• This definition also refers to, or may be applied to, the compliment of a test sequence.
• the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
• the preferred algorithms can account for gaps and the like.
• identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
• Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
• a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
• Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math.
• T is referred to as the neighborhood word score threshold (Altschul et al. , supra).
• These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
• the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
• Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
• Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873- 5787).
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• P(N) the smallest sum probability
• a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
• nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
• a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
• Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
• Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
• Antibodies are large, complex molecules (molecular weight of -150,000 or about 1320 amino acids) with intricate internal structure.
• a natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain.
• Each light chain and heavy chain in turn consists of two regions: a variable (“V”) region, involved in binding the target antigen, and a constant (“C”) region that interacts with other components of the immune system.
• the light and heavy chain variable regions also referred to herein as light chain variable (VL) domain and heavy chain variable (VH) domain, respectively
• VL light chain variable
• VH heavy chain variable domain
• CDRs complementarity determining regions
• the six CDRs in an antibody variable domain fold up together in 3- dimensional space to form the actual antibody binding site which docks onto the target antigen.
• the position and length of the CDRs have been precisely defined by Rabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987.
• the part of a variable region not contained in the CDRs is called the framework (“FR”), which forms the environment for the CDRs.
• FR framework
• the recognized immunoglobulin genes that encode antibodies include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
• Light chains are classified as either kappa or lambda.
• Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
• antigen and “epitope” interchangeably refer to the portion of a molecule (e.g., a polypeptide) which is specifically recognized by a component of the immune system, e.g, an antibody, a T cell receptor, or other immune receptor such as a receptor on natural killer (NK) cells.
• a component of the immune system e.g, an antibody, a T cell receptor, or other immune receptor such as a receptor on natural killer (NK) cells.
• NK receptor on natural killer
• An exemplary immunoglobulin (antibody) structural unit can have a tetramer.
• Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa).
• the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
• variable heavy chain refers to the variable region of an immunoglobulin heavy chain, including an Fv, scFv , dsFv or Fab; while the terms “variable light chain,” “VL” or “VL” refer to the variable region of an immunoglobulin light chain, including an Fv, scFv , dsFv or Fab.
• antibody functional fragments include, but are not limited to, complete antibody molecules, antibody fragments, such as Fv, single chain Fv (scFv), complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), Fab, F(ab)2' and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to target antigen (see, e.g., FUNDAMENTAL IMMUNOLOGY (Paul ed., 4th ed. 2001).
• various antibody fragments can be obtained by a variety of methods, for example, digestion of an intact antibody with an enzyme, such as pepsin; or de novo synthesis.
• Antibody fragments are often synthesized de novo either chemically or by using recombinant DNA methodology.
• the term antibody includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty etal., (1990) Nature 348:552).
• the term "antibody” also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described in, e.g. , Kostelny et al. (1992) J. Immunol.
• the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background.
• Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein.
• polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with the selected antigen and not with other proteins.
• This selection may be achieved by subtracting out antibodies that cross-react with other molecules.
• a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
• solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
• Spike protein or "S protein” as used herein includes any of the recombinant or naturally-occurring forms of Spike glycoprotein, also known as S glycoprotein, E2, peplomer protein, or variants or homologs thereof that maintain Spike protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Spike protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Spike protein.
• the Spike protein is substantially identical to the protein identified by the UniProt reference number P0DTC2.
• Envelope protein or "E protein” as used herein includes any of the recombinant or naturally-occurring forms of Envelope small membrane protein, also known as sM protein, or variants or homologs thereof that maintain Envelope protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Envelope protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g . a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Envelope protein.
• the Envelope protein is substantially identical to the protein identified by the UniProt reference number P0DTC4.
• Membrane protein or "M protein” as used herein includes any of the recombinant or naturally-occurring forms of Membrane protein, or variants or homologs thereof that maintain Membrane protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Membrane protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Membrane protein.
• the Membrane protein is substantially identical to the protein identified by the UniProt reference number P0DTC5.
• Nucleocapsid protein or "N protein” as used herein includes any of the recombinant or naturally-occurring forms of Nucleocapsid protein, also known as Nucleoprotein protein, NC protein, or variants or homologs thereof that maintain Nucleocapsid protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Nucleocapsid protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Nucleocapsid protein.
• the Nucleocapsid protein is substantially identical to the protein identified by the UniProt reference number P0DTC9.
• Glycoprotein G protein or "G protein” as used herein includes any of the recombinant or naturally-occurring forms of Major surface glycoprotein G protein, also known as Membrane-bound glycoprotein, mG protein, or variants or homologs thereof that maintain Glycoprotein G protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Glycoprotein G protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Glycoprotein G protein.
• the Glycoprotein G protein is substantially identical to the protein identified by the UniProt reference number P03423.
• Glycoprotein F protein or "F protein” as used herein includes any of the recombinant or naturally-occurring forms of Fusion glycoprotein protein, also known as Fusion glycoprotein F0, Fusion glycoprotein F2, Intervening segment, Pep27, Peptide 27, Fusion glycoprotein FI, or variants or homologs thereof that maintain Glycoprotein F protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Glycoprotein F protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Glycoprotein F protein.
• the Glycoprotein F protein is substantially identical to the protein identified by the UniProt reference number P03420.
• Glycoprotein SH protein or "SH protein” as used herein includes any of the recombinant or naturally-occurring forms of Small hydrophobic protein, also known as Small protein 1 A, or variants or homologs thereof that maintain Glycoprotein SH protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Glycoprotein SH protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Glycoprotein SH protein.
• the Glycoprotein SH protein is substantially identical to the protein identified by the UniProt reference number P0DOE5.
• Hemagglutinin-neuraminidase protein or "HN protein” as used herein includes any of the recombinant or naturally-occurring forms of Hemagglutinin- neuraminidase, or variants or homologs thereof that maintain Hemagglutinin-neuraminidase protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Hemagglutinin-neuraminidase protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g.
• Fusion glycoprotein protein or "F protein” as used herein includes any of the recombinant or naturally-occurring forms of Fusion glycoprotein 0, or variants or homologs thereof that maintain Fusion glycoprotein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Fusion glycoprotein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Fusion glycoprotein.
• the Fusion glycoprotein is substantially identical to the protein identified by the UniProt reference number P06828.
• Matrix protein or "M protein” as used herein includes any of the recombinant or naturally-occurring forms of Matrix protein, or variants or homologs thereof that maintain Matrix protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Matrix protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Matrix protein.
• the Matrix protein is substantially identical to the protein identified by the UniProt reference number P07873.
• hexon protein or "hexon” as used herein includes any of the recombinant or naturally-occurring forms of hexon protein, or variants or homologs thereof that maintain hexon protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to hexon protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring hexon protein.
• the hexon protein is substantially identical to the protein identified by the UniProt reference number Q9DKL1.
• penton protein or “penton” as used herein includes any of the recombinant or naturally-occurring forms of penton protein, or variants or homologs thereof that maintain penton protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to penton protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring penton protein.
• the penton protein is substantially identical to the protein identified by the UniProt reference number Q2Y0H9.
• Pre-hexon-linking protein Ilia includes any of the recombinant or naturally-occurring forms of Pre-hexon-linking protein Ilia, also known as Capsid vertex-specific component Ilia, CVSC, Protien Ilia, or pllla, or variants or homologs thereof that maintain Pre-hexon-linking protein Ilia activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Pre-hexon-linking protein Ilia).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Pre-hexon-linking protein Ilia.
• the Pre-hexon-linking protein Ilia is substantially identical to the protein identified by the UniProt reference number Q2Y0I0.
• Pre-hexon-linking protein VIII includes any of the recombinant or naturally-occurring forms of Pre-hexon-linking protein VIII, also known as Pre-protein VIII, pVIII, or Protein VIII -N, or variants or homologs thereof that maintain Pre- hexon-linking protein VIII activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Pre-hexon-linking protein VIII).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g.
• the Pre-hexon-linking protein VIII is substantially identical to the protein identified by the UniProt reference number Q71BW3.
• Hexon-interlacing protein includes any of the recombinant or naturally-occurring forms of Hexon-interlacing protein, also known as Protein IX, or variants or homologs thereof that maintain Hexon-interlacing protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Hexon-interlacing protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g.
• Major surface glycoprotein G includes any of the recombinant or naturally-occurring forms of Major surface glycoprotein G, also known as Attachment glycoprotein G, Membrane-bound glycoprotein, mG, or variants or homologs thereof that maintain Major surface glycoprotein G activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Major surface glycoprotein G).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Major surface glycoprotein G.
• the Major surface glycoprotein G protein is substantially identical to the protein identified by the UniProt reference number Q6WB94.
• Fusion glycoprotein F0 includes any of the recombinant or naturally-occurring forms of Fusion glycoprotein F0, also known as Protein F, or variants or homologs thereof that maintain Fusion glycoprotein F0 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Fusion glycoprotein F0).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%,
• the Fusion glycoprotein F0 is substantially identical to the protein identified by the UniProt reference number Q6WB98.
• Nucleoprotein or “Protein N” as used herein includes any of the recombinant or naturally-occurring forms of Nucleoprotein, also known as Nucleocapsid protein, or variants or homologs thereof that maintain Nucleoprotein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Nucleoprotein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Nucleoprotein.
• the Nucleoprotein is substantially identical to the protein identified by the UniProt reference number Q6WB A1.
• Small hydrophobic protein or “SH protein” as used herein includes any of the recombinant or naturally-occurring forms of Small hydrophobic protein, or variants or homologs thereof that maintain Small hydrophobic protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Small hydrophobic protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g . a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Small hydrophobic protein.
• the Small hydrophobic protein is substantially identical to the protein identified by the UniProt reference number Q6WB95.
• Matrix protein or “M protein” as used herein includes any of the recombinant or naturally-occurring forms of Matrix protein, or variants or homologs thereof that maintain Matrix protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Matrix protein).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Matrix protein.
• the Matrix protein is substantially identical to the protein identified by the UniProt reference number Q6WB99.
• Capsid protein VP1 or as used herein includes any of the recombinant or naturally-occurring forms of Capsid protein VP1, or variants or homologs thereof that maintain Capsid protein VP1 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Capsid protein VP1).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Capsid protein VP1.
• the Capsid protein VP1 is substantially identical to the protein identified by the UniProt reference number I0B934.
• Capsid protein VP2 or as used herein includes any of the recombinant or naturally-occurring forms of Capsid protein VP2, or variants or homologs thereof that maintain Capsid protein VP2 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Capsid protein VP2).
• the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Capsid protein VP2.
• the Capsid protein VP2 is substantially identical to the protein identified by the UniProt reference number Q27XI9.
• the terms "disease” or “condition” refer to a state of being or health status of a subject capable of being treated with a compound, pharmaceutical composition, or method provided herein.
• the disease can be an autoimmune, inflammatory, cancer, infectious, metabolic, developmental, cardiovascular, liver, intestinal, endocrine, neurological, or other disease.
• the disease is an infectious disease (e.g. a coronavirus infection).
• infectious disease refers to a disease or condition that can be caused by organisms such as a bacterium, virus, fungi or any other pathogenic microbial agents.
• infectious disease is caused by a pathogenic virus.
• Pathogenic viruses are viruses that can infect and replicate within cells (e.g. human cells) and cause diseases.
• infectious disease is a virus associated disease.
• Non-limiting virus associated diseases include hepatic viral diseases (e.g., hepatitis A, B, C, D, E), herpes virus infection (e.g., HSV-1, HSV-2, herpes zoster), flavivirus infection, Zika virus infection, cytomegalovirus infection, a respiratory viral infection (causing a “pulmonary viral disease”) (e.g., adenovirus infection, influenza, severe acute respiratory syndrome, coronavirus infection (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV, COVID-19, MERS)), a gastrointestinal viral infection (e.g., noro virus infection, rotavirus infection, astro virus infection), an exanthematous viral infection (e.g., measles, shingles, smallpox, rubella), viral hemorrhagic disease (e.g., Ebola, Lassa fever, dengue fever, yellow fever), a neurologic viral infection (e.g.,
• pulmonary viral infection or “pulmonary viral disease” refers to a condition caused by a virus that can infect and replicate within cells and cause diseases or symptoms that affect the respiratory system (e.g. lower respiratory system, upper respiratory system, and lungs).
• the virus that causes a pulmonary viral infection may enter the subject by using the nose and/or mouth as ports of entry.
• the pulmonary viral infection may be caused by viruses including, but not limited to, Human respiratory syncytial virus (HRSV), Human parainfluenza virus (HPV), Human rhinovirus (HRV), Adenovirus (ADV), Human coronavirus (HCoV), Coronavirus associated with SARS (SARS-CoV), Human metapneumovirus (HMPV), or Human bocavirus (HBoV).
• HRSV Human respiratory syncytial virus
• HPV Human parainfluenza virus
• HRV Human rhinovirus
• ADV Human coronavirus
• HMPV Human metapneumovirus
• HMPV Human metapneumovirus
• HMPV Human metapneumovirus
• virus or “virus particle” are used according to their plain ordinary meaning in the biological arts and refer to a particle including a viral genome (e.g. DNA, RNA, single strand, double strand), a protective coat of proteins (e.g. capsid) and associated proteins, and in the case of enveloped viruses (e.g. herpesvirus), an envelope including lipids and optionally components of host cell membranes, and/or viral proteins.
• a viral genome e.g. DNA, RNA, single strand, double strand
• proteins e.g. capsid
• enveloped viruses e.g. herpesvirus
• Human coronavirus or “HCoV” refers to a group of RNA viruses that can enter and replicate in the cells of human and may cause disease (e.g. respiratory tract infections).
• coronaviruses are enveloped viruses with positive-sense single-stranded RNA and a nucleocapsid. Coronaviruses range in size and can be from, for example, 50 to 200 nm in diameter.
• the coronavirus viral envelope is made up of a lipid bilayer and includes the membrane, envelope and spike proteins. In instances, HcoV enters the host cell when the spike protein attaches to a host cell receptor.
• HCoV include Human coronavirus OC43 (HCoV-OC43), Human coronavirus HKU1 (HCoV-HKUl), Human coronavirus 229E (HCoV-229E), Human coronavirus NL63 (HCoV-NL63), Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and Severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2).
• entry of HcoV into a cell may cause SARS, MERS, or COVID-19.
• SARS-CoV-2 refers to the strain of coronavirus that causes coronavirus disease 2019 (COVID-19).
• SARS-CoV-2 is a positive-sense single-stranded RNA virus.
• SARS-CoV-2 belongs to the family of betacoronaviruses, whose members include two other zoonotic viruses that have caused severe disease outbreaks in the new millennium: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV).
• SARS-CoV-2 shows nearly 80 percent genetic similarity to SARS-CoV, which triggered the severe acute respiratory syndrome (SARS) epidemic in 2002-2003.
• SARS-CoV-2 is more distantly related to MERS-CoV, which is responsible for the Middle East respiratory syndrome (MERS) epidemic that began in 2012 and still persists.
• MERS Middle East respiratory syndrome
• SARS-CoV refers to SARS coronavirus.
• SARS-CoV includes any coronovirus, such as SARS-CoV-2, SARS-CoV-1, and MERS-CoV.
• COVID-19 refers to the disease caused by SARS-CoV-2. COVID-19 has an incubation period of 2-14 days, and symptoms include, e.g., fever, tiredness, cough, and shortness of breath (e.g., difficulty breathing).
• SARS-CoV-1 refers to the strain of coronavirus that causes severe acute respiratory syndrome (SARS).
• SARS-CoV-1 is an enveloped, positive-sense, single-stranded RNA virus that infects the epithelial cells within the lungs. In embodiments, the virus enters the host cell by binding to the angiotensin-converting enzyme 2 (ACE2) receptor.
• ACE2 angiotensin-converting enzyme 2
• MERS-CoV refers to Middle Eastern respiratory syndrome-associated coronavirus. See, e.g., Chung et al, Genetic Characterization of Middle East Respiratory Syndrome Coronavirus, South Korea, 2018. Emerging Infectious Diseases, 25(5):958-962 (2019).
• RSV Human respiratory syncytial virus
• RSV also known as human orthopneumovirus refers to a virus that can infect human cells and may cause infections with symptoms affecting the respiratory tract.
• RSV is a negative-sense single- stranded RNA virus.
• RSV can be transmitted through the nose or eys, and in instances can effect the columnar epithelial cells of the upper and lower airway.
• F protein on the surface of RSV may be used to fuse viral and host cell membranes, thus resulting infection of the host cell.
• F and G glycoproteins are used for viral attachment and infection of the host cell.
• Symptoms and syndromes that may be caused by RSV infection include pneumonia, respiratory failure, apnea, respiratory distress, and distant inflammation.
• HPIV Human parainfluenza virus
• HPIVs are single-stranded RNA viruses.
• HPIVs include Human parainfluenza virus type 1, Human parainfluenza virus type 2, Human parainfluenza virus type 3, and Human parainfluenza virus type 4.
• HPIV infects the host cell by attachment and fusion between the virus and host cell lipid membrane.
• HPIV may enter the cell by way of using the Envelope protein and Fusion protein to attach and fuse to the host cell for cell entry.
• Symptoms and syndromes caused by HPIV infection include lower respiratory tract infections, upper respiratory tract infections, bronchiolitis, pneumonia, neurologic disease, and airway inflammation.
• HRV Human rhinovirus
• HRV refers to a virus that infect humans and may cause the common cold. HRV include rhinovirus A, rhinovirus B and rhinovirus C.
• HRV is a single-stranded postivive sense RNA virus.
• HRV is transmitted by aerosols of respiratory droplets or fomites. Syndromes and symptoms caused by HRV infection include the common cold, sore throat, runny nose, nasal congestion, sneezing, cough, muscle aches, fatigue and headache.
• inhibition means negatively affecting (e.g., decreasing or reducing) the activity or function of the molecule relative to the activity or function of the protein in the absence of the inhibition.
• inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein or polynucleotide.
• an “inhibitor” is a compound that inhibits a target bio-molecule (i.e.
• nucleic acid e.g., nucleic acid, peptide, carbohydrate, lipid or any other molecules that can be found from nature), e.g., by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down-regulating activity of the target bio-molecule.
• inhibition refers to reduction of a disease or symptoms of disease (e.g. Covid-19).
• Treating” or “treatment” as used herein also broadly includes any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results.
• beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease’s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable.
• treatment includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease’s spread; relieve the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.
• Treating” and “treatment” as used herein include prophylactic treatment.
• Treatment methods include administering to a subject a therapeutically effective amount of an active agent.
• the administering step may consist of a single administration or may include a series of administrations.
• the length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof.
• the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art.
• chronic administration may be required.
• the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient.
• the treating or treatment is not prophylactic treatment.
• prevention refers to a decrease in the occurrence of a disease or disease symptoms in a patient. As indicated above, the prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur absent treatment.
• a “symptom” of a disease includes any clinical or laboratory manifestation associated with the disease, and is not limited to what a subject can feel or observe.
• a patient refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein.
• Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals.
• a patient is human.
• a “prophylactically effective amount” of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms.
• the full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
• a prophylactically effective amount may be administered in one or more administrations.
• An “activity decreasing amount,” as used herein, refers to an amount of antagonist required to decrease the activity of an enzyme relative to the absence of the antagonist.
• a “function disrupting amount,” as used herein, refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of the antagonist. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).
• therapeutically effective amounts for use in humans can also be determined from animal models.
• a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals.
• the dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.
• a therapeutically effective amount refers to that amount of the therapeutic agent sufficient to ameliorate the disorder, as described above.
• a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.
• Therapeutic efficacy can also be expressed as “-fold” increase or decrease.
• a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.
• Dosages may be varied depending upon the requirements of the patient and the compound being employed.
• the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time.
• the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual’s disease state.
• administering means oral administration, administration as an aerosol, dry powder, nasal spray, suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject.
• Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).
• Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.
• Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.
• the administering does not include administration of any active agent other than the recited active agent.
• compositions described herein are administered at the same time, just prior to, or just after the administration of one or more additional therapies.
• the compounds provided herein can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound).
• the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation).
• the compositions of the present disclosure can be delivered transdermally, by a topical route, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
• compositions of the present invention can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, nanoparticles, pastes, jellies, paints, powders, and aerosols.
• Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.
• Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
• Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions.
• the compositions of the present invention may additionally include components to provide sustained release and/or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates.
• compositions of the present invention can also be delivered as microspheres for slow release in the body.
• microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res.
• an immunogenic composition can include isolated nucleic acid constructs (such as DNA or RNA) that encode one or more immunogenic epitopes of the antigenic polypeptide that can be used to express the epitope(s) (and thus be used to elicit an immune response against this polypeptide or a related polypeptide associated with the targeted pathogen or type of cells).
• isolated nucleic acid constructs such as DNA or RNA
• immunogenic epitopes of the antigenic polypeptide that can be used to express the epitope(s) (and thus be used to elicit an immune response against this polypeptide or a related polypeptide associated with the targeted pathogen or type of cells).
• the subject can be administered an effective amount of one or more of agents, compositions or complexes, all of which are interchangeably used herein, (e.g. complex or vaccine composition including the same) provided herein.
• effective amount and “effective dosage” are used interchangeably.
• effective amount is defined as any amount necessary to produce a desired effect (e.g., expressing an immunogentic peptide expressed by a nucleic acid and exhibiting intended outcome of the immunogenic peptide).
• Effective amounts and schedules for administering the agent can be determined empirically by one skilled in the art.
• the dosage ranges for administration are those large enough to produce the desired effects, e.g.
• contacting may include allowing two species to react, interact, or physically touch, wherein the two species may be a compound as described herein and a protein or enzyme. In some embodiments contacting includes allowing a compound described herein to interact with a protein or enzyme that is involved in a signaling pathway.
• conjugated when referring to two moieties means the two moieties (e.g. nanoparticle outer layer and nucleic acid) are bonded, wherein the bond or bonds connecting the two moieties may be covalent or non-covalent.
• the two moieties are covalently bonded to each other (e.g., directly or through a covalently bonded intermediary).
• the two moieties are non-covalently bonded (e.g., through ionic bond(s), van der Waals bond(s)/interactions, hydrogen bond(s), polar bond(s), or combinations or mixtures thereof).
• bioconjugate and “bioconjugate linker” refers to the resulting association between atoms or molecules of “bioconjugate reactive groups” or “bioconjugate reactive moieties”.
• the association can be direct or indirect.
• a conjugate between a first bioconjugate reactive group e.g., -NH2, -C(0)0H, -N- hydroxysuccinimide, or -maleimide
• a second bioconjugate reactive group e.g., sulfhydryl, sulfur-containing amino acid, amine, amine sidechain containing amino acid, or carboxylate
• covalent bond or linker e.g.
• bioconjugate reactive groups including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition).
• nucleophilic substitutions e.g., reactions of amines and alcohols with acyl halides, active esters
• electrophilic substitutions e.g., enamine reactions
• additions to carbon-carbon and carbon-heteroatom multiple bonds e.g., Michael reaction, Diels-Alder addition.
• the first bioconjugate reactive group e.g., -sulfo-N-hydroxysuccinimide moiety
• the second bioconjugate reactive group e.g. an amine
• biotin conjugate can react with avidin or strepavidin to form a avi din-biotin complex or streptavidin-biotin complex.
• oligomers can have 1 to about 10 monomers, 1 to about 20 monomers, 1 to about 30 monomers, 1 to about 40 monomers, 1 to about 50 monomers, 1 to about 100 monomers, 1 to about 150 monomers, 1 to about 200 monomers, 1 to about 250 monomers, 1 to about 300 monomers, 1 to about 350 monomers, 1 to about 400 monomers, 1 to about 450 monomers or 1 to about 500 monomers is in length.
• oligomers can have less than about 500 monomers, less than about 450 monomers, less than about 400 monomers, less than about 350 monomers, less than about 300 monomers, less than about 250 monomers, less than about 200 monomers, less than about 150 monomers, less than about 100 monomers, less than about 50 monomers, less than about 40 monomers, less than about 30 monomers, less than about 20 monomers or less than about 10 monomers in length.
• the number of monomers in polymers is generally more than the number of monomers in oligomers.
• polymers can have about 500 to about 1000 monomers, about 500 to about 2000 monomers, about 500 to about 3000 monomers, about 500 to about 4000 monomers, about 500 to about 5000 monomers, about 500 to about 6000 monomers, about 500 to about 7000 monomers, about 500 to about 8000 monomers, about 500 to about 9000 monomers, about 500 to about 10000 monomers, or more than 10000 monomers in length.
• the polymer is a biopolymer.
• biopolymer refers to a polymer produced in the cells of living organisms.
• the polymer is a polysaccharide.
• the polymer is a cationic polysaccharide.
• block copolymer is used in accordance with its ordinary meaning and refers to two or more portions (e.g., blocks) of polymerized monomers linked by a covalent bond.
• a block copolymer is a repeating pattern of polymers.
• the block copolymer includes two or more monomers in a periodic (e.g., repeating pattern) sequence.
• a diblock copolymer has the formula: -B-B-B-B- B- B-B-A-A-A-A-A-A-A-, where ⁇ ’ is a first subunit and ‘A’ is a second subunit covalently bound together.
• a triblock copolymer therefore is a copolymer with three distinct blocks, two of which may be the same (e.g., -A-A-A-A-A-B-B-B-B-B-B-A-A-A-A-) or all three are different (e.g., -A-A-A-A-A-B-B-B-B-B-B-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-) where ‘A’ is a first subunit, ‘B’ is a second subunit, and ‘C’ is a third subunit, covalently bound together.
• nanoparticle refers to the weight average molecular weight of a polymer as determined by gel permeation chromatography (also known as GPC or size exclusion chromatography (SEC)) using tetrahydrofuran (THF) as the solvent and using a molecular weight calibration curve using polystyrene standards.
• a “nanoparticle,” as used herein, is a particle wherein the longest diameter is less than or equal to 1000 nanometers. The longest dimension of the nanoparticle may be referred to herein as the length of the nanoparticle. The shortest dimension of the nanoparticle may be referred to herein refer as the width of the nanoparticle. Nanoparticles may be composed of any appropriate material.
• the outer layer of the nanoparticle may include moieties that attach to one or more biomolecules (e.g. pulmonary viral protein, nucleic acid encoding said pulmonary viral protein)
• Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and the like.
• inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic,
• salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see. for example, Berge el al. , “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19).
• Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
• “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient.
• Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethy cellulose, polyvinyl pyrrolidine, and colors, and the like.
• Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
• auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
• auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
• preparation is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.
• carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.
• cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
• a “synergistic amount” as used herein refers to the sum of a first amount (e.g., an amount of a compound provided herein) and a second amount (e.g., a therapeutic agent) that results in a synergistic effect (i.e. an effect greater than an additive effect). Therefore, the terms “synergy”, “synergism”, “synergistic”, “combined synergistic amount”, and “synergistic therapeutic effect” which are used herein interchangeably, refer to a measured effect of the compound administered in combination where the measured effect is greater than the sum of the individual effects of each of the compounds provided herein administered alone as a single agent.
• a synergistic amount may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
• Clinical syndromes caused by pulmonary viruses may include the common cold, acute and chronic bronchitis, bronchiolitis, croup, pneumonia, asthma, bronchiectasis, pneumonia, pulmonary embolism, pulmonary hypertension, sarcoidosis, sleep apnea, and distant inflammatory effects.
• a pulmonary virus causes one or more syndromes as listed in Table 2.
• “pulmonary viral protein” refers to a protein (e.g. spike protein, etc.) that is encoded within the genome of a pulmonary virus (e.g. human coronaviruses, adenoviruses, etc.).
• the complex provided herein including embodiments thereof includes a nanoparticle, wherein the nanoparticle includes a gold core.
• the gold core is amenable to attaching (e.g. by indirect or direct attachment) a viral protein or a nucleic acid encoding the viral protein to the nanoparticle.
• the viral protein or nucleic acid may be indirectly attached to the nanoparticle gold core, for example, though a covalent linker (e.g. a peptide linker, a chemical linker, a bioconjugate linker etc.) or through attachment with the nanoparticle outer layer.
• a covalent linker e.g. a peptide linker, a chemical linker, a bioconjugate linker etc.
• a complex including: (a) a nanoparticle including a gold core; and (b) a pulmonary viral protein or fragment thereof, or a nucleic acid encoding the pulmonary viral protein or fragment thereof, wherein the pulmonary viral protein or nucleic acid is attached to the nanoparticle.
• the complex includes at least one pulmonary viral protein or fragment thereof and at least one nucleic acid encoding a pulmonary viral protein or fragment thereof.
• the complex includes a plurality of pulmonary viral proteins or fragments thereof.
• the plurality of pulmonary viral proteins include different pulmonary viral proteins.
• the complex includes a plurality of nucleic acids encoding the pulmonary viral protein or fragment thereof.
• the plurality of nucleic acids encode different pulmonary viral proteins or fragments thereof.
• the complex includes a plurality of pulmonary viral proteins or fragments thereof and a plurality of nucleic acids encoding pulmonary viral proteins or fragments thereof.
• the pulmonary viral protein is a protein from human respiratory syncytial virus (HRSV), human parainfluenza virus (HPV), human rhinovirus (HRV), adenovirus (ADV), human coronavirus (HCoV), coronavirus associated with SARS (SARS- CoV), human metapneumovirus (HMPV), or human bocavirus (HBoV).
• the pulmonary viral protein is a protein from human respiratory syncytial virus (HRSV).
• the pulmonary viral protein is a protein from human parainfluenza virus (HPV).
• the pulmonary viral protein is a protein from human rhinovirus (HRV).
• the pulmonary viral protein is a protein from adenovirus (ADV).
• the pulmonary viral protein is a protein from human coronavirus (HCoV).
• the pulmonary viral protein is a protein from coronavirus associated with SARS (SARS-CoV). In embodiments, the pulmonary viral protein is a protein from human metapneumovirus (HMPV). In embodiments, the pulmonary viral protein is a protein from human bocavirus (HBoV).
• the pulmonary viral protein from HRSV is Glycoprotein G (receptor binding), Glycoprotein F (membrane fusion), or Glycoprotein SH.
• the pulmonary viral protein from HRSV is Glycoprotein G.
• the pulmonary viral protein from HRSV is Glycoprotein F.
• the pulmonary viral protein from HRSV is Glycoprotein SH.
• the pulmonary viral protein from HPV is HN-Tetramer, F- Protein trimer, or Matrix protein (M).
• the pulmonary viral protein from HPV is HN-Tetramer.
• the pulmonary viral protein from HPV is F- Protein trimer.
• the pulmonary viral protein from HPV is Matrix protein (M).
• the pulmonary viral protein from HRV is Viral capsid glycoprotein VP1, Viral capsid glycoprotein VP2, Viral capsid glycoprotein VP3, or Viral capsid glycoprotein VP4.
• the pulmonary viral protein from HRV is Viral capsid glycoprotein VP1.
• the pulmonary viral protein from HRV is Viral capsid glycoprotein VP2.
• the pulmonary viral protein from HRV is Viral capsid glycoprotein VP3.
• the pulmonary viral protein from HRV is Viral capsid glycoprotein VP4.
• the pulmonary viral protein from ADV is Hexon,
• the pulmonary viral protein from ADV is Penton. In embodiments, the pulmonary viral protein from ADV is Fiber. In embodiments, the pulmonary viral protein from ADV is Ilia. In embodiments, the pulmonary viral protein from ADV is VIII. In embodiments, the pulmonary viral protein from ADV is IX. In embodiments, the pulmonary viral protein from HCoV is Envelop, Membrane, Spike Protein, or Nucleocapsid protein. In embodiments, the pulmonary viral protein from HCoV is Envelop protein. In embodiments, the pulmonary viral protein from HCoV is Membrane protein. In embodiments, the pulmonary viral protein from HCoV is Spike Protein.
• the pulmonary viral protein from HCoV is Nucleocapsid protein.
• the pulmonary viral protein from SARS-CoV is Envelop, Membrane, Spike Protein, or Nucleocapsid protein.
• the pulmonary viral protein from SARS-CoV is Envelop protein.
• the pulmonary viral protein from SARS-CoV is Membrane protein.
• the pulmonary viral protein from SARS-CoV is Spike Protein.
• the pulmonary viral protein from SARS-CoV is Nucleocapsid protein.
• the pulmonary viral protein from HMPV is Glycoprotein-G, Fusion protein-F, Nucleoprotein- N, SH-Protein, or Matrix protein.
• the pulmonary viral protein from HMPV is Glycoprotein-G. In embodiments, the pulmonary viral protein from HMPV is Fusion protein-F. In embodiments, the pulmonary viral protein from HMPV is Nucleoprotein- N. In embodiments, the pulmonary viral protein from HMPV is SH-Protein. In embodiments, the pulmonary viral protein from HMPV is Matrix protein. In embodiments, the pulmonary viral protein from HBoV is Viral capsid protein 1 (VP2) or and Viral capsid protein 2 (VP2). In embodiments, the pulmonary viral protein from HBoV is Viral capsid protein 1 (VP2). In embodiments, the pulmonary viral protein from HBoV is Viral capsid protein 2 (VP2).
• the pulmonary virus is SARS-CoV-2.
• the pulmonary viral protein or fragment thereof is S protein, N protein, M protein, or E protein.
• the pulmonary viral protein or fragment thereof is S protein. In embodiments, the pulmonary viral protein or fragment thereof is N protein. In embodiments, the pulmonary viral protein or fragment thereof is M protein. In embodiments, the pulmonary viral protein or fragment thereof is E protein.
• the nucleic acid encodes S protein, N protein, M protein, or E protein. In embodiments, the nucleic acid encodes S protein. In embodiments, the nucleic acid encodes N protein. In embodiments, the nucleic acid encodes M protein. In embodiments, the nucleic acid encodes E protein.
• the gold core is a gold-iron oxide core.
• the iron-oxide component may be used for imaging or diagnosing methods (e g. MRI).
• the nanoparticle is modified by attaching an outer layer to the gold core.
• the nanoparticle includes an outer layer.
• the outer layer of the nanoparticle may include a compound (e.g. polymer, cationic polysaccharide) with a functional group by which a viral protein or a nucleic acid encoding the viral protein may be attached (e.g. by way of electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond) or hydrophobic interactions, by covalent conjugation chemistry) to the nanoparticle.
• covalent conjugation methods may be used which are well known in the art and described herein.
• the outer layer of the nanoparticle is covalently attached to the nanoparticle core (e.g. by attachment of a thiol group (-SH) on the outer layer to the surface of the gold core).
• the outer layer of the nanoparticle is non-covalently attached to the nanoparticle core (e.g. ionic electrostatic interactions with the gold core).
• the negatively charged surface of the gold core may attach to a positively charged outer surface (e.g. chitosan, chitosan-cyclodextrin) through ionic interactions.
• the outer layer includes a polymer.
• the polymer is Polyethylene glycol (PEG), polyethylenimine (PEI) or polyamidoamine (PAMAM).
• the polymer is Polyethylene glycol (PEG).
• the polymer is polyethylenimine (PEI).
• the polymer is polyamidoamine (PAMAM).
• the outer layer includes a cationic polysaccharide.
• the cationic polysaccharide includes chitosan.
• the cationic polysaccharide includes chitosan-cyclodextrin.
• the outer layer is a chitosan polysaccharide attached to the nanoparticle gold core by non-covalent interactions.
• the outer layer is a chitosan-cyclodextrin polysaccharide attached to the nanoparticle gold core by non-covalent interactions.
• the outer layer includes an amino acid including a primary amine group.
• the outer layer includes a thiol (-SH) group.
• the thiol group on the outer layer may be used to attach the outer layer to the nanoparticle core.
• the pulmonary viral protein or nucleic acid encoding the protein is covalently attached to the nanoparticle.
• a pulmonary viral protein or nucleic acid functionalized with a thiol may adsorb to the nanoparticle gold core.
• the pulmonary viral protein or nucleic acid is covalently attached to the outer layer of the nanoparticle.
• the nanoparticle outer layer may include a first reactive moiety that is able to form a covalent bond with a second reactive moiety on the viral protein or nucleic acid encoding said viral protein.
• a thiol group on the nanoparticle outer layer may react with a thiol group on the viral protein or nucleic acid to form a covalent disulfide bond.
• a nucleic acid or protein modified with a covalent reactive moiety may be covalently attached to the nanoparticle outer layer using a variety of conjugation methods described herein and known in the art.
• the pulmonary viral protein or nucleic acid is non-covalently attached to the nanoparticle.
• the pulmonary viral protein or nucleic acid may attach to the nanoparticle by non-covalent bonds (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), hydrophobic interactions).
• a functionalized or non-functionalized protein may attach to the nanoparticle gold core through hydrophobic interactions.
• the pulmonary viral protein or nucleic acid is non-covalently attached to the outer layer of the nanoparticle.
• a negatively charged nucleic acid may attach to a positively charged outer layer (e.g. chitosan) by non- covalent bonds (e.g. ionic electrostatic interactions).
• the nucleic acid is deoxyribonucleic acid. In embodiments, the nucleic acid is ribonucleic acid.
• the nucleic acid may be chemically modified, such as to increase stability and/or cell penetration.
• the nucleic acids provided herein may include one or more reactive moieties, e.g., a covalent reactive moiety.
• a reactive moiety may be attached to the nucleic acid using any appropriate linker, such as a polymer linker known in the art (e.g. a polyethylene glygcol linker or equivalent).
• the term “covalent reactive moiety” refers to a chemical moiety capable of chemically reaction with a second covalent reactive moiety (e.g.
• the pulmonary viral protein provided herein including embodiments thereof may be modified (e.g. to increase stability and/or cell penetration).
• the pulmonary viral protein may be modified to include reactive moieties (e.g. thiol groups, etc.) as a means to attach to the nanoparticle (e.g. nanoparticle core, nanoparticle outer layer).
• the complex provided herein includes a detectable moiety.
• the detectable moiety can be any known in the art and described herein.
• the detectable moiety is an enzyme, biotin, digoxigenin, a paramagnetic molecule, a contrast agent, gadolinium, a radioisotope, radionuclide, fluorodeoxyglucose, barium sulfate, thorium dioxide, gold, a fluorophore, a hapten, a protein, a fluorescent moiety, or a combination of two or more thereof.
• the contrast agent is a magnetic resonance imaging contrast agent, an X-ray contrast agent, or an iodinated contrast agent.
• the detectable agent is a fluorophore (e.g., fluorescein, rhodamine, coumarin, cyanine, or analogs thereof).
• the detectable agent is a chemiluminescent agent.
• the detectable agent is a radionuclide.
• the detectable agent is a radioisotope.
• the detectable agent is a paramagnetic molecule or a paramagnetic nanoparticle.
• the detectable moiety can be attached to the nanoparticle core, the outer layer of the nanoparticle, the pulmonary viral protein, or nucleic acid.
• the detectable moiety is an enzyme (e.g. a detectable protein (e.g.
• the complex includes a detectable protein (e.g. luciferase), wherein the protein is attached to the nanoparticle.
• the complex includes a nucleic acid (e.g. DNA, mRNA) encoding the detectable protein (e.g. luciferase), wherein the nucleic acid is attached to the nanoparticle.
• the complex provided herein including embodiments thereof may further be characterized by size.
• the size of the complex e.g. the nanoparticle (e.g. core, core and outer layer) and pulmonary viral protein or nucleic acid
• the size of the complex is the average diameter of the complex.
• the size of the complex is from about 20 nm to about 80 nm.
• the size of the complex is from about 25 nm to about 80 nm.
• the size of the complex is from about 30 nm to about 80 nm.
• the size of the complex is from about 35 nm to about 80 nm.
• the size of the complex is from about 40 nm to about 80 nm.
• the size of the complex is from about 45 nm to about 80 nm. In embodiments, the size of the complex is from about 50 nm to about 80 nm. In embodiments, the size of the complex is from about 55 nm to about 80 nm. In embodiments, the size of the complex is from about 60 nm to about 80 nm. In embodiments, the size of the complex is from about 65 nm to about 80 nm. In embodiments, the size of the complex is from about 70 nm to about 80 nm. In embodiments, the size of the complex is from about 75 nm to about 80 nm.
• the size of the complex is from about 20 nm to about 35 nm. In embodiments, the size of the complex is from about 20 nm to about 30 nm. In embodiments, the size of the complex is from about 20 nm to about 25 nm. In embodiments, the size of the complex is about 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 or 80 nm.
• the size of the complex is about 20 nm. In embodiments, the size of the complex is 20 nm. In embodiments, the size of the complex is about 25 nm. In embodiments, the size of the complex is 25 nm. In embodiments, the size of the complex is about 30 nm. In embodiments, the size of the complex is 30 nm. In embodiments, the size of the complex is about 35 nm. In embodiments, the size of the complex is 35 nm. In embodiments, the size of the complex is about 40 nm. In embodiments, the size of the complex is 40 nm. In embodiments, the size of the complex is about 45 nm. In embodiments, the size of the complex is 45 nm. In embodiments, the size of the complex is about 50 nm.
• the size of the complex is 50 nm. In embodiments, the size of the complex is about 55 nm. In embodiments, the size of the complex is 55 nm. In embodiments, the size of the complex is about 60 nm. In embodiments, the size of the complex is 60 nm. In embodiments, the size of the complex is about 65 nm. In embodiments, the size of the complex is 65 nm. In embodiments, the size of the complex is about 70 nm. In embodiments, the size of the complex is 70 nm. In embodiments, the size of the complex is about 75. In embodiments, the size of the complex is 75. In embodiments, the size of the complex is about 80 nm. In embodiments, the size of the complex is 80 nm.
• the size of the complex is from about 30 nm to about 50 nm. In embodiments, the size of the complex is from about 32 nm to about 50 nm. In embodiments, the size of the complex is from about 34 nm to about 50 nm. In embodiments, the size of the complex is from about 36 nm to about 50 nm. In embodiments, the size of the complex is from about 38 nm to about 50 nm. In embodiments, the size of the complex is from about 40 nm to about 50 nm. In embodiments, the size of the complex is from about 42 nm to about 50 nm. In embodiments, the size of the complex is from about 44 nm to about 50 nm. In embodiments, the size of the complex is from about 46 nm to about 50 nm. In embodiments, the size of the complex is from about 48 nm to about 50 nm.
• the size of the complex is from about 30 nm to about 48 nm. In embodiments, the size of the complex is from about 30 nm to about 46 nm. In embodiments, the size of the complex is from about 30 nm to about 44 nm. In embodiments, the size of the complex is from about 30 nm to about 42 nm. In embodiments, the size of the complex is from about 30 nm to about 40 nm. In embodiments, the size of the complex is from about 30 nm to about 38 nm. In embodiments, the size of the complex is from about 30 nm to about 36 nm. In embodiments, the size of the complex is from about 30 nm to about 34 nm.
• the size of the complex is from about 30 nm to about 32 nm. In embodiments, the size of the complex is about 30 nm, 32 nm, 34 nm, 38 nm, 40 nm, 42 nm, 44 nm, 48 nm, or 50 nm. In embodiments, the size of the complex is about 30 nm. In embodiments, the size of the complex is 30 nm. In embodiments, the size of the complex is about 32 nm. In embodiments, the size of the complex is 32 nm. In embodiments, the size of the complex is about 34 nm. In embodiments, the size of the complex is 34 nm. In embodiments, the size of the complex is about 36 nm.
• the size of the complex is 36 nm. In embodiments, the size of the complex is about 38 nm. In embodiments, the size of the complex is 38 nm. In embodiments, the size of the complex is about 40 nm. In embodiments, the size of the complex is 40 nm. In embodiments, the size of the complex is about 42 nm. In embodiments, the size of the complex is 42 nm. In embodiments, the size of the complex is about 44 nm. In embodiments, the size of the complex is 44 nm. In embodiments, the size of the complex is about 46 nm. In embodiments, the size of the complex is 46 nm. In embodiments, the size of the complex is about 48 nm. In embodiments, the size of the complex is 48 nm. In embodiments, the size of the complex is about 50 nm. In embodiments, the size of the complex is 50 nm. In embodiments, the size of the complex is 50 nm. In embodiments, the size of the complex is 50
• the size (e.g. average diameter) of the nanoparticle core is about 4 nm to about 30 nm. In embodiments, the size of the nanoparticle core is about 6 nm to about 30 nm. In embodiments, the size of the nanoparticle core is about 8 nm to about 30 nm. In embodiments, the size of the nanoparticle core is about 10 nm to about 30 nm. In embodiments, the size of the nanoparticle core is about 12 nm to about 30 nm. In embodiments, the size of the nanoparticle core is about 14 nm to about 30 nm. In embodiments, the size of the nanoparticle core is about 16 nm to about 30 nm.
• the size of the nanoparticle core is about 18 nm to about 30 nm. In embodiments, the size of the nanoparticle core is about 20 nm to about 30 nm. In embodiments, the size of the nanoparticle core is about 22 nm to about 30 nm. In embodiments, the size of the nanoparticle core is about 24 nm to about 30 nm. In embodiments, the size of the nanoparticle core is about 26 nm to about 30 nm. In embodiments, the size of the nanoparticle core is about 28 nm to about 30 nm.
• the size of the nanoparticle core is about 4 nm to about 12 nm. In embodiments, the size of the nanoparticle core is about 4 nm to about 10 nm. In embodiments, the size of the nanoparticle core is about 4 nm to about 8 nm. In embodiments, the size of the nanoparticle core is about 4 nm to about 6 nm. In embodiments, the size of the nanoparticle core is about 4 nm, 6 nm, 8 nm, 10 nm, 12, nm, 14 nm, 16 nm, 18 nm, 20 nm,
• the size of the nanoparticle core is about 12 nm. In embodiments, the size of the nanoparticle core is 12 nm. In embodiments, the size of the nanoparticle core is about 14 nm. In embodiments, the size of the nanoparticle core is 14 nm. In embodiments, the size of the nanoparticle core is about 16 nm. In embodiments, the size of the nanoparticle core is 16 nm. In embodiments, the size of the nanoparticle core is about 18 nm. In embodiments, the size of the nanoparticle core is 18 nm. In embodiments, the size of the nanoparticle core is about 20 nm. In embodiments, the size of the nanoparticle core is 20 nm.
• the size of the nanoparticle core is about 22 nm. In embodiments, the size of the nanoparticle core is 22 nm. In embodiments, the size of the nanoparticle core is about 24 nm. In embodiments, the size of the nanoparticle core is 26 nm. In embodiments, the size of the nanoparticle core is about 28 nm. In embodiments, the size of the nanoparticle core is 28 nm. In embodiments, the size of the nanoparticle core is about 30 nm. In embodiments, the size of the nanoparticle core is 30 nm.
• the vaccine composition further includes one or more of a stabilizer, an adjuvant, and a preservative.
• the vaccine composition includes a stabilizer.
• the vaccine composition includes an adjuvant.
• the vaccine composition includes a preservative.
• the complex is administered by an intranasal route. In embodiments, the complex is administered by an oro-nasal route. In embodiments, the complex is administered to the lungs.
• the composition including the complex described herein is used for a prophylactic purpose, especially in a subject who is considered predisposed of infection but presently does not have the viral disease.
• the prophylactic vaccine can be administered to the predisposed subject and prevent or reduce a likelihood of the occurrence of the viral disease in the subject.
• the composition has a therapeutic effect such that the composition can be used to treat a disease (e.g., a pulmonary vial disease) or condition.
• the composition can exhibit one or more anti-viral activity, e.g. reduction of viral particle number, reduction and/or inhibition of viral replication and infectivity.
• the composition including the complex provided herein can provide both therapeutic and prophylactic effects by delivering two separate pulmonary viral proteins or nucleic acids sequences encoding for said proteins in a single composition.
• the composition can (1) induce a more immediate treatment effect to the existing pulmonary viral infection or condition with the first immunogenic viral protein or fragment thereof, and (2) induce adaptive immunity in the subject with the second immunogenic viral protein or fragment thereof for future occurrence of a different pulmonary viral disease or condition.
• the composition includes a complex including two or more different pulmonary viral proteins or nucleic acids encoding the same wherein each protein independently exhibits a therapeutic or prophylactic effect, respectively.
• the composition provided herein including embodiments thereof are provided as a pulmonary pharmaceutical composition comprising a pulmonary pharmaceutical excipient.
• pulmonary pharmaceutical composition and the like refer to pharmaceutical compositions intended for pulmonary administration (e.g. intranasal route, oro-nasal route).
• pulmonary administration and the like refer, in the usual and customary sense, to administration to achieve inhalation therapy (e.g. intranasal route, oro-nasal route).
• inhalation therapy and the like refer to direct delivery of medications to the lungs by inhalation.
• the complexes provided herein including embodiments thereof are effective when delivered directly to the lung by an inhaled drug delivery system.
• pulmonary pharmaceutical liquid refers to a pulmonary pharmaceutical composition which is a liquid.
• pulmonary pharmaceutical solid “pulmonary pharmaceutical solid” and the like refer to a pulmonary pharmaceutical composition which is a solid (e.g., a powder).
• the dosage and frequency (single or multiple doses) administered to a subject can vary depending upon a variety of factors, for example, whether the subject suffers from another disease, its route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated, kind of concurrent treatment, complications from the disease being treated or other health- related problems.
• Other therapeutic regimens or agents can be used in conjunction with the methods and compositions described herein including embodiments thereof. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the ability of those skilled in the art.
• the composition is administered in a dose wherein about 2 ug to about 48 ug of nucleic acid is delivered to a subject. In embodiments, the composition is administered in a dose wherein about 2 ug to about 46 ug of nucleic acid is delivered to a subject. In embodiments, the composition is administered in a dose wherein about 2 ug to about 44 ug of nucleic acid is delivered to a subject. In embodiments, the composition is administered in a dose wherein about 2 ug to about 42 ug of nucleic acid is delivered to a subject. In embodiments, the composition is administered in a dose wherein about 2 ug to about 40 ug of nucleic acid is delivered to a subject.
• the composition is administered in a dose wherein about 2 ug, 4 ug, 6 ug, 8 ug, 10 ug, 12 ug, 14 ug, 16 ug, 18 ug, 20 ug, 22 ug, 24 ug, 26 ug, 28 ug, 30 ug, 32 ug, 34 ug, 36 ug, 38 ug, 40 ug, 42 ug, 44 ug, 46 ug, 48 ug or 50 ug of nucleic acid is delivered to a subject.
• the composition is administered in a dose wherein 2 ug of nucleic acid is delivered to a subject.
• the composition is administered in a dose wherein 40 ug of nucleic acid is delivered to a subject. In embodiments, the composition is administered in a dose wherein 42 ug of nucleic acid is delivered to a subject. In embodiments, the composition is administered in a dose wherein 44 ug of nucleic acid is delivered to a subject. In embodiments, the composition is administered in a dose wherein 46 ug of nucleic acid is delivered to a subject. In embodiments, the composition is administered in a dose wherein 48 ug of nucleic acid is delivered to a subject. In embodiments, the composition is administered in a dose wherein 50 ug of nucleic acid is delivered to a subject.
• a nanoparticle including a plurality of nucleic acids attached thereto and plurality of proteins atached thereto, wherein each of the plurality of nucleic acids encode for a different SARS-CoV-2 viral protein, and each of said plurality of proteins is a different SARS-CoV-2 viral protein.
• the nanoparticle is biocompatible.
• the nanoparticle includes a core, wherein said core includes gold.
• the core of the nanoparticle is a gold-iron oxide core.
• the nanoparticle includes a core and an outer layer, wherein said outer layer includes chitosan-cyclodextrin polymers.
• the plurality of nucleic acids and the plurality of proteins are atached to the outer layer of the nanoparticle.
• the one or more nucleic acids includes an RNA sequence encoding the SARS- CoV-2 viral proteins.
• the RNA sequence is an mRNA sequence.
• the SARS-CoV-2 viral proteins are selected from the group consisting of an S protein, N protein, M protein, and E protein.
• a vaccine formulation including a nanoparticle as provided herein including embodiments thereof, and a pharmaceutically acceptable excipient.
• the vaccine formulation further includes a vaccine excipient.
• the vaccine excipient is a stabilizer, an adjuvant or a preservative.
• the vaccine formulation includes a plurality of nanoparticles.
• a method of preventing or treating COVID-19 in a subject including administering a composition including an effective amount of a vaccine as provided herein including embodiments thereof, or a nanoparticle provided herein including embodiments thereof, to a subject in need thereof.
• delivery can be through intranasal or oro-nasal delivery.
• airway targeting of multiple mRNAs in one anti-SARS-CoV-2 vaccine is performed.
• provided herein are combined nano-biotechnological and theranostic strategy for vaccine development against COVID-19.
• provided hererin is an RNA vaccine against SARS-CoV-2 delivered directly into the airways via the IN or oro-nasal route.
• RNA multivalent vaccine produced by robust expression of mRNA encoding a plurality of SARS-CoV-2 surface antigens (e.g. S, N, M and E proteins) or fragments thereof and a plurality of surface antigens (e.g. S, N, M, and E proteins) or fragments thereof.
• SARS-CoV-2 surface antigens e.g. S, N, M and E proteins
• surface antigens e.g. S, N, M, and E proteins
• an engineered polyfunctional NP with multiple components tailored specifically for IN or oro-nasal administration and subsequent targeting of respiratory airway columnar ciliated cells (and their progenitor cells) expressing ACE2 receptors, to deliver synthetic mRNA sequences of ‘viral’ antigens, as well as a plurality of S, N, M, and E proteins.
• provided herein is an in vitro evaluation of the uptake and functional effects of SARS-CoV-2 antigens by measuring their expression, cell surface display, and antibody recognition after transfection in mammalian lung cells using PolyGION-CD-CS hybrid polymer NPs.
• provided herein is an evaluation in mice of an mRNA multivalent vaccine incorporating all four surface antigens of SARS-CoV- 2, as well as the S, N, M, and E proteins, using PolyGION-CD-CS hybrid polymer NPs via the IN or oro-nasal route to evaluate their in vivo immune response in mouse models.
• P Embodiment 3 The nanoparticle of P embodiment 2, wherein the core of the nanoparticle is a gold-iron oxide core.
• P Embodiment 4 The nanoparticle of P embodiment 1, wherein said nanoparticle comprises a core and an outer layer, where said outer layer comprises chitosan-cyclodextrin polymers.
• P Embodiment 6 The nanoparticle of P embodiment 5, wherein the one or more nucleic acids comprise an RNA sequence encoding said SARS-CoV-2 viral proteins.
• P Embodiment 7 The nanoparticle of P embodiment 5, wherein the SARS-CoV-2 viral proteins are selected from the group consisting of an S protein, N protein, M protein, and E protein.
• P Embodiment 8 A vaccine formulation comprising the nanoparticle of one of P embodiments 1-7 and a pharmaceutically acceptable excipient.
• P Embodiment 10 The vaccine formulation of P embodiment 9, wherein the vaccine excipient is a stabilizer, an adjuvant or a preservative.
• P Embodiment 13 The method of P embodiment 11, wherein the composition is administered via the oro-nasal route.
• P Embodiment 15 A method of preventing or treating a SARS-CoV-2 viral infection in a subject in need thereof, the method comprising administering a composition comprising an effective amount of the vaccine of one of P embodiments 8-10 or the nanoparticle of one of P embodiments 1-7 to a subject in need thereof.
• Embodiment 1 A complex comprising: (a) a nanoparticle comprising a gold core; and (b) a pulmonary viral protein or fragment thereof, or a nucleic acid encoding said pulmonary viral protein or fragment thereof, wherein said pulmonary viral protein or nucleic acid is attached to said nanoparticle.
• Embodiment 2 The complex of embodiment 1, comprising a plurality of pulmonary viral proteins or fragments thereof.
• Embodiment 3 The complex of embodiment 2, wherein said plurality of pulmonary viral proteins comprise different pulmonary viral proteins.
• Embodiment 4 The complex of embodiment 1, comprising a plurality of nucleic acids encoding said pulmonary viral protein or fragment thereof.
• Embodiment 6 The complex of any one of embodiments 1 to 5, wherein said pulmonary virus is human respiratory syncytial virus (HRSV), human parainfluenza virus (HPV) Human rhinovirus (HRV), Adenovirus (ADV), Human coronavirus (HCoV), Coronavirus associated with SARS (SARS-CoV), Human metapneumovirus (HMPV) or Human bocavirus (HBoV).
• HRSV human respiratory syncytial virus
• HPV Human rhinovirus
• ADV Human coronavirus
• HMPV Human metapneumovirus
• HMPV Human metapneumovirus
• HMPV Human metapneumovirus
• Embodiment 7 The complex of embodiment 6 wherein said pulmonary virus is SARS-CoV-2.
• Embodiment 8 The complex of embodiment 7, wherein said pulmonary viral protein or fragment thereof is S protein, N protein, M protein, or E protein.
• Embodiment 9 The complex of any one of embodiments 1 to 8, wherein said gold core is a gold-iron oxide core.
• Embodiment 11 The complex of embodiment 10, wherein said outer layer is covalently attached to said gold core.
• Embodiment 12 The complex of embodiment 10, wherein said outer layer is non- covalently attached to said gold core.
• Embodiment 13 The complex of any one of embodiments 10 to 12, wherein said outer layer comprises a polymer.
• Embodiment 14 The complex of any one of embodiments 10 to 13, wherein said outer layer comprises a cationic polysaccharide.
• Embodiment 15 The complex of embodiment 14, wherein said cationic polysaccharide comprises chitosan.
• Embodiment 16 The complex of embodiment 14, wherein said cationic polysaccharide comprises chitosan-cyclodextrin.
• Embodiment 17 The complex of any one of embodiments 1 to 16, wherein said pulmonary viral protein or nucleic acid is covalently attached to said nanoparticle.
• Embodiment 18 The complex of embodiment 17, wherein said pulmonary viral protein or nucleic acid is covalently attached to said outer layer of said nanoparticle.
• Embodiment 19 The complex of any one of embodiments 1 to 16, wherein said pulmonary viral protein or nucleic acid is non-covalently attached to said nanoparticle.
• Embodiment 20 The complex of embodiment 19, wherein said pulmonary viral protein or nucleic acid is non-covalently attached to said outer layer of said nanoparticle.
• Embodiment 21 The complex of any one of embodiments 1 to 20, wherein said nucleic acid is deoxyribonucleic acid.
• Embodiment 22 The complex of any one of embodiments 1 to 20, wherein said nucleic acid is ribonucleic acid.
• Embodiment 23 The complex of any one of embodiments 1 to 22, wherein said complex is from about 20 nm to about 80 nm in diameter.
• Embodiment 24 The complex of any one of embodiments 1 to 23, wherein said complex is about 40 nm in diameter.
• Embodiment 25 A vaccine composition comprising the complex of any one of embodiments 1 to 24 and a pharmaceutically acceptable excipient.
• Embodiment 26 The vaccine composition of embodiment 25, further comprising one or more of a stabilizer, an adjuvant, and a preservative.
• Embodiment 27 The vaccine composition of embodiment 25 or 26, wherein said composition is formulated for nasal administration.
• Embodiment 28 A method of treating or preventing a pulmonary viral disease in a subject in need of such treatment or prevention, said method comprising administering a therapeutically or prophylactically effective amount of the complex of any one of embodiments 1 to 24 to said subject.
• Embodiment 29 The method of embodiment 28, wherein the complex is administered by an intranasal route.
• Embodiment 30 The method of embodiment 28, wherein the complex is administered by an oro-nasal route.
• Embodiment 31 The method of embodiment 28, wherein the complex is administered to the lungs.
• Embodiment 32 A method of treating or preventing a pulmonary viral disease in a subject in need of such treatment or prevention, said method comprising administering a therapeutically or prophylactically effective amount of the vaccine composition of any one of embodiments 25 to 27 to said subject.
• Embodiment 33 The method of embodiment 32, wherein the composition is administered via the intra-nasal route.
• Embodiment 34 The method of embodiment 32, wherein the composition is administered via the oro-nasal route.
• Embodiment 35 The method of embodiment 32, wherein the composition is administered to the lungs.
• Embodiment 36 A method for immunizing a subject susceptible to a pulmonary viral disease, the method comprising administering the complex of any one of embodiments 1 to 24 to said subject, under conditions such that antibodies that bind to said pulmonary viral protein or fragment thereof are produced.
• Embodiment 37 The method of embodiment 36, wherein said antibodies are IgG, IgA or IgM antibodies.
• Nucleic acid vaccines are safe and easy to develop because their production involves making genetic material only, and not the virus itself.
• Anti-COVID-19 nucleic acid vaccines entail delivery of specific portions of viral RNA that code for individual proteins or protein fragments, into human cells, which then produce copies of the viral proteins against the delivered RNA.
• the mRNA vaccines are capable of inducing antigen-specific T- and B- cell responses. 17 18 By April 30, 2020, at least 20 teams were studying the use of SARS-CoV- 2 DNA or RNA to prompt an immune response.
• RNA vaccine When placed in contrast to traditional or DNA vaccines, an RNA vaccine has several important benefits: (1) An RNA vaccine is not made with pathogen particles, so they are non-infectious, and the messenger RNA (mRNA) strand is rapidly degraded once the protein is made. (2) Unlike pDNA that relies on cell and nuclear membrane poration to reach the nucleus for transcription and further translation into proteins, it is sufficient for the RNA strand to gain access to the cytosol for translation. (3) DNA expression cassettes carry the theoretical risks of genome integration, insertional mutagenesis, long-term expression, and the induction of anti-DNA antibodies. (4) Some of the early clinical trial results indicate that an RNA vaccine can generate a reliable immune response and is well-tolerated by healthy individuals, with negligible side effects.
• nucleic acid vaccines encode the S protein alone.
• a multivalent (multi-antigen) vaccine strategy for the robust expression of mRNA encoding two or more surface antigens (e.g. S, N, M and E proteins) of SARS- CoV-2.
• SARS-CoV-2 possesses significant homology with other coronaviruses, it also possesses substantial variations in some antigens. These variations are clearly reflected in SARS-CoV-2 by differences in the mode of its infection, pathogenicity, spread, and severity of the disease.
• prediction-based approaches of possible viral protein(s) for designing epitope vaccines may not be promising owing to their poor success rate.
• a multivalent mRNA vaccine expressing two or more surface antigens provides better success.
• vaccines that can be therapeutic for infected individuals (e.g. by inducing immune antibodies while preventing further infection by blocking ACE2 receptors) and/or prophylactic for uninfected individuals.
• Engineering mRNA sequences has rendered synthetic mRNAs more translatable than ever before — the in vivo half-life of mRNA can be regulated through use of various base modifications and delivery methods.
• efficient in vivo delivery is achieved by formulating mRNA into/onto carrier vehicles, allowing rapid uptake and expression in host cell cytoplasm.
• intranasal (IN) delivery is combined with a novel nanocarrier for a multivalent mRNA vaccine strategy against COVID-19 in the clinical setting.
• RNA vaccines via the IN route to express antigens against SARS-CoV-2.
• the use of an efficient delivery system for IN delivery can dramatically reduce the doses needed to generate potent immune responses, without an additional conventional adjuvant.
• nanoformulations carrying synthetic mRNAs encoding two or more of SARS-CoV-2 S, N are described herein.
• M, and E proteins or fragments thereof as independent transcripts, or two or more of S, N, M, and E proteins or fragments thereof.
• Loading mRNA and proteins on a biocompatible nanoparticle (NP) and coupling this with a clinically practical delivery method provides an easy, safe, minimally invasive, and tissue-specific method for successful expression of a multivalent SARS-CoV-2 vaccine for immunization against COVID-19.
• GBM glioblastoma
• This nanoformulation includes a 50 nm polyfunctional gold-iron oxide NP (termed polyGION) used to deliver therapeutic microRNAs to mouse GBMs via IN delivery.
• PolyGIONs surface functionalized with cyclodextrin-chitosan (CD-CS) hybrid polymers provide an efficient platform for surface loading of negatively charged RNAs through electrostatic interactions. 21 Moreover, to be of value as therapeutic agents, the targeted delivery of polyGIONs, and visualization of their trafficking would be essential in pre-clinical studies, at least.
• the non-invasive respiratory mucosal targeted delivery of viral RNAs specific to each antigen using our polyGION-CD-CS nanoformulation provides a new anti-SARS-CoV-2 multivalent vaccine to directly target the airways and lungs — precisely the main initial target organs for COVID-19 disease, to activate pulmonary immune responses.
• a similar approach using recombinant adenovirus-based vaccine expressing S protein of MERS-CoV was found to induce significant immune responses when administered IN to BALB/c mice. 12
• gold NPs are non-toxic. 22 24 .
• the iron oxide component from the polyGION-CD-CS nanoformulation can be removed.
• the advantages of the IN and respiratory airway route include avoidance of circulating blood, reduced systemic side effects and hepatic/renal clearance, creating airway and lung resident memory T cell responses, and the possibility of practical repeated or chronic vaccine administration.
• its non-invasiveness, painless and convenient administration to individuals as a nasal spray or inhaler with high compliance, and rapid onset of action provide novel features for an anti-SARS-CoV-2 vaccine.
• PolvGION-CD-CS NPs serve as a biocompatible non-toxic platform for IN delivery of therapeutics.
• FLuc-mRNA delivery facilitates monitoring delivery, stability, and expression of delivered RNA in lungs by using bioluminescence imaging (BLI).
• BLI bioluminescence imaging
• the PolyGION-CD-CS-FLuc-mRNA complex was investigated for IN delivery to mice. 5 pi of NP complex was administered 4 times in each nostril (a total of 20 m ⁇ for each dose; 2 pg of mRNA equivalent). The NP complex was delivered in awake mice. BLI was obtained at 24 hr time points after delivery, and dosage administration was continued each day. After 6 days, mice were BLI imaged in vivo and ex vivo after sacrifice. The mice showed strong BLI signals in the trachea after the first dose, and strong signals in the lungs 48 hr later. The signals in the lungs were strikingly strong after five doses (FIG. 3). Pre-sacrifice BLI showed localized signals in the lungs (FIG. 4B).
• Example 5 Gold-Nanostar-Chitosan Mediated Delivery of a SARS-CoV-2 DNA Vaccine for Respiratory Mucosal Immunization
• intranasal delivery of vaccine is preferred for respiratory infections to achieve both humoral and innate immune responses, while also producing sterilizing immunity in the respiratory tract and lungs.
• IN delivery requires a nanocarrier that can transport the loaded nucleic acid vaccine across the nasal cavity and down into lungs.
• An efficient nanoparticle (NP) delivery system is also key to mount an effective DNA/RNA vaccine immune response. Any ideal delivery system needs to demonstrate a combination of high loading capacity, stability, and biocompatibility. In that respect, apart from liposomes, a cationic polysaccharide and natural biopolymer, such as chitosan, has been used as an adjuvant in vaccine delivery systems.
• S protein mediates viral transduction via interaction with angiotensin-converting enzyme 2 (ACE2) receptors followed by endocytosis.
• ACE2 angiotensin-converting enzyme 2
• vaccines based on the S protein could induce antibodies to block virus binding and fusion with respiratory airway columnar ciliated cells (and their progenitor cells) expressing ACE2 receptors, or neutralize the virus infection. 22
• the S protein appears to be the main immunogenic protein to induce both cellular and humoral immunity against virus infection.
• the pristine AuNS had a surface potential of -5.6 mV ( ⁇ 2.89 mV) that shifted to a cationic surface potential of ⁇ 35.8 mV ( ⁇ 3.59 mV) upon capping with cationic chitosan polymer.
• pDNA the coding sequence of SC2 S protein
• Intranasal vaccination boosts cross-variant humoral immune response against mutant variants of SC2.
• a cell-mediated immune response plays a critical role in combating viral infections. 41 It is comprised of T cell responses that fundamentally differ from antibody (humoral) responses in that they result in infection control. Cell-mediated immunity is primarily driven by mature T cells, macrophages, DCs, NK cells, and the released cytokines, in response to antigen delivery. 42 In order to deduce the role of cell-mediated immunity after IN DNA vaccination, we performed immunophenotyping of leukocytes collected from lungs, spleen, thymus, and lymph nodes of mice delivered using control DNA and DNA coding for S protein of SC2 using AuNS-chitosan NPs.
• MZ B cells are the major constituent of the marginal zone, together with myeloid, dendritic, and stromal cells. These MZ B cells are the main producers of IgM antibodies against S antigen. The enrichment and mobilization of MZ B cells indicate the characteristics of splenic immune response.
• the subcapsular sinus macrophages direct target specific immune responses to a variety of lymph-borne pathogens by relaying antigens to B cells, producing cytokine signaling cascades to cause influx of DCs, neutrophils, NK cells, or in some conditions, presenting antigens to T cells, and SC2 vaccinated mice showed presence of such subcapsular sinus macrophages and DCs (data not shown).
• Activation of B cells is often triggered by binding of S antigen to the B cell receptor, which can occur through B cell recognition of S antigens captured on cell membranes of APC in the lymph nodes.
• the nodes are also where cytotoxic T cells are trained with S antigen presented by professional APCs, especially the DCs. 65
• Our FACS analysis results indicated this modulation of CD4+ T cells in S vaccinated mice compared to that of pDNA vaccinated mice (FIGS. 16B-16E).
• the histology of lungs from vaccinated mice indicated the successful delivery and expression of S antigen and also captured the interaction of DCs with these S expressing cells (data not shown).
• lymph nodes from C57BL/6J-DR mice vaccinated using SC2-spike indicated a characteristic feature of antigen activation.
• T and B cells were compartmentalized into specific locations, with T cells residing primarily in the deeper paracortex of nodes, while B cells were in the follicles (data not shown).
• S antigen carried to nodes by lymph or lymphatic-migrating APCs arrive at the subcapsular sinus of the draining lymph node.
• B cells that receive initial signaling by binding to S antigens enter specialized subregions of the follicles (the GCs). Such dynamic mobilization of B cells was evident in the vaccinated mice when compared to control mice (data not shown).
• GCs develop in the B cell follicles of secondary lymphoid tissues during T cell -dependent antibody responses.
• the B cells that give rise to GCs initially have to be activated outside the follicles, i.e., in the T cell rich zones in association with interdigitating cells and T helper cell.
• FDCs Follicular DCs
• FDCs Follicular DCs
• B cells can acquire antigens from FDCs that are processed and presented on class II MHC molecules.
• the activated B cells can exit the GC to become short-lived antibody producing cells termed plasmablasts.
• the results shown from histology staining illustrate the migration of B cells from the GC into the light zone as evidence for the movement of antibody producing plasmablasts in response to the SC2 DNA vaccine.
• the amount of antigen accumulated in lymph nodes directly correlates with the number of T follicular helper cells and GC B cells that develop in immunized lymph nodes. 72
• the FDCs are located along with B cells in the follicles of any secondary lymphoid organs.
• FDCs have very important functions regarding the generation and the selection of high affinity plasmacytes, i.e., memory B lymphocytes, during the adaptive immune response.
• One key property of FDCs is their ability to trap and display antigens as immune complexes in a highly stimulatory way to proliferating B cells.
• lymph nodes there is a network of stromal cells that includes the FDCs.
• FDCs were first identified as “antigen retaining reticular cells”.
• FDCs have been recognized for their unique ability to retain antigens for prolonged periods. This property of FDCs is critical to several immune functions, including GC formation and long-term immune memory.
• IFNy has a critical role in recognizing and eliminating pathogens, it has been identified as a prognostic marker for vaccine response.
• Type I IFNs are pleiotropic antiviral cytokines that can affect nearly every step of the immune response to SC2 vaccination, ranging from S protein expression, DC activation, to T cell differentiation. Unsurprisingly, type I IFNs have been found to be central mediators of T and B cell responses to SC2 vaccines. We also noticed an increased expression of INFy by T cells in the lymph nodes and blood (FIGS. 17A-17E). 78 The increase in splenic CD8+ T cells expressing IFNy, which is a signature cytokine of both innate and adaptive immune systems, was evident only in SC2 vaccinated mice but not the pDNA-control vector treated mice.
• AuNS-chitosan showed a robust delivery of FLuc mRNA in the lungs of mice upon IN delivery as measured using bioluminescence imaging.
• the antibody response results in high levels of IgG and IgA, which show a strong neutralizing effect against pseudoviruses expressing different spike variants of SC2 (Wuhan, D614G and SA mutant).
• Additional evaluation using immunostaining-based FACS and confocal microscopy for cell-mediated immune response shows an effective activation of T and B cell responses in the lungs and lymph nodes, which are similar to immune responses normally observed against infectious diseases.
• Our findings highlight the merits of using AuNS-chitosan as an efficient in vitro and in vivo nanoformulation to deliver DNA and synthetic mRNA, and also its role in stabilizing nucleic acids for functional in vivo transfection for future mRNA vaccine development and applications.
• chitosan dissolved in 0.2% acetic acid was microfluidized using a LV1- microfluidics system (Microfluidics, Westwood, MA) at 30,000 psi.
• a LV1- microfluidics system Microfluidics, Westwood, MA
• the complexes were loaded on 0.7% agarose gel and the electrophoresis run at 40 V for 45 min. After the run, the gel was imaged in BioRad Gel Doc XR+ Gel Documentation system (Bio Rad, Hercules, CA, USA) to further quantify and analyze the extent of DNA encapsulation.
• the optimized SC2 plasmid or pcDNA loaded AuNS-chitosan was adapted for subsequent in vitro and in vivo studies.
• the plasmid DNA loaded complexes were administered in 20 pL dosages at each time point of study.
• mice were purchased 6-8 weeks old BALB/c female mice, from Charles River Laboratories (Wilmington, MA); and C57BL/6J mice, as well as mice carrying the Ccr2 RFP Cx3crl GFP dual-reporter from the Jackson Laboratory (Bar Harbor, ME). Mice were maintained under specific pathogen-free conditions. We performed all animal experiments under the guidance of the Administrative Panel on Laboratory Animal Care (APLAC) of our university. We immunized mice IN with 10 mg of SC2 DNA or pcDNA vaccine in solution. We performed the IN delivery of the NP formulation in mice under mild sedation, using isoflurane gas anesthesia, to enable animal recovery within a couple of minutes.
• APIAC Administrative Panel on Laboratory Animal Care
• Virus neutralization by antibodies is an important prognostic factor in many viral diseases.
• SC2 spike D614G pseudotyped lentiviruses were produced using SC2 Spike (Genbank Accession #QHD43416.1; with D614G mutation) as the envelope glycoproteins instead of the commonly used VSV-G.
• SC2 Spike Genebank Accession #QHD43416.1; with D614G mutation
• These pseudo virions contain the FLuc gene driven by a CMV promoter; therefore, the spike-mediated cell entry can be conveniently determined via FLuc imaging.
• the SC2 Spike D614G pseudotyped lentivirus can be used to measure the activity of neutralizing antibody against SC2 in a Biosafety Level 2 facility.
• B.1.351 was first identified in the fall of 2020 in the Republic of South Africa. This South African variant, also known as 501Y.V2, has many mutations which may lead to higher transmissibility and infectivity.
• the Spike (B.1.351 Variant) (SC2) pseudotyped lentiviruses were produced using SC2 B.1.351 Variant Spike (Genbank Accession #QHD43416.1 with B.1.351 mutations; as the envelope glycoproteins instead of the commonly used VSV-G.
• the membrane was blocked with 5% non-fat dry milk in tris-buffered saline containing 0.01% Tween-20 (TBS-T, pH 7.6) for 30 min and incubated with the anti-rabbit SPK monoclonal antibody overnight at 4 °C on a rocking platform.
• TBS-T tris-buffered saline containing 0.01% Tween-20
• the blots were developed with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, USA) and imaged and quantified with the IVIS Lumina III In- Vivo Imaging System.
• the tissues were blocked using 1% bovine serum albumin in PBS for 30 min at room temperature and then incubated with antigen specific anti-mouse fluorophore tagged antibody (CD4- FITC, CD8-Alexa-700, CD19 Alexa-700) and incubated overnight at 2-8 °C. After the incubation time, we washed the antibodies three times for 15 minutes in wash buffer. We then incubated the slides in 300 pL of the diluted solution of Hoechst 33342 and incubated for 5 min at room temperature. The slide were finally rinsed once with PBS and mounted with an anti-fade mounting media and visualized using a Leica DMi8 confocal microscope under respective filters.
• Flow cytometry immunophenotyping We performed cell surface marker based immune cell analysis using flow cytometry for lungs, spleen, lymph nodes, thymus, and blood samples. Briefly, we prepared single-cell suspensions from tissues using mechanical dissociation, and red blood cells were removed using ACK lysing buffer. After the final wash, we filtered cells through a 70-pm cell strainer and viability was checked using 0.1% trypan blue.
• cell surface marker specific anti-mouse antibody labelled with fluorochrome i.e., CD45-Pac-Blue, CD3/CD4/CD8 PE- CY7/FITC/Alexa-700, CD45/CDllb PacBlue/APC-Cy7, CD45/CDllc PacBlue/PE-Cy7, CD45/CD86 Pac Blue / PE, CD 19 Alexa-700, CD22 Alexa-700 (Biolegend).
• Isotype antibodies were included for gating and compensation. Following addition of antibodies, we kept cells in the dark for 30 min. We washed cells using PBS and suspended in fresh PBS, then analyzed for 20,000 events using a Guava® easyCyteTM Flow Cytometer.
• Table 1 Complex size and surface characteristics. Size is shown in diameter. For complexes without outer layer and/or nucleic acid, size is shown for the nanoparticle core.
• Table 2 Viruses and antigens targeted by the complexes provided herein.
• Mycobacterium bovis BCG vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis. Infect Immun 2004, 72 (1), 238-46.
• Nanocarriers targeting dendritic cells for pulmonary vaccine delivery Pharm Res 2013, 30 (2), 325-41.
• Follicular dendritic cells help establish follicle identity and promote B cell retention in germinal centers. J Exp Med 2011, 208 (12), 2497-510.

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