WO2024073552A2 - Combination exosomal immunogenic compositions and methods - Google Patents

Combination exosomal immunogenic compositions and methods Download PDF

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Publication number
WO2024073552A2
WO2024073552A2 PCT/US2023/075343 US2023075343W WO2024073552A2 WO 2024073552 A2 WO2024073552 A2 WO 2024073552A2 US 2023075343 W US2023075343 W US 2023075343W WO 2024073552 A2 WO2024073552 A2 WO 2024073552A2
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polypeptide
protein
fusion protein
seq
cell
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PCT/US2023/075343
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French (fr)
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WO2024073552A3 (en
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Minghao SUN
Kristi ELLIOTT
Li-En HSIEH
Mafalda CACCIOTTOLO
Michael LECLAIRE
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Capricor, Inc.
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Publication of WO2024073552A2 publication Critical patent/WO2024073552A2/en
Publication of WO2024073552A3 publication Critical patent/WO2024073552A3/en

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    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16034—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—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
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • SARS-CoV-2 acute respiratory syndrome coronavirus 2
  • influenza influenza
  • RSV respiratory syncytial virus
  • SARS-CoV-2 many vaccines have been developed, which try to achieve immunity to the virus, mainly directing the immune response to surface spike protein, which binds to the host cell receptor angiotensin-converting enzyme 2 (ACE2), mediating viral cell entry and infection.
  • ACE2 angiotensin-converting enzyme 2
  • the spike (S) protein is a class I fusion glycoprotein, the major surface protein on the SARS-CoV-2 virus and the primary target for neutralizing antibodies. Spike is also the primary site of mutations identified in SARS-CoV-2 viruses, reducing the efficacy of the immune-response induced by vaccines. Since the beginning of the CO VID-19 pandemic, five variants of concern (VOC) and 8 variants of interest (VOI) have been reported, which poses challenges to current vaccines and creating the need for more effective vaccines. Vaccines directed against SARS-CoV-2 nucleocapsid protein, which is an internal soluble coronavirus protein, have heretofore been largely unsuccessful.
  • a problem to which the present invention may be directed includes the production of SARS-CoV-2 variants of concern having more infectivity and less immunogenicity than some variants of lesser concern. This creates an ongoing public health and medical problem in which vaccines are no longer effective against new strains, and new strains that are less immunogenic (long term and short-term immunogenicity).
  • a safe (LNP-free, adjuvant-free) and effective combination vaccine that confers long term humoral and cell-mediated immunity against multiple, emerging, and recalcitrant variants of SARS-CoV-2, influenza, and/or RSV which includes an extracellular vesicle that displays a spike protein or nucleocapsid protein from a single variant of SARS-CoV-2, which confers robust humoral and/or cellular immunity against several SARS-CoV-2 variants of concern or interest, combined with an extracellular vesicle that displays a hemagglutinin protein from a single variant of influenza, which also confers immunity against an influenza variant of interest, and or combined with an extracellular vesicle that displays an RSV protein from a single variant of RSV, which also confers immunity against an RSV variant of interest.
  • an immunogenic composition contains a combination of two or more vesicle-expressing virus antigen substances.
  • the immunogenic composition contains (i) a plurality of vesicles that express on their surface a SARS-CoV-2 protein polypeptide or antigen, and (ii) a plurality of vesicles that express on their surface an influenza protein polypeptide or antigen.
  • an immunogenic composition contains a combination of two or more vesicle-expressing virus antigen substances.
  • the immunogenic composition contains (i) a plurality of vesicles that express on their surface a SARS-CoV-2 protein polypeptide or antigen, and (ii) a plurality of vesicles that express on their surface an RSV protein polypeptide or antigen.
  • an immunogenic composition contains a combination of two or more vesicle-expressing virus antigen substances.
  • the immunogenic composition contains (i) a plurality of vesicles that express on their surface an influenza protein polypeptide or antigen, and (ii) a plurality of vesicles that express on their surface an RSV protein polypeptide or antigen.
  • an immunogenic composition contains a combination of two or more vesicle-expressing virus antigen substances.
  • the immunogenic composition contains (i) a plurality of vesicles that express on their surface a SARS-CoV-2 protein polypeptide or antigen, (ii) a plurality of vesicles that express on their surface an influenza protein polypeptide or antigen, and (iii) a plurality of vesicles that express on their surface an RSV protein polypeptide or antigen.
  • the SARS-CoV-2 protein is a spike glycoprotein, such as, for example, a SARS-CoV-2 delta spike protein or engineered variant thereof.
  • the SARS-CoV-2 protein is a nucleocapsid protein or engineered variant thereof.
  • the influenza protein is a hemagglutinin protein or engineered variant of a hemagglutinin protein, such as, for example, an H3 glycoprotein or engineered variant thereof.
  • the influenza protein is a neuraminidase protein or engineered variant thereof.
  • the RSV protein is an RSV fusion (F) protein, such as, for example, RSV F protein or prefusion F protein (see, e.g., Simoes et al., N Engl J Med 2022; 386: 1615-1626).
  • the vesicle is an exosome.
  • virus protein polypeptide or antigen is fused to an exosomal protein, such as, for example, a tetraspanin, or more specifically, a CD9 protein or engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.
  • exosomal protein such as, for example, a tetraspanin, or more specifically, a CD9 protein or engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.
  • the immunogenic composition contains a plurality of vesicles containing the synthetic fusion protein at a concentration of about 2E9 vesicles/mL - 3E13 vesicles/mL.
  • each vesicle type may be provided at a concentration of about 1E9 vesicles/mL - 2E13 vesicles/mL or 2E9 vesicles/mL - 3E13 vesicles/mL.
  • the immunogenic composition contains a plurality of vesicles containing the synthetic fusion protein at a concentration of about 0.3 ng/mL - 3 pg/mL of the synthetic fusion protein.
  • each vesicle type may be provided at a fusion protein concentration of about 0.1 ng/mL - 1.5 pg/mL or about 0.3 ng/mL - 3 pg/mL.
  • the immunogenic composition further contains one or more pharmaceutically acceptable excipients. In one embodiment, the immunogenic composition does not contain an adjuvant.
  • a vesicle of the plurality of vesicles of the immunogenic composition has an average diameter of about 50-500 nm.
  • the vesicle is a synthetic vesicle.
  • the vesicle is produced by a cell.
  • the vesicle is an extracellular vesicle.
  • the vesicle is a microvesicle.
  • the vesicle is an exosome.
  • the vesicle is an apoptotic body.
  • the vesicle expresses a CD81 protein on its surface.
  • a method of eliciting an immune response in a subject is provided by administering to the subject a dose of an immunogenic composition described in the preceding aspects and embodiments.
  • the subject is administered more than one dose, such as a second dose administered a period of time after the first dose, and/or subsequent booster doses.
  • the period of time e.g., between doses is 14 days - 1 year.
  • a dose contains about 100 pL - 1 mL of the immunogenic composition.
  • the immunogenic composition contains about 0.1 ng/mL - 3 pg/mL of synthetic fusion protein in a form expressed on the surface of vesicles.
  • the immunogenic composition contains about 2.81E9 vesicles/mL - 2.81E13 vesicles/mL that express synthetic fusion protein on their surfaces.
  • the immune response that is elicited in the subject is the production of neutralizing antibodies against a virus, such as SARS-CoV-2, influenza, RSV, and the like.
  • the immune response that is elicited in the subject is the production of neutralizing antibodies against two or more SARS-CoV-2 variants, such as for example a delta variant, an omicron variant, or other variant of interest or concern now known or yet to be discovered.
  • the immune response that is elicited in the subject is the production of anti-spike antibodies. In some embodiments, the immune response that is elicited in the subject is the production of anti-nucleocapsid antibodies. In some embodiments, the immune response that is elicited in the subject is a spike-specific T cell response, such as CD4+ and/or CD8+ response. In some embodiments, the immune response that is elicited in the subject is a nucleocapsid-specific T cell response, such as CD4+ and/or CD8+ response. [0021 ] In some embodiments, the immune response that is elicited in the subject is the production of anti-hemagglutinin antibodies.
  • the immune response that is elicited in the subject is a spike-specific T cell response, such as CD4+ and/or CD8+ response. In some embodiments, the immune response that is elicited in the subject is a hemagglutininspecific T cell response, such as CD4+ and/or CD8+ response.
  • the immune response that is elicited in the subject is the production of anti-RSV F antibodies.
  • the immune response that is elicited in the subject is a spike-specific T cell response, such as CD4+ and/or CD8+ response.
  • the immune response that is elicited in the subject is a RSV F-specific T cell response, such as CD4+ and/or CD8+ response.
  • a method for vaccinating a subject against influenza and SARS-CoV-2 is provided.
  • a subject is administered a composition containing (i) a plurality of vesicles that express on their surface a SARS-CoV-2 protein polypeptide or antigen, and (ii) a plurality of vesicles that express on their surface an influenza protein polypeptide or antigen.
  • the SARS-CoV-2 protein is a spike glycoprotein, such as, for example, a SARS-CoV-2 delta variant spike protein or an engineered variant thereof.
  • the SARS-CoV-2 protein is a nucleocapsid protein or an engineered variant thereof.
  • influenza protein is a hemagglutinin protein, such as, for example, an H3 glycoprotein or an engineered variant thereof.
  • influenza protein is a neuraminidase protein or an engineered variant thereof.
  • the vesicle is an exosome.
  • virus protein polypeptide or antigen is fused to an exosomal protein, such as, for example, a tetraspanin, or more specifically, a CD9 protein or engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.
  • a method for vaccinating a subject against RSV and SARS-CoV-2 is provided.
  • a subject is administered a composition containing (i) a plurality of vesicles that express on their surface a SARS-CoV-2 protein polypeptide or antigen, and (ii) a plurality of vesicles that express on their surface an RSV protein polypeptide or antigen.
  • the SARS-CoV-2 protein is a spike glycoprotein, such as, for example, a SARS- CoV-2 delta variant spike protein or an engineered variant thereof.
  • the SARS-CoV-2 protein is a nucleocapsid protein or an engineered variant thereof.
  • the RSV protein is a F-protein or prefusion F-protein or an engineered variant thereof.
  • the vesicle is an exosome.
  • the virus protein polypeptide or antigen is fused to an exosomal protein, such as, for example, a tetraspanin, or more specifically, a CD9 protein or engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.
  • a method for vaccinating a subject against influenza and RSV is provided.
  • a subject is administered a composition containing (i) a plurality of vesicles that express on their surface an RSV protein polypeptide or antigen, and (ii) a plurality of vesicles that express on their surface an influenza protein polypeptide or antigen.
  • the RSV protein is a F-protein or prefusion F-protein or an engineered variant thereof.
  • the influenza protein is a hemagglutinin protein, such as, for example, an H3 glycoprotein or an engineered variant thereof.
  • the influenza protein is a neuraminidase protein or an engineered variant thereof.
  • the vesicle is an exosome.
  • the virus protein polypeptide or antigen is fused to an exosomal protein, such as, for example, a tetraspanin, or more specifically, a CD9 protein or engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.
  • a method for vaccinating a subject against influenza and RSV is provided.
  • a subject is administered a composition containing (i) a plurality of vesicles that express on their surface a SARS-CoV-2 protein polypeptide or antigen, (ii) a plurality of vesicles that express on their surface an influenza protein polypeptide or antigen, and (iii) a plurality of vesicles that express on their surface an RSV protein polypeptide or antigen.
  • the SARS-CoV-2 protein is a spike glycoprotein, such as, for example, a SARS-CoV-2 delta variant spike protein or an engineered variant thereof.
  • the SARS-CoV-2 protein is a nucleocapsid protein or an engineered variant thereof.
  • influenza protein is a hemagglutinin protein, such as, for example, an H3 glycoprotein or an engineered variant thereof.
  • influenza protein is a neuraminidase protein or an engineered variant thereof.
  • the RSV protein is a F-protein or prefusion F-protein or an engineered variant thereof.
  • the vesicle is an exosome.
  • the virus protein polypeptide or antigen is fused to an exosomal protein, such as, for example, a tetraspanin, or more specifically, a CD9 protein or engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.
  • an exosomal protein such as, for example, a tetraspanin, or more specifically, a CD9 protein or engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.
  • a synthetic fusion protein contains polypeptide sequences of a SARS-CoV-2 protein fused to polypeptide sequences of an exosomal tetraspanin protein.
  • the fusion protein is designed and made such that a SARS-CoV-2 protein antigen can be expressed on the surface of an exosome to enable the eliciting of an immune response when administered to a subject.
  • the SARS-CoV-2 protein is a spike protein or an engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is a nucleocapsid protein or an engineered variant thereof.
  • the spike protein polypeptide is positioned N- terminal to the CD9 protein polypeptide.
  • a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide, as depicted in Fig. 1A.
  • the SARS-CoV-2 protein is a nucleocapsid protein and the exosomal tetraspanin protein is a CD9 protein
  • the nucleocapsid protein polypeptide is positioned N-terminal to the CD9 protein polypeptide.
  • a signal peptide is positioned at the N-terminal end of the nucleocapsid protein polypeptide.
  • a hinge peptide, a transmembrane domain peptide, and a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide, as depicted in Fig. 2A.
  • a synthetic fusion protein contains polypeptide sequences of a SARS-CoV-2 protein fused to polypeptide sequences of an exosomal tetraspanin protein.
  • the fusion protein is designed and made such that a SARS-CoV-2 protein antigen can be expressed on the surface of an exosome to enable the eliciting of an immune response when administered to a subject.
  • the SARS-CoV-2 protein is a spike protein or an engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is a nucleocapsid protein or an engineered variant thereof.
  • the SARS-CoV-2 protein is a spike protein (e.g., SEQ ID NO:1) and the exosomal tetraspanin protein is a CD9 protein (SEQ ID NO: 10)
  • the spike protein polypeptide is positioned N-terminal to the CD9 protein polypeptide.
  • a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO:2), as depicted in Fig. 1A.
  • the SARS-CoV-2 spike protein polypeptide contains one or more mutations, such as, e.g., a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and/or a di-proline substitution (2P [986KV987-to-986PP987]).
  • CSM furin cleavage site mutation
  • 2P 986KV987-to-986PP987
  • the spike-containing fusion protein has an amino acid sequence as set forth in SEQ ID NO:3.
  • nucleic acid encoding a spike-CD9 fusion protein is provided.
  • the nucleic acid encoding a spike-CD9 fusion protein has a nucleic acid sequence of SEQ ID NO:4.
  • the SARS-CoV-2 protein is a nucleocapsid protein (e.g., SEQ ID NO:5) and the exosomal tetraspanin protein is a CD9 protein (e.g., SEQ ID NO: 10)
  • the nucleocapsid protein polypeptide is positioned N-terminal to the CD9 protein polypeptide.
  • a signal peptide is positioned at the N-terminal end of the nucleocapsid protein polypeptide.
  • a hinge peptide, a transmembrane domain peptide (e.g., SEQ ID NO:9), and a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO:6), depicted in Fig. 2A.
  • the nucleocapsid-containing fusion protein has an amino acid sequence as set forth in SEQ ID NO:7.
  • nucleic acid encoding a nucleocapsid-CD9 fusion protein is provided.
  • the nucleic acid encoding a nucleocapsid-CD9 fusion protein has a nucleic acid sequence of SEQ ID NO: 8.
  • a synthetic fusion protein that contains polypeptide sequences of an influenza protein fused to polypeptide sequences of an exosomal tetraspanin protein.
  • the fusion protein is designed and made such that influenza protein antigen can be expressed on the surface of an exosome to enable the eliciting of an immune response when administered to a subject.
  • the influenza protein is a hemagglutinin protein or an engineered variant thereof.
  • the influenza protein is a hemagglutinin 3 (H3) protein or an engineered variant thereof (e.g., SEQ ID NO: 14).
  • the H3 protein polypeptide is positioned N-terminal to the CD9 protein polypeptide.
  • a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO: 15), as depicted in Fig. 3 A.
  • the H3-containing fusion protein has an amino acid sequence of SEQ ID NO: 16.
  • nucleic acid encoding an H3-CD9 fusion protein is provided.
  • the nucleic acid encoding an H3-CD9 fusion protein has a nucleic acid sequence of SEQ ID NO: 17.
  • a synthetic fusion protein contains polypeptide sequences of a respiratory syncytial virus (RSV) protein fused to polypeptide sequences of an exosomal tetraspanin protein.
  • the fusion protein is designed and made such that an RSV protein antigen can be expressed on the surface of an exosome to enable the eliciting of an immune response when administered to a subject.
  • RSV respiratory syncytial virus
  • the RSV protein is a prefusion (F) protein or an engineered variant thereof (e.g., SEQ ID NO: 18).
  • the RSV protein is a prefusion protein (RSV F) and the exosomal tetraspanin protein is a CD9 protein (e.g., SEQ ID NO: 10)
  • the RSV F protein polypeptide is positioned N-terminal to the CD9 protein polypeptide.
  • a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO: 19), as depicted in Fig. 21 A.
  • the RSV F-containing fusion protein has an amino acid sequence of SEQ ID NO:20.
  • a nucleic acid encoding an RSV F-CD9 fusion protein is provided.
  • the nucleic acid encoding an RSV F-CD9 fusion protein has a nucleic acid sequence of SEQ ID NO:21.
  • a polynucleotide is provided that encodes a synthetic fusion protein that contains polypeptide sequences of a SARS-CoV-2 protein fused to polypeptide sequences of an exosomal tetraspanin protein.
  • the encoded fusion protein is designed and made such that a SARS-CoV-2 protein antigen can be expressed in a cell and sorted onto the surface of an exosome to enable the eliciting of an immune response when that exosome is administered to a subject.
  • the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide.
  • the encoded SARS-CoV-2 spike protein polypeptide contains one or more mutations, such as, e.g., a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and/or a di-proline substitution (2P [986KV987-to- 986PP987]).
  • the spike protein has an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 or is essentially identical to SEQ ID NOT.
  • the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 11 or is essentially identical to SEQ ID NOT E
  • the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide
  • the encoded spike protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein.
  • a linker peptide is encoded by the polynucleotide to be positioned between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 1A.
  • the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NOTO.
  • the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13.
  • the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NOT or is essentially identical to SEQ ID NOT.
  • the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:4 or is essentially identical to SEQ ID NO:4.
  • the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide.
  • the nucleocapsid protein has an amino acid sequence that is at least 80% identical to SEQ ID NO: 5 or is essentially identical to SEQ ID NO:4.
  • the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 12 or is essentially identical to SEQ ID NO: 12.
  • the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide
  • the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide
  • the encoded nucleocapsid protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide.
  • a signal peptide is positioned at the N- terminal end of the nucleocapsid protein polypeptide.
  • a hinge peptide, a transmembrane domain peptide, and a linker peptide are encoded by the polynucleotide to be positioned between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 2A.
  • the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10.
  • the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13.
  • the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:7 or is essentially identical to SEQ ID NO:7.
  • the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 8 or is essentially identical to SEQ ID NO:8.
  • a polynucleotide that encodes a synthetic fusion protein that contains polypeptide sequences of an influenza protein fused to polypeptide sequences of an exosomal tetraspanin protein.
  • the encoded fusion protein is designed and made such that an influenza protein antigen can be expressed in a cell and sorted onto the surface of an exosome to enable the eliciting of an immune response when that exosome is administered to a subject.
  • the encoded influenza protein polypeptide is a hemagglutinin protein polypeptide.
  • the encoded hemagglutinin protein polypeptide is an H3 protein polypeptide.
  • the encoded H3 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 14 or is essentially identical to SEQ ID NO: 14.
  • the polynucleotide encoding the H3 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:22 or is essentially identical to SEQ ID NO:22.
  • the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide.
  • the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10 or is essentially identical to SEQ ID NO: 10.
  • the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13.
  • the encoded influenza protein polypeptide is a hemagglutinin 3 (H3) protein polypeptide and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide
  • the encoded hemagglutinin protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein.
  • a linker peptide is encoded by the polynucleotide to be positioned between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 3A.
  • the H3-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO: 16 or is essentially identical to SEQ ID NO: 16.
  • the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 17 or is essentially identical to SEQ ID NO: 17.
  • a polynucleotide that encodes a synthetic fusion protein that contains polypeptide sequences of a respiratory syncytial virus (RSV) protein fused to polypeptide sequences of an exosomal tetraspanin protein.
  • the encoded fusion protein is designed and made such that an RSV protein antigen can be expressed in a cell and sorted onto the surface of an exosome to enable the eliciting of an immune response when that exosome is administered to a subject.
  • RSV respiratory syncytial virus
  • the encoded RSV protein polypeptide is a prefusion protein polypeptide (RSV F).
  • the encoded RSV F protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 18 or is essentially identical to SEQ ID NO: 18.
  • the polynucleotide encoding the RSV F protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:23 or is essentially identical to SEQ ID NO:23.
  • the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide.
  • the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10.
  • the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13.
  • the encoded RSV F protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein.
  • a linker peptide is encoded by the polynucleotide to be positioned between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 21 A.
  • the RSV F-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:20 or is essentially identical to SEQ ID NO:20.
  • the polynucleotide encoding the spike- CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:21 or is essentially identical to SEQ ID NO:21.
  • a cell contains a polynucleotide that encodes a synthetic fusion protein that contains polypeptide sequences of a SARS-CoV-2 fused to polypeptide sequences of an exosomal tetraspanin protein.
  • the encoded fusion protein is designed and made such that a SARS-CoV-2 protein antigen can be expressed in the cell and sorted onto the surface of an exosome to enable the eliciting of an immune response when that exosome is administered to a subject.
  • the cell is a metazoan cell.
  • the cell is a vertebrate cell.
  • the cell is a mammalian cell.
  • the cell is a primate cell.
  • the cell is a human cell.
  • the cell is a primary cell.
  • the cell is of an established cell line.
  • the cell is a human embryonic kidney cell.
  • the cell is a HEK293 cell.
  • the cell is a 293F cell.
  • the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide.
  • the encoded SARS-CoV-2 spike protein polypeptide contains one or more mutations, such as, e.g., a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and/or a di-proline substitution (2P [986KV987-to- 986PP987]).
  • the spike protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:1 or is essentially identical to SEQ ID NO: 1.
  • the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 11 or is essentially identical to SEQ ID NO:11.
  • the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide
  • the encoded spike protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein.
  • a linker peptide is encoded by the polynucleotide to be positioned between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 1A.
  • the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10.
  • the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13.
  • the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:3 or is essentially identical to SEQ ID NO:3.
  • the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:4 or is essentially identical to SEQ ID NO:4.
  • the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide.
  • the nucleocapsid protein has an amino acid sequence that is at least 80% identical to SEQ ID NO: 5 or is essentially identical to SEQ ID NO:4.
  • the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 12 or is essentially identical to SEQ ID NO: 12.
  • the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide
  • the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide
  • the encoded nucleocapsid protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide.
  • a signal peptide is positioned at the N- terminal end of the nucleocapsid protein polypeptide.
  • a hinge peptide, a transmembrane domain peptide, and a linker peptide are encoded by the polynucleotide to be positioned between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 2A.
  • the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10.
  • the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13.
  • the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:7 or is essentially identical to SEQ ID NO:7.
  • the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80%> identical to SEQ ID NO:8 or is essentially identical to SEQ ID NO:8.
  • a cell contains a polynucleotide that encodes a synthetic fusion protein that contains polypeptide sequences of an influenza virus fused to polypeptide sequences of an exosomal tetraspanin protein.
  • the encoded fusion protein is designed and made such that an influenza SARS-CoV-2 protein antigen can be expressed in the cell and sorted onto the surface of an exosome to enable the eliciting of an immune response when that exosome is administered to a subject.
  • the cell is a metazoan cell.
  • the cell is a vertebrate cell.
  • the cell is a mammalian cell.
  • the cell is a primate cell.
  • the cell is a human cell.
  • the cell is a primary cell.
  • the cell is of an established cell line.
  • the cell is a human embryonic kidney cell.
  • the cell is a HEK293 cell.
  • the cell is a 293F cell.
  • the encoded influenza protein polypeptide is a hemagglutinin protein polypeptide.
  • the encoded hemagglutinin protein polypeptide is an H3 protein polypeptide.
  • the encoded H3 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 14 or is essentially identical to SEQ ID NO: 14.
  • the polynucleotide encoding the H3 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:22 or is essentially identical to SEQ ID NO:22.
  • the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide.
  • the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10 or is essentially identical to SEQ ID NO: 10.
  • the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13.
  • the encoded influenza protein polypeptide is a hemagglutinin 3 (H3) protein polypeptide and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide
  • the encoded hemagglutinin protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein.
  • a linker peptide is encoded by the polynucleotide to be positioned between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 3A.
  • the H3-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO: 16 or is essentially identical to SEQ ID NO: 16.
  • the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:17 or is essentially identical to SEQ ID N0: 17.
  • a cell contains a polynucleotide that encodes a synthetic fusion protein that contains polypeptide sequences of a respiratory syncytial virus (RSV) fused to polypeptide sequences of an exosomal tetraspanin protein.
  • the encoded fusion protein is designed and made such that an RSV protein antigen can be expressed in the cell and sorted onto the surface of an exosome to enable the eliciting of an immune response when that exosome is administered to a subject.
  • RSV respiratory syncytial virus
  • the cell is a metazoan cell. In some embodiments, the cell is a vertebrate cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a primate cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is of an established cell line. In some embodiments, the cell is a human embryonic kidney cell. In some embodiments, the cell is a HEK293 cell. In some embodiments, the cell is a 293F cell. [0069] In one embodiment, the encoded RSV protein polypeptide is a prefusion protein polypeptide (RSV F).
  • RSV F prefusion protein polypeptide
  • the encoded RSV F protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 18 or is essentially identical to SEQ ID NO: 18.
  • the polynucleotide encoding the RSV F protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:23 or is essentially identical to SEQ ID NO:23.
  • the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide.
  • the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10.
  • the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13.
  • the encoded RSV F protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein.
  • a linker peptide is encoded by the polynucleotide to be positioned between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 21 A.
  • the RSV F-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:20 or is essentially identical to SEQ ID NO:20.
  • the polynucleotide encoding the spike- CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:21 or is essentially identical to SEQ ID NO:21.
  • Figure 1A depicts a linear cartoon of a SARS-CoV-2 spike CD9 fusion protein oriented from left-to-right amino-terminus-to-carboxy -terminus with a linker sequence positioned between the spike protein polypeptide and the CD9 protein polypeptide.
  • Figure IB depicts a cartoon of a SARS-CoV-2 spike CD9 fusion protein of Fig. 1A relative to a vesicle membrane oriented from left-to-right amino-terminus-to-carboxy-terminus.
  • the spike protein polypeptide spans the membrane once with its amino terminus oriented on the outside of the vesicle.
  • the linker is positioned on the luminal side of the membrane.
  • the CD9 protein polypeptide spans the membrane 4 times with its carboxy terminus oriented in the lumen of the vesicle.
  • Figure 2A depicts a linear cartoon of a SARS-CoV-2 nucleocapsid CD9 fusion protein oriented from left-to-right amino-terminus-to-carboxy -terminus with an amino terminal signal peptide fused to a nucleocapsid protein polypeptide fused to a hinge region peptide fused to a transmembrane domain peptide fused to a linker peptide fused to a CD9 protein polypeptide.
  • Figure 2B depicts a cartoon of a SARS-CoV-2 nucleocapsid CD9 fusion protein of Fig. 2A relative to a vesicle membrane oriented from left-to-right amino-terminus-to-carboxy- terminus.
  • the nucleocapsid protein polypeptide with the amino terminal signal sequence is positioned on the outside (cytoplasmic or external side) with a linker sequence connecting the nucleocapsid protein polypeptide to the transmembrane domain peptide, which spans the membrane and which in turn connects to the linker positioned in the lumen and which connects to the CD9 protein polypeptide which spans the membrane 4 times with its carboxy terminus oriented in the lumen of the vesicle.
  • Figure 3A depicts a linear cartoon of an influenza hemagglutinin CD9 fusion protein oriented from left-to-right amino-terminus-to-carboxy-terminus with a linker sequence positioned between the spike protein polypeptide and the CD9 protein polypeptide.
  • Figure 3B depicts a cartoon of an influenza hemagglutinin CD9 fusion protein of Fig. 3 A relative to a vesicle membrane oriented from left-to-right amino-terminus-to-carboxy-terminus.
  • the hemagglutinin protein polypeptide spans the membrane once with its amino terminus oriented on the outside of the vesicle.
  • the linker is positioned on the luminal side of the membrane.
  • the CD9 protein polypeptide spans the membrane 4 times with its carboxy terminus oriented in the lumen of the vesicle.
  • Figure 4A is a flow chart depicting the elements and steps for producing cells expressing a spike, nucleocapsid, influenza hemagglutinin, or other antigen protein polypeptide fusion protein using a packaging cell (301) and a host cell (311) mediated by a lentivirus vector (304).
  • Figure 4B is a histogram depicting relative fluorescent intensity flow analysis of host cells expressing spike protein on their surface.
  • Figure 5A is a graph depicting the concentration of spike-expressing exosomes per milliliter as a function of exosome diameter in nanometers.
  • Figure 5B is a western blot stained for SARS-CoV-2 spike protein.
  • the first column from left to right depicts lane loaded with size markers
  • second column represents lane loaded with non-transduced 293F (host cell) protein
  • third column represents lane loaded with protein from exosomes derived from non-transduced 293F cells
  • fourth column represents lane loaded with protein from 293F cells constitutively expressing spike fusion protein
  • fifth column represents lane loaded with protein from exosomes derived from 293F cells constitutively expressing spike fusion protein
  • sixth column represents lane loaded with spike fusion protein.
  • Figure 5C is a histogram depicting relative fluorescent intensity flow analysis of exosomes with spike expressed on the surface.
  • the left curve represents exosomes derived from 293F cells that do not express a spike-CD9 fusion protein.
  • the right curve represents exosomes derived from 293F cells expressing a spike-CD9 fusion protein.
  • Figure 6A depicts a transmission electron micrograph of exosomes expressing SARS- CoV-2 spike protein. Inset is a blow up showing an exosome decorated with spike protein. Arrows point to SARS-CoV-2 spikes around the circumference of exosomes.
  • Figure 6B depicts a higher magnification transmission electron micrograph of exosomes expressing SARS-CoV-2 spike protein. Arrows point to SARS-CoV-2 spikes around the circumference of exosomes.
  • Figure 7A is a graph depicting the concentration of nucleocapsid-expressing exosomes per milliliter as a function of exosome diameter in nanometers.
  • Figure 7B is a western blot stained for SARS-CoV-2 nucleocapsid protein.
  • the first column from left to right depicts lane loaded with size markers
  • second column represents lane loaded with nucleocapsid protein
  • third column represents lane loaded with protein from exosomes derived from non-transduced 293F cells
  • fourth column represents lane loaded with protein from non-transduced 293F cells
  • fifth column represents lane loaded with protein from exosomes derived from 293F cells expressing nucleocapsid fusion protein.
  • Figure 7C is a histogram depicting relative fluorescent intensity flow analysis of exosomes with or without nucleocapsid protein expressed on the surface.
  • the first curve represents exosomes derived from unmodified 293F cells
  • the second curve represents exosomes derived from uninduced 293F cells harboring a nucleocapsid-CD9 fusion protein-encoding polynucleotide
  • the third curve represents exosomes derived from 293F cells expressing nucleocapsid-CD9 fusion protein
  • the smaller third curve represents exosomes derived from 293F cells expressing nucleocapsid-CD9 fusion protein under tet induction
  • the larger third curve represents exosomes derived from 293F cells harboring a CD9 knock-out and expressing nucleocapsid-CD9 fusion protein.
  • Figures 8A and 8B are histograms depicting relative fluorescent intensity flow analysis of 293F cells with or without influenza H3 hemagglutinin protein expressed on the surface. Moving from left to right on each panel, the first curve represents unmodified 293F cells, the second curve represents 293F cells harboring and expressing an influenza H3-CD9 fusion proteinencoding polynucleotide. Panel A represents adherent 293F cells. Panel B represents 293F cells in suspension.
  • Figure 8C is a western blot stained for influenza H3 protein (panel 1) or CD9 (panel 2).
  • first column from left to right depicts lane loaded with size markers
  • second column represents lane loaded with unmodified 293F (host cell) protein
  • third column represents lane loaded with protein from exosomes derived from unmodified 293F cells
  • fourth column represents lane loaded with protein from 293F cells expressing H3-CD9 fusion protein
  • fifth column represents lane loaded with protein from exosomes derived from 293F cells expressing H3-CD9 fusion protein.
  • Each sample was loaded at 0.6mg/ml total protein.
  • Figure 8D is a flow cytometry dot plot of H3 -expressing exosomes depicting CD81 surface expression as a function of H3 surface expression.
  • about 54.2% of the flowed exosomes express both CD81 and H3, thus demonstrating that the expression of a hemagglutinin polypeptide or antigen at the surface of a vesicle when fused to an exosomal protein such as a tetraspanin like CD9.
  • Figure 9 is a timeline depicting a process for generating am immune response from administering an immunogenic composition containing exosomes expressing SARS-CoV-2 fusion proteins on their surface.
  • Figure 10A is a histogram depicting fold change in murine day-14 l : 100-diluted serum antibody titer against spike protein as a function of exosome-expressing spike dosage in nanograms.
  • the left-most bar represents PBS control; the second bar represents a 10 ng dose of spike expressing exosomes (STX-S); the third bar represents a 32 ng dose of STX-S; the right most bar represents sera from mice injected with 32 ng spike protein dose combined with adjuvant.
  • Figure 10B is a histogram depicting fold change in murine day-35 l : 100-diluted serum antibody titer against spike protein as a function of exosome-expressing spike dosage in nanograms.
  • the left-most bar represents PBS control; the second bar represents a 10-ng dose of spike expressing exosomes (STX-S); the third bar represents a 32 ng dose of STX-S; the right most bar represents sera from mice injected with 32 ng spike protein dose combined with adjuvant.
  • Figure 10C is a bar graph depicting an ELISpot assay showing the number of IL4 producing spots responding to spike protein as a function of antigen dose.
  • the first bar represents non-spike-containing wells from PBS control splenocytes;
  • the second bar represents spike protein reactivity from PBS control splenocytes;
  • the third bar represents non-spike-containing wells from splenocytes of 10 ng STX-S dosed subject;
  • the fourth bar represents spike protein reactivity from splenocytes of 10 ng STX-S dosed subject;
  • the fifth bar represents non-spike-containing wells from splenocytes of 32 ng STX-S dosed subject;
  • the sixth bar represents spike protein reactivity from splenocytes of 32 ng STX-S dosed subject;
  • the seventh bar represents non-spike-containing wells from splenocytes of 32 ng spike protein with adjuvant dosed subject;
  • the eighth bar represents spike protein
  • Figure 10D is a bar graph depicting an ELISpot assay showing the number of IFNy producing spots responding to spike protein as a function of antigen dose.
  • the first bar represents non-spike-containing wells from PBS control splenocytes;
  • the second bar represents spike protein reactivity from PBS control splenocytes;
  • the third bar represents non-spike-containing wells from splenocytes of 10 ng STX-S dosed subject;
  • the fourth bar represents spike protein reactivity from splenocytes of 10 ng STX-S dosed subject;
  • the fifth bar represents non-spike-containing wells from splenocytes of 32 ng STX-S dosed subject;
  • the sixth bar represents spike protein reactivity from splenocytes of 32 ng STX-S dosed subject;
  • the seventh bar represents non-spike-containing wells from splenocytes of 32 ng spike protein with adjuvant dosed subject;
  • the eighth bar represents spike protein
  • FIGS 11, panels A-C Line graphs depicting percent SARS-CoV-2 neutralization as a function of immune sera at escalating serum dilution. The graphs depict the production of neutralizing antibodies after STX-S injection. Data are shown as mean ⁇ SEM.
  • COV-02-Delta plasma from a patient immunized with Modema’s mRNA vaccine with a breakthrough SARS- CoV-2 delta spike infection.
  • Panel 11 A STX-S vaccine generated strong neutralization against SARS-CoV-2 delta spike (B.1.617.2)
  • Panel 11B STX-S vaccine resulted in neutralization of SARS-CoV-2 spike Omicron BA. l
  • Panel 11C STX-S vaccine resulted in neutralization of SARS-CoV-2 spike Omicron BA.5.2.1.
  • Figure 12A is a histogram depicting OD of day-14 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3), and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).
  • Figure 12B is a histogram depicting fold change in levels of day-14 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3), and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).
  • Figure 12C is a histogram depicting the loglO titer of day-14 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3), and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).
  • Figure 12D is a histogram depicting OD of day-14 serum anti-spike IgG as a function of injected material dose, which is, from left to right, PBS control, and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).
  • Figure 12E is a histogram depicting fold change in levels of day-14 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3), and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).
  • Figure 12FE is a histogram depicting the loglO titer of day-14 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3), and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).
  • Panels 13A and 13B Histograms depicting anti-nucleocapsid antibody titers of day-35 post immunization sera as a function of dose.
  • STX-N vaccine induced modest expression of SARS-CoV-2 Nucleocapsid antibodies in two sample bins (N 1 and N 2).
  • PBS was used as a vehicle control.
  • Panels 13C and 13D Histograms depicting anti-nucleocapsid IFNy ELISpot positive wells of day-40 post immunization splenocytes as a function of dose.
  • Figure 14 depicts the expression of SARS-CoV-2 spike protein on 293 cells.
  • Panel 14A is a histogram depicting relative fluorescent intensity flow analysis of 293F cells with SARS-CoV2 spike expressed on the surface.
  • the left curve represents 293F cells that do not express a spike-CD9 fusion protein.
  • the right curve represents 293F cells expressing a spike-CD9 fusion protein.
  • Panel 14B is a western blot stained for SARS-CoV-2 spike protein.
  • the first column from left to right depicts lane loaded with size markers
  • second column represents lane loaded with non-transduced 293F (host cell) protein
  • third column represents lane loaded with protein from exosomes derived from non-transduced 293F cells
  • fourth column represents lane loaded with protein from 293F cells expressing spike fusion protein
  • fifth column represents lane loaded with protein from exosomes derived from 293F cells expressing spike fusion protein
  • sixth column represents lane loaded with spike fusion protein.
  • Panel 15A is a histogram depicting relative fluorescent intensity flow analysis of 293F cells with influenza hemagglutinin 3 (H3) expressed on the surface.
  • the left curve represents 293F cells that do not express an H3-CD9 fusion protein.
  • the right curve represents 293F cells expressing an H3-CD9 fusion protein.
  • Panel 15B is a western blot stained for influenza hemagglutinin 3 (H3) protein.
  • the first column from left to right depicts lane loaded with size markers
  • second column represents lane loaded with non-transduced 293F (host cell) protein
  • third column represents lane loaded with protein from exosomes derived from non-transduced 293F cells
  • fourth column represents lane loaded with protein from 293F cells expressing H3 fusion protein
  • fifth column represents lane loaded with protein from exosomes derived from 293F cells expressing H3 fusion protein.
  • Figure 16 are histograms depicting expression of surface markers on 293F-derived exosomes.
  • Panel 16A is a histogram depicting relative fluorescent intensity flow analysis of exosomes derived from 293F cells with naturally-occurring CD81 expressed on the surface.
  • the left curve represents 293F-derived exosomes decorated with isotype control antibody.
  • the right curve represents 293F-derived exosomes decorated with anti-CD81 antibody.
  • Panel 16B is a histogram depicting relative fluorescent intensity flow analysis of exosomes derived from 293F cells with spike expressed on the surface.
  • the left curve represents exosomes derived from 293F cells that do not express a spike-CD9 fusion protein.
  • the right curve represents exosomes derived from 293F cells expressing a spike-CD9 fusion protein.
  • Panel 16C is a histogram depicting relative fluorescent intensity flow analysis of exosomes derived from 293F cells transfected with hemagglutinin 3 (H3) expressed on the surface.
  • the left curve represents exosomes derived from 293F cells that do not express an H3- CD9 fusion protein.
  • the right curve represents exosomes derived from 293F cells expressing an H3-CD9 fusion protein.
  • Figure 17 presents histograms depicting fold-change in antigen-specific IgG production.
  • Panel 17A is a histogram depicting fold change in levels of day-14 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, and a combination of exosomes expressing hemagglutinin 3 (STX-H3) and exosomes expressing SARS-CoV-2 spike (STX-S).
  • STX-H3 hemagglutinin 3
  • STX-S SARS-CoV-2 spike
  • Panel 17B is a histogram depicting fold change in levels of day-14 serum anti-spike IgG as a function of injected material dose, which is, from left to right, PBS control, and a combination of exosomes expressing hemagglutinin 3 (STX-H3) and exosomes expressing SARS-CoV-2 spike (STX-S).
  • STX-H3 hemagglutinin 3
  • STX-S SARS-CoV-2 spike
  • Figure 18 presents histograms depicting fold-change in antigen-specific IgG production.
  • Panel 18A is a histogram depicting fold change in levels of day-35 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, and a combination of exosomes expressing hemagglutinin 3 (STX-H3) and exosomes expressing SARS-CoV-2 spike (STX-S).
  • Panel 18B is a histogram depicting fold change in levels of day-35 serum anti-spike IgG as a function of injected material dose, which is, from left to right, PBS control, and a combination of exosomes expressing hemagglutinin 3 (STX-H3) and exosomes expressing SARS-CoV-2 spike (STX-S).
  • STX-H3 hemagglutinin 3
  • STX-S SARS-CoV-2 spike
  • Figure 19 presents ELISpot histograms of IFNg production.
  • Panel 19A is a histogram depicting IFNg production by splenocytes as a function of immunogen and antigen. From left to right, the first histogram represents splenocytes from animals immunized with PBS and not exposed to antigen, the second histogram represents splenocytes from animals immunized with PBS and exposed to H3 antigen, the third histogram represents splenocytes from animals immunized with a combination of exosomes that express spike and exosomes that express H3 and not exposed to antigen, and the fourth histogram represents splenocytes from animals immunized with a combination of exosomes that express spike and exosomes that express H3 and exposed to H3 antigen.
  • Panel 19A is a histogram depicting IFNg production by splenocytes as a function of immunogen and antigen. From left to right, the first histogram represents splenocytes from animals immunized with PBS and not exposed to antigen, the second histogram represents splenocytes from animals immunized with PBS and exposed to H3 antigen, the third histogram represents splenocytes from animals immunized with a combination of exosomes that express spike and exosomes that express H3 and not exposed to antigen, and the fourth histogram represents splenocytes from animals immunized with a combination of exosomes that express spike and exosomes that express H3 and exposed to H3 antigen.
  • Panel 19B is a histogram depicting IFNg production by splenocytes as a function of immunogen and antigen. From left to right, the first histogram represents splenocytes from animals immunized with PBS and not exposed to antigen, the second histogram represents splenocytes from animals immunized with PBS and exposed to spike antigen, the third histogram represents splenocytes from animals immunized with a combination of exosomes that express spike and exosomes that express H3 and not exposed to antigen, and the fourth histogram represents splenocytes from animals immunized with a combination of exosomes that express spike and exosomes that express H3 and exposed to spike antigen.
  • Figure 20 depicts graphic representations of respiratory syncytial virus fusion protein (RSFV F) coding sequence.
  • Bar A represents non-engineered RSV F.
  • Bar B represents engineered RSV F version 1 (VI) which is DS-Cavl .
  • Bar C represents engineered RSV F version 2 (V2) which is DS-Cavl with polyA signals deleted.
  • Bar D represents engineered RSV F version 3 (V3) which is DS-Cavl with polyA signals and two furin cleavage sites deleted.
  • Bar D represents engineered RSV F version 3 (V4) which is DS-Cavl with polyA signals and the N- terminal most furin cleavage site deleted.
  • SP signal peptide
  • F2 RSV fusion protein subunit 2
  • p27 RSV fusion protein p27 subunit
  • FP hydrophobic fusion peptide (FP)
  • Fl RSV fusion protein subunit 1
  • TM transmembrane domain.
  • Figures 22 are histograms depicting anti-RSV antibody titer fold change as a function of antigen (RSV version 3).
  • the Y-axes represent antibody titer fold change, and the X-axes represent antigen, the left bars are PBS control, the right bars are RSV F antigen.
  • Panel 22A represents antibody fold change at 14 days post injection.
  • Panel 22B represents antibody fold change at 35 days post injection and after a booster at day -21.
  • Figures 23 are histograms depicting anti-RSV antibody titer fold change as a function of antigen (RSV version 4).
  • the Y-axes represent antibody titer fold change, and the X-axes represent antigen.
  • Panel 23 A represents antibody fold change at 14 days post injection.
  • Panel 23B represents antibody fold change at 35 days post injection and after a booster at day-21.
  • Figure 24 is a graphic representing a combination of exosomes expressing SARS-CoV2 spike-tetraspanin (STX-S) and exosomes expressing influenza H3 -tetraspanin (STX-H3).
  • Figure 25 is a graphic representing a combination of exosomes expressing SARS-CoV2 spike-tetraspanin (STX-S) and exosomes expressing respiratory syncytial virus fusion protein- tetraspanin (STX-F).
  • STX-S SARS-CoV2 spike-tetraspanin
  • STX-F respiratory syncytial virus fusion protein- tetraspanin
  • Figure 26 is a graphic representing a combination of exosomes expressing influenza H3- tetraspanin (STX-H3) and exosomes expressing respiratory syncytial virus fusion protein- tetraspanin (STX-F).
  • Figure 27 is a graphic representing a combination of exosomes expressing SARS-CoV2 spike-tetraspanin (STX-S), exosomes expressing influenza H3 -tetraspanin (STX-H3), and exosomes expressing respiratory syncytial virus fusion protein-tetraspanin (STX-F).
  • Figures 28 are histograms depicting antibody titer fold change as a function of antigen.
  • the Y-axes represent antibody titer fold change, and the X-axes represent antigen.
  • Panel 28A represents anti-spike antibody fold change at 14 days post injection.
  • Panel 28B represents anti- 143 antibody fold change at 14 days post injection.
  • Panel 28C represents anti-RSV antibody fold change at 14 days post injection.
  • Figures 29 are histograms depicting antibody titer fold change as a function of antigen.
  • the Y-axes represent antibody titer fold change, and the X-axes represent antigen.
  • Panel 29 A represents anti-spike antibody fold change at 35 days post injection and after booster at day-21.
  • Panel 28B represents anti-H3 antibody fold change at 35 days post injection and after booster at day-21.
  • Panel 28C represents anti-RSV antibody fold change at 35 days post injection and after booster at day-21.
  • DETAILED DESCRIPTION OF EMBODIMENTS
  • Treatment, prevention and prophylaxis can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, increase in survival time or rate, etc. Treatment, prevention, and prophylaxis can be complete or partial.
  • the term “prophylactic” means not only “prevent”, but also minimize illness and disease.
  • a “prophylactic” agent can be administered to a subject, e.g., a human subject, to prevent infection, or to minimize the extent of illness and disease caused by such infection. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment.
  • the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects, the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques.
  • a treatment can be considered “effective,” as used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 2%, 3%, 4%, 5%, 10%, or more, following treatment according to the methods described herein.
  • Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (e.g., progression of the disease is halted).
  • Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms.
  • An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease.
  • Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. One skilled in the art can monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters.
  • the term “effective amount” as used herein refers to the amount of a composition or an agent needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of therapeutic composition to provide the desired effect.
  • the term “therapeutically effective amount” refers to an amount of a composition or therapeutic agent that is sufficient to provide a particular effect when administered to a typical subject.
  • An effective amount as used herein, in various contexts, can include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease.
  • a therapeutically effective amount will show an increase or decrease of therapeutic effect at least any of 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 any of a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.
  • the therapeutically effective amount may be administered in one or more doses of the therapeutic agent.
  • the therapeutically effective amount may be administered in a single administration, or over a period of time in a plurality of doses.
  • administering can include any suitable routes of administering a therapeutic agent or composition as disclosed herein. Suitable routes of administration include, without limitation, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection or topical administration. Administration can be local or systemic.
  • the term “pharmaceutically acceptable” refers to a carrier that is compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the term is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”.
  • a pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a dose refers to the amount of active ingredient given to an individual at each administration.
  • the dose can refer to the concentration of the extracellular vesicles or associated components, e.g., the amount of therapeutic agent or dosage of radiolabel.
  • the dose will vary depending on a number of factors, including frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; the route of administration; and the imaging modality of the detectable moiety (if present).
  • the term “dosage form” refers to the particular format of the pharmaceutical and depends on the route of administration.
  • a dosage form can be in a liquid, e.g., a saline solution for injection.
  • Subject “Subject,” “patient,” “individual” and like terms are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species.
  • mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species.
  • the term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision.
  • a patient can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc.
  • an element includes a combination of two or more elements, notwithstanding use of other terms and phrases for one or more elements, such as “one or more.”
  • the term “or” is, unless indicated otherwise, non-exclusive, i.e., encompassing both “and” and “or.”
  • the term “any of’ between a modifier and a sequence means that the modifier modifies each member of the sequence. So, for example, the phrase “at least any of 1 , 2 or 3” means “at least 1, at least 2 or at least 3”.
  • the phrase “at least one” includes “a plurality”.
  • the term “native form” corresponds to the polypeptide as it is understood to be encoded by the infectious agent’s genome.
  • the term “exosomal form” corresponds to any derivative of the protein that, in whole or in part, is fused to an exosome-associated protein.
  • the term “cytoplasmic form” corresponds to any derivative of the protein that, in whole or in part, is configured, or designed, to be expressed within the cytoplasm of the cell, rather than entering the canonical secretory pathway.
  • a certain protein is “configured, or designed, to be expressed” in a certain way means that its nucleotide sequence encodes certain a particular amino acid sequence such that when that protein is expressed in a cell, that protein will be in its native form, exosomal form, or cytoplasmic form by virtue of that particular amino acid sequence.
  • a spike protein S
  • it is configured, or designed, to induce a humoral or cellular immune response by virtue of the fact that it is a transmembrane protein with an extracellular domain.
  • extracellular vesicle refers to lipid bilayer-delimited particles that are naturally released from cells. EVs range in diameter from around 20-30 nanometers to about 10 microns or more. EVs can comprise proteins, nucleic acids, lipids and metabolites from the cells that produced them. EVs include exosomes (about 50 to about 200 nm), microvesicles (about 100 to about 300 nm), ectosomes (about 50 to about 1000 nm), apoptotic bodies (about 50 to about 5000 nm) and lipid-protein aggregates of the same dimensions.
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), as well as chemically modified nucleic acids such as morpholino (PMO), peptide nucleic acid (PNA), 2'O-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate, and phosphorothioate. Nucleic acids may be of any size.
  • Nucleic acids include but are not limited to genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules such as aptamers, plasmids, anti-sense DNA strands, shRNA, ribozymes, nucleic acids conjugated and oligonucleotides.
  • a nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule.
  • a nucleic acid might be employed for introduction into, e.g., transfection of, cells, e g., in the form of RNA which can be prepared by in vitro transcription from a DNA template.
  • the RNA can moreover be modified before application by stabilizing sequences, capping, and polyadenylation.
  • nucleic acid can be extracted, isolated, amplified, or analyzed by a variety of techniques such as those described by, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY 2,028 pages (2012).
  • peptide refers to any chain of at least two amino acids, linked by a covalent chemical bound.
  • a peptide can refer to the complete amino acid sequence coding for an entire protein or to a portion thereof.
  • a “protein coding sequence” or a sequence that “encodes” a particular polypeptide or peptide is a nucleic acid sequence that is transcribed (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • a coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • protein polypeptide means a polypeptide sequence of or derived from a protein.
  • a CD9 protein polypeptide may be any polypeptide of the CD9 protein, such as, e.g., a full length CD9 protein, a transmembrane domain polypeptide of a CD9 protein, a C-terminal stretch of a CD9 protein, an extracellular loop region of a CD9 protein, the intracellular (intralumenal) loop region of a CD9 protein, a C-terminal stretch of a CD9 protein, combinations thereof, and/or the like.
  • a protein polypeptide may be at least 10 amino acids long.
  • spike protein includes any SARS-CoV-2 spike glycoprotein, fragment of a SARS-CoV-2 spike glycoprotein, monomer of a SARS-CoV-2 spike glycoprotein, trimer of SARS-CoV-2 spike glycoprotein monomers, variant of a SARS-CoV-2 spike glycoprotein, fusion protein or chimeral protein containing a SARS-CoV-2 spike glycoprotein sequence and another non-SARS-CoV-2 spike glycoprotein sequence, SARS-CoV-2 spike glycoproteins having one or more deletions, additions, or substitutions of one or more amino acids, and conservatively substituted variations of a SARS-CoV-2 spike glycoprotein having at least 80% amino acid sequence identity of e.g., at least the stem region of an S2 subunit, membrane-proximal stem helix region, or the receptor binding domain, or other like domains.
  • a fragment of a SARS-CoV-2 spike glycoprotein includes peptide or polypeptides the encompass, comprise, consist of, or overlap with e.g., antigenic epitopes, specific domains like receptor binding domain (RBD) in up or down conformational states, a receptor-binding fragment SI, fusion fragment S2, N-terminal domain (NTD), receptor-binding domain (RBD) C- terminal domain 1 (CTD1), C-terminal domain 2 (CTD2), fusion peptide (FP), fusion-peptide proximal region (FPPR), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), heptad repeat 2 (HR2), transmembrane segment (TM), the cytoplasmic tail (CT), and the like.
  • RCD receptor binding domain
  • a variant of a SARS-CoV-2 spike glycoprotein includes any known or yet to be discovered, including alpha, beta, gamma, delta, epsilon, eta, iota, kappa, 1.617.3, mu, zeta, omicron, or their subvariants, lineages, and conservatively substituted spike protein sequence.
  • SARS-CoV-2 spike glycoprotein may have additions, deletions, substitution, point mutations.
  • a spike protein may have a deletion of several (2-20) amino acids from its C-terminus (see Johnson et al., 2020 and Xiong et al., 2020), or furin cleavage site change (see e.g., Johnson et al., 2020).
  • nucleocapsid protein means a soluble coronavirus structural protein that binds and forms complexes with RNA and viral membrane protein (M) and is critical for viral genome pacjkaging.
  • Nucleocapsid protein contains (from amino terminus to carboxy terminus) an intrinsically disordered region (IDR), and N terminal domain containing an RNA binding domain (NTD), a serine-arginine rich linker region (LKR), a C terminal domain containing an RNA binding and dimerization domain and a nuclear localization signal, followed by a C terminal IDR.
  • IDR intrinsically disordered region
  • NTD RNA binding domain
  • LLR serine-arginine rich linker region
  • C terminal domain containing an RNA binding and dimerization domain and a nuclear localization signal
  • Nucleocapsid protein is generally described, e.g., in McBride et al., “The Coronavirus Nucleocapsid Is a Multifunctional Protein,” Viruses. 2014 Aug; 6(8): 2991-3018; Cubuk et al., “The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA,” Nature Communications volume 12, Article number: 1936 (2021); and references cited therein.
  • Fusion proteins or chimeral proteins of SARS-CoV-2 spike glycoprotein or SARS-CoV-2 nucleocapsid protein includes fusions of spike or nucleocapsid protein sequences and sequences of another protein to effect a particular outcome, such as improved sorting or targeting to endosomes and resultant extracellular vesicles such as exosomes.
  • spike protein may be a fusion of spike or nucleocapsid protein sequences and other glycoproteins known to sort to exosomes or useful in the production of pseudovirions, such as VSV glycoproteins, lentivirus glycoprotein, and the like.
  • Spike or nucleocapsid protein may be a fusion of spike protein sequences and other proteins known to sort to exosomes such as various tetraspanins (CD9, CD63, and CD81). Fusion proteins may contain mostly SARS-CoV-2 sequences with short (i.e., dipeptides to peptides of 100 amino acids) sequences of the other protein.
  • tetraspanin or “tetraspanin protein” means any member (or chimera thereof) of a family of proteins having four transmembrane domains and in some cases present in exosome membranes. Tetraspanin proteins are known to regulate trafficking and cell and membrane compartmentalization. Tetraspanins include inter alia CD9, CD37, CD63, CD81, CD82, CD151, TSPAN7, TSPAN8, TSPAN12, TSPAN33, peripherin, UPla/lb, TSP-15, TSP- 12, TSP3A, TSP86D, TSP26D, TSP-2, and analogs, orthologs, and homologs thereof.
  • useful tetraspanins may include chimeras of any one or more canonical tetraspanins, such as, e.g., a CD9/CD81 chimera or the like.
  • Tetraspanins are generally described in Charrin et al., “Tetraspanins at a glance,” J Cell Sci (2014) 127 (17): 3641-3648; Kummer et al., “Tetraspanins: integrating cell surface receptors to functional microdomains in homeostasis and disease,” Med Microbiol Immunol. 2020; 209(4): 397-405; and references cited therein.
  • the CD9 member of the tetraspanin superfamily is generally described in Umeda et al., “Structural insights into tetraspanin CD9 function,” Nature Communications volume 11, Article number: 1606 (2020), and references cited therein.
  • sequence identity refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877.
  • Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.
  • PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • the default parameters of the respective programs e.g., of XBLAST and NBLAST
  • the default parameters of the respective programs can be used (see, e.g., the NCBI website).
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
  • percentage sequence identities can be determined when antibody sequences are maximally aligned by IMGT. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, multiplied by 100 to convert to percentage.
  • a subject antibody region e.g., the entire mature variable region of a heavy or light chain
  • Percent amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).
  • NCBI-BLAST2 sequence comparison program may be obtained from the National Institute of Health, Bethesda, Md.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • nucleic acid sequence refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages and includes cDNA. The term also includes modified or substituted sequences comprising non- naturally occurring monomers or portions thereof.
  • the nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.
  • the sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. It is understood that polynucleotides comprising non-transcribable nucleotide bases may be useful as probes in, for example, hybridization assays.
  • nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term “nucleic acid” includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents.
  • antibody refers to immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds an antigen.
  • Antibodies are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains.
  • the light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (X), based on the amino acid sequences of their constant domains.
  • K kappa
  • X lambda
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, 8, s, y, and p, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • the antibody may have one or more effector functions which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region or any other modified Fc region) of an antibody.
  • effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor (BCR); and cross-presentation of antigens by antigen presenting cells or dendritic cells).
  • neutralizing antibody refers an antibody that defends a cell from a pathogen or infectious particle by neutralizing any effect it has biologically. Neutralization renders the particle no longer infectious or pathogenic.
  • Neutralizing antibodies are part of the humoral response of the adaptive immune system against viruses, intracellular bacteria and microbial toxin. By binding specifically to surface antigen on an infectious particle, neutralizing antibodies prevent the particle from interacting with its host cells it might infect and destroy.
  • Immunity due to neutralizing antibodies is also known as sterilizing immunity, as the immune system eliminates the infectious particle before any infection took place.
  • an antigen refers to any substance that will elicit an immune response.
  • an antigen relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T-lymphocytes (T cells).
  • T cells T-lymphocytes
  • the term “antigen” comprises any molecule which comprises at least one epitope.
  • an antigen is a molecule which, optionally after processing, induces an immune reaction.
  • any suitable antigen may be used, which is a candidate for an immune reaction, wherein the immune reaction may be a cellular immune reaction.
  • the antigen may be presented by a cell, which results in an immune reaction against the antigen.
  • an antigen is a product which corresponds to or is derived from a naturally occurring antigen.
  • antigens include, but are not limited to, SARS-CoV-2 structural proteins S, N, M, and E, and any variants or mutants thereof.
  • composition refers to a formulation comprising an active ingredient, and optionally a pharmaceutically acceptable carrier, diluent or excipient.
  • active ingredient can interchangeably refer to an “effective ingredient,” and is meant to refer to any agent that is capable of inducing a sought-after effect upon administration.
  • pharmaceutically acceptable it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof, nor to the activity of the active ingredient of the formulation.
  • Pharmaceutically acceptable carriers, excipients or stabilizers are well known in the art, for example Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).
  • Pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine
  • excipient examples include, but are not limited to, anti-adherents such as magnesium stearate, binders such as saccharides and their derivatives (sucrose, lactose, starches, cellulose, sugar alcohols and the like) protein like gelatin and synthetic polymers, lubricants such as talc and silica, and preservatives such as antioxidants, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate and parabens.
  • diluent include, but are not limited to, water, alcohol, saline solution, glycol, mineral oil and dimethyl sulfoxide (DMSO).
  • the term “vaccine” or “immunogenic composition” relates to a pharmaceutical preparation (pharmaceutical composition) or product that upon administration induces an immune response, e.g., a cellular immune response, which recognizes and attacks a pathogen or a diseased cell.
  • an immune response refers to an integrated bodily response to an antigen and refers to a cellular immune response and/or a humoral immune response. The immune response may be protective/preventive/prophy lactic and/or therapeutic.
  • cellular immune response or “cell-mediated immune response” describes any adaptive immune response in which antigen-specific T cells have the main role. It is defined operationally as all adaptive immunity that cannot be transferred to a native recipient by serum antibody. In contrast, the term “humoral immune response” describes immunity due to antibodies.
  • the cellular response relates to cells called T cells or T-lymphocytes which act as either “helpers” or “killers”.
  • the helper T cells also termed CD4+ T cells
  • the killer cells also termed cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs kill diseased cells such as cancer cells, preventing the production of more diseased cells.
  • immunoreactive cell refers to a cell which exerts effector functions during an immune reaction.
  • An “immunoreactive cell” preferably is capable of binding an antigen or a cell characterized by presentation of an antigen, or an antigen peptide derived from an antigen and mediating an immune response.
  • such cells secrete cytokines and/or chemokines, secrete antibodies, recognize cancerous cells, and optionally eliminate such cells.
  • immunoreactive cells comprise T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells.
  • adjuvant refers to a pharmacological or immunological agent that modifies the effect of other agents.
  • An adjuvant may be added to the vaccine composition of the invention to boost the immune response to produce more antibodies and longer-lasting immunity, thus minimizing the dose of antigen needed.
  • adjuvants may also be used to enhance the efficacy of a vaccine by helping to modify the immune response to particular types of immune system cells: for example, by activating T cells instead of antibody-secreting B cells depending on the purpose of the vaccine.
  • Immunologic adjuvants are added to vaccines to stimulate the immune system's response to the target antigen, but do not provide immunity themselves.
  • adjuvants include, but are not limited to analgesic adjuvants; inorganic compounds such as alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide; mineral oil such as paraffin oil; bacterial products such as killed bacteria (Bordetella pertussis, Mycobacterium bovis, toxoids); nonbacterial organics such as squalene; delivery systems such as detergents (Quil A); plant saponins from Quillaja, soybean, or Polygala senega; cytokines such as IL-1, IL- 2, IL- 12; combination such as Freund’s complete adjuvant, Freund’s incomplete adjuvant; foodbased oil such as Adjuvant 65, which is based on peanut oil.
  • analgesic adjuvants such as alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide; mineral oil such as paraffin oil; bacterial products such as killed bacteria (Bordetella pertussis, Mycobacterium bovis, to
  • Embodiments provided include:
  • Embodiment 1 An immunogenic composition comprising a first vesicle and a first fusion protein, and a second vesicle and a second fusion protein, wherein said first fusion protein comprises a first virus polypeptide and an exosomal polypeptide and said second fusion protein comprises a second virus polypeptide and an exosomal polypeptide.
  • Embodiment 2 An immunogenic composition comprising a first vesicle and a first fusion protein, a second vesicle and a second fusion protein, and a third vesicle and a third fusion protein, wherein said first fusion protein comprises a first virus polypeptide and an exosomal polypeptide, said second fusion protein comprises a second virus polypeptide and an exosomal polypeptide, and said third fusion protein comprises a third virus polypeptide and an exosomal polypeptide.
  • Embodiment 3 An immunogenic composition of embodiment 1 or embodiment 2 further comprising an excipient.
  • Embodiment 4 An immunogenic composition of embodiment 3, wherein the excipient comprises a buffer.
  • Embodiment 5 An immunogenic composition of embodiment 3 or 4, wherein the excipient comprises a cryoprotectant.
  • Embodiment 6 An immunogenic composition of any one of embodiments 1-5 not comprising an adjuvant.
  • Embodiment 7 An immunogenic composition of any one of embodiments 1-4, wherein said first fusion protein and said second fusion protein are present in a membrane of the each respective vesicle.
  • Embodiment 8 An immunogenic composition of any one of embodiments 1-7, wherein a part or all of each virus polypeptide is present at or on an outer surface of the vesicle.
  • Embodiment 9 An immunogenic composition of any one of embodiments 1-8, wherein each fusion protein is present in the composition at a concentration of about 1 ng/100 pL to about 50 ng/100 pL.
  • Embodiment 10 An immunogenic composition of any one of embodiments 1-9, wherein the first virus polypeptide is a SARS-CoV-2 polypeptide and the second virus polypeptide is an influenza polypeptide.
  • Embodiment 1 An immunogenic composition of any one of embodiments 1-10, wherein the first virus polypeptide is a SARS-CoV-2 spike protein polypeptide.
  • Embodiment 12 An immunogenic composition of any one of embodiments 1-11, wherein the first virus polypeptide is a SARS-CoV-2 delta variant spike protein polypeptide.
  • Embodiment 13 An immunogenic composition of any one of embodiments 1-12, wherein the first virus polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO SEQ ID NO: 1, or identical to SEQ ID NO:1.
  • Embodiment 14 An immunogenic composition of any one of embodiments 1-10, wherein the first virus polypeptide is a SARS-CoV-2 nucleocapsid protein polypeptide.
  • Embodiment 15 An immunogenic composition of embodiment 14, wherein the first virus polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO SEQ ID NO:5, or identical to SEQ ID NO:5.
  • Embodiment 16 An immunogenic composition of any one of embodiments 1-15, wherein the second virus polypeptide is an influenza hemagglutinin protein polypeptide.
  • Embodiment 17 An immunogenic composition of any one of embodiments 1-16, wherein the second virus polypeptide is an influenza hemagglutinin 3 (H3) protein polypeptide.
  • H3 influenza hemagglutinin 3
  • Embodiment 18 An immunogenic composition of any one of embodiments 1-17, wherein the second virus polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO: 14, or identical to SEQ ID NO: 14.
  • Embodiment 19 An immunogenic composition of any one of embodiments 2-18, wherein the third virus polypeptide is a respiratory syncytial virus (RSV) protein polypeptide.
  • RSV respiratory syncytial virus
  • Embodiment 20 An immunogenic composition of any one of embodiments 2-19, wherein the third virus polypeptide is an RSV fusion (RSV F) protein polypeptide.
  • RSV F RSV fusion
  • Embodiment 21 An immunogenic composition of any one of embodiments 2-20, wherein the third virus polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO: 18, or identical to SEQ ID NO: 18.
  • Embodiment 22 An immunogenic composition of any one of embodiments 1-21, wherein the exosomal polypeptide is a tetraspanin protein polypeptide.
  • Embodiment 23 An immunogenic composition of any one of embodiments 1-22, wherein the exosomal polypeptide is a CD9 protein polypeptide.
  • Embodiment 24 An immunogenic composition of any one of embodiments 1-23, wherein the exosomal polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO SEQ ID NO:10, or identical to SEQ ID NO:10.
  • Embodiment 25 An immunogenic composition of any one of embodiments 1-13 and 17-24, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO:2, or identical to SEQ ID NO:2.
  • Embodiment 26 An immunogenic composition of any one of embodiments 1-13 and 17-24, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO:3, or identical to SEQ ID NO:3.
  • Embodiment 27 An immunogenic composition of any one of embodiments 1-10 and 14-23, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO:6, or identical to SEQ ID NO:6.
  • Embodiment 28 An immunogenic composition of any one of embodiments 1-10, 14-23 and 27, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO:7, or identical to SEQ ID NO:7.
  • Embodiment 29 An immunogenic composition of any one of embodiments 1-28, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO: 15, or identical to SEQ ID NO: 15.
  • Embodiment 30 An immunogenic composition of any one of embodiments 1-29, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO: 16, or identical to SEQ ID NO:16.
  • Embodiment 31 An immunogenic composition of any one of embodiments 2-30, wherein the third fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO:19, or identical to SEQ ID NO:19.
  • Embodiment 32 An immunogenic composition of any one of embodiments 2-31, wherein the third fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO:20, or identical to SEQ ID NO:20.
  • Embodiment 33 An immunogenic composition of any one of embodiments 1-32, wherein an immunogenic dose of said composition comprises about 1 ng to about 50 ng of the first fusion protein or first virus polypeptide, and about 1 ng to about 50 ng of the second fusion protein or second virus polypeptide.
  • Embodiment 34 An immunogenic composition of any one of embodiments 2-33, wherein an immunogenic dose of said composition comprises about 1 ng to about 50 ng of the third fusion protein or third virus polypeptide.
  • Embodiment 35 A method of immunizing a subject against a virus infection comprising administering to the subject an immunogenically effective dose of an immunogenic composition of any one of embodiments 1-34.
  • Embodiment 36 A method of embodiment 35, wherein said immunogenically effective dose elicits protective immunity in said subject against a first virus and a second virus.
  • Embodiment 37 A method of embodiment 35 or 36, wherein said immunogenically effective dose elicits protective immunity in said subject against a first virus, a second virus, and a third virus.
  • Embodiment 38 A method of embodiment 35 or 36, wherein the immunogenic composition comprises a SARS-CoV-2 spike protein polypeptide and an influenza hemagglutinin polypeptide, and the first virus is a SARS-CoV2 and the second virus is an influenza virus.
  • Embodiment 39 A method of any one of embodiments 35-38, wherein the immunogenic composition comprises a SARS-CoV-2 spike protein polypeptide, an influenza hemagglutinin polypeptide, and a respiratory syncytial virus (RSV) fusion protein polypeptide, and the first virus is a SARS-CoV2, the second virus is an influenza virus, and the third virus is an RSV.
  • Embodiment 40 A method of any one of embodiments 35-39, wherein said immunogenically effective dose elicits protective immunity in said subject against a SARS-CoV- 2 delta variant and a SARS-CoV-2 omicron variant.
  • Embodiment 41 A method of any one of embodiments 35-40, wherein the immunogenically effective dose comprises about 1 ng to about 50 ng of said first fusion protein, about 1 ng to about 50 ng of said second fusion protein, and about 1 ng to about 50 ng of said third fusion protein.
  • Embodiment 42 A method of any one of embodiments 35-41 further comprising administering to the subject a second effective dose of the immunogenic composition of any one of embodiments 1-34.
  • Embodiment 43 A method of any one of embodiments 35-42, wherein the elicited protective immunity comprises (a) a high antibody titer directed against the first, second, and third virus, (b) a CD4+ T-cell response against the first, second, and third virus, and (c) a CD8+ cytotoxic T-cell response against the first, second, and third virus.
  • Embodiment 44 A synthetic fusion protein comprising a virus polypeptide and an exosomal polypeptide.
  • Embodiment 45 A synthetic fusion protein of embodiment 44 further comprising a linker polypeptide positioned between the virus polypeptide and the exosomal polypeptide.
  • Embodiment 46 A synthetic fusion protein of embodiment 44 or 45 further comprising a hinge polypeptide positioned between the virus polypeptide and the exosomal polypeptide.
  • Embodiment 47 A synthetic fusion protein of any one of embodiments 44-46 further comprising a transmembrane domain polypeptide positioned between the virus polypeptide and the exosomal polypeptide.
  • Embodiment 48 A synthetic fusion protein of any one of embodiments 44-47, wherein the exosomal polypeptide is a tetraspanin polypeptide.
  • Embodiment 49 A synthetic fusion protein of any one of embodiments 44-48, wherein the exosomal polypeptide is a CD9 polypeptide.
  • Embodiment 50 A synthetic fusion protein of any one of embodiments 44-49, wherein the virus polypeptide is a SARS-CoV-2 structural protein polynucleotide.
  • Embodiment 51 A synthetic fusion protein of any one of embodiments 44-50, wherein the virus polypeptide is a SARS-CoV-2 spike protein polypeptide.
  • Embodiment 52 A synthetic fusion protein of embodiment 51, wherein said SARS- CoV-2 spike protein polypeptide comprises a one of more of a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and a di-proline substitution (2P [986KV987-to- 986PP987]).
  • CSM furin cleavage site mutation
  • 2P di-proline substitution
  • Embodiment 53 A synthetic fusion protein of any one of embodiments 44-52, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, a SARS-CoV-2 spike protein polypeptide, a linker polypeptide, and a CD9 polypeptide.
  • Embodiment 54 A synthetic fusion protein of any one of embodiments 44-53, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:3.
  • Embodiment 55 A synthetic fusion protein of any one of embodiments 44-54, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:3.
  • Embodiment 56 A synthetic fusion protein of any one of embodiments 44-50, wherein the virus polypeptide in a SARS-CoV-2 nucleocapsid protein polypeptide.
  • Embodiment 57 A synthetic fusion protein of any one of embodiments 44-50 and 56, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, a signal peptide, a SARS-CoV-2 nucleocapsid protein polypeptide, a hinge region, a transmembrane domain polypeptide, a linker polypeptide, and a CD9 polypeptide.
  • Embodiment 58 A synthetic fusion protein of any one of embodiments 44-50, 56 and 57, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:7.
  • Embodiment 59 A synthetic fusion protein of any one of embodiments 44-50 and 56-58, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:7.
  • Embodiment 60 A synthetic fusion protein of any one of embodiments 44-49, wherein the virus polypeptide is an influenza protein polypeptide.
  • Embodiment 61 A synthetic fusion protein of embodiment 60, wherein said influenza protein polypeptide comprises a hemagglutinin.
  • Embodiment 62 A synthetic fusion protein of embodiment 60 or 61, wherein said influenza protein polypeptide comprises a hemagglutinin 3 (H3).
  • H3 hemagglutinin 3
  • Embodiment 63 A synthetic fusion protein of any one of embodiments 60-62, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, a hemagglutinin protein polypeptide, a linker polypeptide, and a CD9 polypeptide.
  • Embodiment 64 A synthetic fusion protein of any one of embodiments 60-63, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16.
  • Embodiment 65 A synthetic fusion protein of any one of embodiments 60-64, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 16.
  • Embodiment 66 A synthetic fusion protein of any one of embodiments 44-49, wherein the virus polypeptide is a respiratory syncytial virus (RSV) protein polypeptide.
  • RSV respiratory syncytial virus
  • Embodiment 67 A synthetic fusion protein of embodiment 66, wherein said RSV protein polypeptide comprises an RSV fusion (RSV F) protein.
  • RSV F RSV fusion
  • Embodiment 68 A synthetic fusion protein of embodiment 66 or 67, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, an RSV F protein polypeptide, a linker polypeptide, and a CD9 polypeptide.
  • Embodiment 69 A synthetic fusion protein of any one of embodiments 66-68, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 20.
  • Embodiment 70 A synthetic fusion protein of any one of embodiments 60-64, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:20.
  • Embodiment 71 A synthetic polynucleotide encoding a synthetic fusion protein of any one of embodiments 44-70.
  • Embodiment 72 A synthetic polynucleotide of embodiment 71 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 13.
  • Embodiment 73 A synthetic polynucleotide of embodiment 71 or 72 comprising a nucleic acid sequence set forth in SEQ ID NO: 13.
  • Embodiment 74 A synthetic polynucleotide of any one of embodiments 71-73 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 11.
  • Embodiment 75 A synthetic polynucleotide of any one of embodiments 71-74 comprising a nucleic acid sequence set forth in SEQ ID NO: 11.
  • Embodiment 76 A synthetic polynucleotide of any one of embodiments 71-75 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:4.
  • Embodiment 77 A synthetic polynucleotide of any one of embodiments 71-76 comprising a nucleic acid sequence set forth in SEQ ID NO:4.
  • Embodiment 78 A synthetic polynucleotide of any one of embodiments 71-73 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 12.
  • Embodiment 79 A synthetic polynucleotide of any one of embodiments 71-73 and 78 comprising a nucleic acid sequence set forth in SEQ ID NO: 12.
  • Embodiment 80 A synthetic polynucleotide of any one of embodiments 71-73, 78 and 79 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:8.
  • Embodiment 81 A synthetic polynucleotide of any one of embodiments 71-73 and 78-80 comprising a nucleic acid sequence set forth in SEQ ID NO:8.
  • Embodiment 82 A synthetic polynucleotide of any one of embodiments 71-73 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:22.
  • Embodiment 83 A synthetic polynucleotide of any one of embodiments 71-73 and 82 comprising a nucleic acid sequence set forth in SEQ ID NO:22.
  • Embodiment 84 A synthetic polynucleotide of any one of embodiments 71-73, 82 and 83 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 17.
  • Embodiment 85 A synthetic polynucleotide of any one of embodiments 71-73 and 82-84 comprising a nucleic acid sequence set forth in SEQ ID NO: 17.
  • Embodiment 86 A synthetic polynucleotide of any one of embodiments 71-73 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:23.
  • Embodiment 87 A synthetic polynucleotide of any one of embodiments 71-73 and 86 comprising a nucleic acid sequence set forth in SEQ ID NO:23.
  • Embodiment 88 A synthetic polynucleotide of any one of embodiments 71-73, 86 and 87 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:21.
  • Embodiment 89 A synthetic polynucleotide of any one of embodiments 71-73 and 86-88 comprising a nucleic acid sequence set forth in SEQ ID NO:21.
  • Embodiment 90 A cell comprising a synthetic polynucleotide of any one of embodiments 71-89.
  • Embodiment 91 A cell of embodiment 90, wherein the cell is a metazoan cell.
  • Embodiment 92 A cell of embodiment 90 or 91 wherein the cell is a vertebrate cell.
  • Embodiment 93 A cell of any one of embodiments 90-92, wherein the cell is a mammalian cell.
  • Embodiment 94 A cell of any one of embodiments 90-93, wherein the cell is a primate cell.
  • Embodiment 95 A cell of any one of embodiments 90-94, wherein the cell is a human cell.
  • Embodiment 96 A cell of any one of embodiments 90-95, wherein the cell is a primary cell.
  • Embodiment 97 A cell of any one of embodiments 90-95, wherein the cell is a human embryonic kidney cell.
  • Embodiment 98 A cell of embodiment 97, wherein the cell is a 293 cell.
  • Embodiment 99 A cell of any one of embodiments 90-98, wherein said cell is produced by transducing the cell with a lentivirus comprising the synthetic polynucleotide.
  • Embodiment 100 A cell of any one of embodiments 90-99, wherein said cell comprises a synthetic fusion protein of any one of embodiments 44-70.
  • Embodiment 101 A vesicle comprising a synthetic fusion protein of any one of embodiments 44-70.
  • Embodiment 102 A vesicle of embodiment 101, wherein said vesicle is an exosome.
  • Embodiment 103 A vesicle of embodiment 101 or 102, wherein said vesicle has a diameter of about 50-500 nm.
  • Embodiment 104 A vesicle of any one of embodiments 101-103, wherein a SARS- CoV-2 spike protein polypeptide is expressed on an outer surface of said vesicle.
  • Embodiment 105 A vesicle of any one of embodiments 101-103, wherein said vesicle expresses a SARS-CoV-2 nucleocapsid protein polypeptide on its surface.
  • Embodiment 106 A vesicle of any one of embodiments 101-103, wherein said vesicle expresses an influenza hemagglutinin protein polypeptide on its surface.
  • Embodiment 107 A vesicle of any one of embodiments 101-103, wherein said vesicle expresses a respiratory syncytial virus fusion protein polypeptide on its surface.
  • Embodiment 108 A method of making a vesicle of any one of embodiments 101-107 comprising culturing a cell of any one of embodiments 90-100 in a cell culture medium, collecting the cell culture medium, and purifying a plurality of vesicles comprising said vesicle from the cell culture medium.
  • Embodiment 109 A method of embodiment 108 further comprising inducing expression of a synthetic polynucleotide of any one of embodiments 71-89 to produce a synthetic fusion protein of any one of embodiments 44-70.
  • Embodiment 110 A method of embodiment 105, wherein said inducing comprises contacting the cell with tetracycline, doxycycline, or analogs thereof.
  • Embodiment 1 11 A method of embodiment 105, wherein said inducing comprises removing from the cell tetracycline, doxycycline, or analogs thereof.
  • Embodiment 112. A method of eliciting an immune response in a subject comprising administering to said subject a first dose of an immunogenic composition comprising a plurality of vesicles comprising a vesicle of any one of embodiments 101-107 or a vesicle produced according to a method of any one of embodiments 108-111, wherein a synthetic fusion protein of any one of embodiments 44-70 is expressed on an outer surface of said vesicle.
  • Embodiment 113 A method of embodiment 112 further comprising administering to said subject a second dose of the immunogenic composition a period of time after administering the first dose.
  • Embodiment 114 A method of embodiment 113, wherein the period of time is 14 days - 1 year.
  • Embodiment 115 A method of any one of embodiments 112-114, wherein the first dose or the second dose comprises about 100 pL - 1 mL of the immunogenic composition, wherein said immunogenic composition comprises about 0.3 ng/mL - 3 pg/mL of said synthetic fusion protein.
  • Embodiment 116 A method of any one of embodiments 112-115, wherein the first dose or the second dose comprises about 100 pL - 1 mL of the immunogenic composition which comprises about 2E9 vesicles/mL - 3E13 vesicles/mL.
  • Embodiment 117 A method of any one of embodiments 112-115, wherein said synthetic fusion protein comprises a SARS-CoV-2 spike protein polypeptide.
  • Embodiment 118 A method of any one of embodiments 112-117, wherein said synthetic fusion protein comprises a SARS-CoV-2 nucleocapsid protein polypeptide.
  • Embodiment 119 A method of any one of embodiments 112-117, wherein said synthetic fusion protein comprises an influenza hemagglutinin protein polypeptide.
  • Embodiment 120 A method of any one of embodiments 112-117, wherein said synthetic fusion protein comprises a respiratory syncytial virus fusion (RSV F) protein polypeptide.
  • RSV F respiratory syncytial virus fusion
  • Embodiment 121 A method of any one of embodiments 1 12-120, wherein said immunogenic composition comprises vesicles that express on the outer surface SARS-CoV-2 spike protein polypeptides, vesicles that express on the outer surface influenza hemagglutinin protein polypeptides, and vesicles that express on the outer surface RSV F protein polypeptides.
  • Embodiment 122 A method of any one of embodiments 112-121, wherein the elicited immune response comprises producing neutralizing antibodies against an antigen present in the synthetic fusion protein.
  • Embodiment 123 A method of any one of embodiments 112-123, wherein the elicited immune response comprises producing anti-spike antibodies.
  • Embodiment 124 A method of any one of embodiments 112-123, wherein the elicited immune response comprises a spike-specific T cell response.
  • Embodiment 125 A method of any one of embodiments 112-124, wherein the elicited immune response comprises producing anti-nucleocapsid antibodies.
  • Embodiment 126 A method of any one of embodiments 112-125, wherein the elicited immune response comprises a nucleocapsid-specific T cell response.
  • Embodiment 127 A method of any one of embodiments 112-126, wherein the elicited immune response comprises producing anti-hemagglutinin antibodies.
  • Embodiment 128 A method of any one of embodiments 112-127, wherein the elicited immune response comprises a hemagglutinin -specific T cell response.
  • Embodiment 129 A method of any one of embodiments 112-128, wherein the elicited immune response comprises producing anti-RSV F antibodies.
  • Embodiment 130 A method of any one of embodiments 112-129, wherein the elicited immune response comprises a RSV F -specific T cell response.
  • Embodiment 131 A method of any one of embodiments 112-130, wherein the immune response persists in the subject for up to nine months.
  • Embodiment 132 A method of any one of embodiments 112-130, wherein the immune response persists in the subject for at least nine months.
  • Extracellular Vesicles and Exosomes
  • a variety of host cells are known in the art and suitable for proteins expression and extracellular vesicles production.
  • Non-limiting examples of typical cell used for transfection include, but are not limited to, a bacterial cell, a eukaryotic cell, a yeast cell, an insect cell, or a plant cell.
  • a bacterial cell eukaryotic cell
  • yeast cell eukaryotic cell
  • insect cell eukaryotic cell
  • plant cell e.g. human embryonic kidney 293 (HEK293), E. coli, Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SJ9, CHO, COS (e g. COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H, or 293F.
  • COS e g. COS-7
  • 3T3-F442A HeLa
  • Extracellular vesicles are lipid bound vesicles secreted by cells into the extracellular space.
  • the three main subtypes of EVs are microvesicles (MVs), exosomes, and apoptotic bodies, which are differentiated based upon their biogenesis, release pathways, size, content, and function.
  • MVs microvesicles
  • exosomes lipid bound vesicles secreted by cells into the extracellular space.
  • apoptotic bodies which are differentiated based upon their biogenesis, release pathways, size, content, and function.
  • Exosomes include small, secreted vesicles of about 20-200 nm in diameter that are released by inter alia mammalian cells, and made either by budding into endosomes or by budding from the plasma membrane of a cell.
  • exosomes have a characteristic buoyant density of approximately 1.1 -1.2 g/mL, and a characteristic lipid composition.
  • Their lipid membrane is typically rich in cholesterol and contains sphingomyelin, ceramide, lipid rafts and exposed phosphatidylserine.
  • Exosomes express certain marker proteins, such as integrins and cell adhesion molecules, but generally lack markers of lysosomes, mitochondria, or caveolae.
  • the exosomes contain cell-derived components, such as, but not limited to, proteins, DNA and RNA (e.g., microRNA [miR] and noncoding RNA).
  • exosomes can be obtained from cells obtained from a source that is allogeneic, autologous, xenogeneic, or syngeneic with respect to the recipient of the exosomes.
  • RNA e.g., microRNA (miRNA)
  • miRNAs function as post-transcriptional regulators, often through binding to complementary sequences on target messenger RNA transcripts (mRNAs), thereby resulting in translational repression, target mRNA degradation and/or gene silencing.
  • Useful exosomes can be obtained from any cell source, including prokaryotes, plants, fungi, metazoans, vertebrate, mammalian, primate, human, autologous cells and allogeneic cells. See, e.g., Kim et al., “Platform technologies and human cell lines for the production of therapeutic exosomes,” Extracell Vesicles Circ Nucleic Acids 2021;2:3-17.
  • exosomes may be derived from mesenchymal stem cells, embryonic stem cells, iPS cells, immune cells, PBMCs, neural stem cells, HEK293 cells, which are described in e.g., Dumont et al., “Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives,” Crit Rev Biotechnol 2016;36: 1110-22, HEK293T cells, which are described in e.g., Li et al., “Identification and characterization of 293T cell-derived exosomes by profiling the protein, mRNA and MicroRNA components,” PLoS One 2016; 1 l:e0163043, 293F cells, Stenkamp et al., “Exosomes represent a novel mechanism of regulatory T cell suppression (P1079),” J Immunol May 1, 2013, 190 (1 Supplement) 121.11, amniotic cells, CAR-T cells, cardiospheres and cardiosphere-derived cells (CDCs), which are described in,
  • methods for preparing exosomes can include the steps of: culturing cells in media, isolating the cells from the media, purifying the exosome by, e.g., sequential centrifugation, and optionally, clarifying the exosomes on a density gradient, e.g., sucrose density gradient.
  • a density gradient e.g., sucrose density gradient.
  • the isolated and purified exosomes are essentially free of non-exosome components, such as cellular components or whole cells.
  • Exosomes can be resuspended in a buffer such as a sterile PBS buffer containing 0.01-1% human serum albumin. The exosomes may be frozen and stored for future use.
  • Exosomes can be collected, concentrated and/or purified using methods known in the art.
  • differential centrifugation has become a leading technique wherein secreted exosomes are isolated from the supernatants of cultured cells. This approach allows for separation of exosomes from larger extracellular vesicles and from most non-particulate contaminants by exploiting their size.
  • Exosomes can be prepared as described in a wide array of papers, including but not limited to, Fordjour et al., “A shared pathway of exosome biogenesis operates at plasma and endosome membranes”, bioRxiv , preprint posted February 11, 2019, at https://www.biorxiv.org/content/10.1101/545228vl; Booth et ah, “Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane”, J Cell BioL, 172:923-935 (2006); and, Fang et ah, “Higher-order oligomerization targets plasma membrane proteins and HIV gag to exosomes”, PLoS Biol., 5 :el58 (2007).
  • Exosomes using a commercial kit such as, but not limited to the ExoSpinTM Exosome Purification Kit, Invitrogen® Total Exosome Purification Kit, PureExo® Exosome Isolation Kit, and ExoCapTM Exosome Isolation kit.
  • Methods for isolating exosome from stem cells are found in, e.g., Tan et ah, Journal of Extracellular Vesicles, 2:22614 (2013); Ono et ah, Sci Signal, 7(332):ra63 (2014) and U.S. Application Publication Nos. 2012/0093885 and 2014/0004601.
  • Size exclusion allows for their separation from biochemically similar, but biophysically different microvesicles, which possess larger diameters of up to 1,000 nm. Differences in flotation velocity further allows for separation of differentially sized exosomes. In general, exosome sizes will possess a diameter ranging from 30-200 nm, including sizes of 40-100 nm. Further purification may rely on specific properties of the particular exosomes of interest. This includes, e.g., use of immunoadsorption with a protein of interest to select specific vesicles with exoplasmic or outward orientations.
  • differential centrifugation is the most commonly used for exosome isolation.
  • This technique utilizes increasing centrifugal force from 2,000xg to 10,000xg to separate the medium- and larger-sized particles and cell debris from the exosome pellet at 100,000xg.
  • Centrifugation alone allows for significant separation/collection of exosomes from a conditioned medium, although it may be insufficient to remove various protein aggregates, genetic materials, particulates from media and cell debris that are common contaminants.
  • Enhanced specificity of exosome purification may deploy sequential centrifugation in combination with ultrafiltration, or equilibrium density gradient centrifugation in a sucrose density gradient, to provide for the greater purity of the exosome preparation (flotation density 1.1 - 1.2 g/mL) or application of a discrete sugar cushion in preparation.
  • Ultrafiltration can be used to purify exosomes without compromising their biological activity.
  • Membranes with different pore sizes - such as 100 kDa molecular weight cutoff (MWCO) and gel filtration to eliminate smaller particles - have been used to avoid the use of a nonneutral pH or non-physiological salt concentration.
  • MWCO molecular weight cutoff
  • THF tangential flow filtration
  • HPLC can also be used to purify exosomes to more uniformly sized particle preparations and preserve their biological activity as the preparation is maintained at a physiological pH and salt concentration.
  • F1FFF Flow field-flow fractionation
  • exosomes may be produced via 293F cells.
  • the 293F cells may be transfected with (or transduced with a lentivirus bearing) a polynucleotide that encodes a spike protein or a nucleocapsid protein or an influenza hemagglutinin protein, or chimeral fusions thereof, as described herein (see Fig. 4A), and expressing the spike protein or nucleocapsid protein, such that the spike protein or nucleocapsid is sorted into and displayed in or on the exosomes isolated therefrom.
  • An example procedure for making exosomes from 293F cells may include steps as follows: 293F cells (GibcoTM, Cat.# 51- 0029, ThermoFisher Scientific, Waltham, MA) may be tested for pathogens and found to be free of viral (cytomegalovirus, human immunodeficiency virus I and II, Epstein Barr virus, hepatitis B virus, and parvovirus Bl 9) and bacterial ( Mycoplasma ) contaminants. Cells may be maintained in FreeStyleTM 293 Expression Medium (Gibco, Cat.# 12338-018, ThermoFisher Scientific, Waltham, MA) and incubated at 37°C in 8% CO2.
  • 293F cells may be seeded at a density of 1.5E6 cells/ml in shaker flasks in a volume of about 1/4 the flask volume and grown at a shaking speed of about 110 rpm.
  • HEK293 cells may be grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum.
  • DMEM Dulbecco’s modified Eagle’s medium
  • the 293F cells may be cultured in shaker flasks for a period of three days. Cells and large cell debris may be removed by centrifugation at 300 x g for 5 minutes followed by 3,000 x g for 15 minutes. The resulting supernatant may be passed through a 0.22 pm sterile filtration filter unit (Thermo Fisher, Cat.# 566-0020) to generate a clarified tissue culture supernatant (CTCS).
  • the CTCS may be concentrated by centrifugal filtration (Centricon Plus-70, Ultracel-PL Membrane, 100 kDa size exclusion, Millipore Sigma, Cat.# UFC710008, St.
  • CTCS size exclusion chromatography
  • lx PBS qEV original columns/35 nm: Izon Science, Cat.# SP5
  • Purified exosomes may be reconcentrated using Amicon® Ultra-4 100 kDa cutoff spin columns (Cat.# UFC810024).
  • This process may yield a population of exosomes/small EVs that have the expected ultrastructure and size distribution profile of human exosomes and contain the exosomal marker proteins CD9 and CD63, at a concentrating effect of about 500-fold, to a final concentration of 1E10 - 2E12 exosomes/ml.
  • concentration and size of the isolated extracellular vesicles may be measured using NANOSIGHT nanoparticle tracking analysis system (Malvern Panalytical, Malvern, UK).
  • membrane-bound vesicles that contain one or more populations of SARS- CoV-2 structural proteins.
  • contain what is meant is that the contained protein may be within the lumen of the vesicle, displayed on the surface of the vesicle, or both within the lumen and on the surface.
  • those fusion proteins containing a tetraspanin protein polypeptide sequence are mostly displayed on the surface of the vesicle.
  • the SARS-CoV-2 structural protein is a spike glycoprotein (S), a nucleocapsid (N) protein, a membrane (M) protein, or an envelope (E) protein, or any combination thereof. See Satarker and Nampoothiri, “Structural Proteins in Severe Acute Respiratory Syndrome Coronavirus-2,” Arch Med Res. 2020 Aug;
  • Figs. 1 A, IB, 2A, and 2C depict spike/CD9 and nucleocapsid/CD fusion proteins for expression of spike or nucleocapsid antigen on the surface of the exosomes.
  • the antigenic protein is SARS-CoV-2 spike glycoprotein (a.k.a. spike protein or simply “spike”).
  • the spike protein can be of any variant of SARS-CoV-2, such as, e.g., the Wuhan-1 strain, an omicron variant (e.g., BA.2 variant), a delta variant (e.g., B.1.617.2, AYA, AY.103, AY.44, AY.43 variant, or the like), and epsilon variant (e.g., B.1.427 or B.1.429 variant), or any variant now known or yet to be discovered.
  • the term spike refers to any SARS-CoV-2 spike glycoprotein, chimera, or fragment thereof unless otherwise specified.
  • the SARS-CoV-2 spike protein is the Wuhan-1 strain SARS-CoV-
  • the invention provides extracellular vesicles that express on their surface (a.k.a. “display”) spike protein or nucleocapsid that are useful as a vaccine against multiple variants of SARS-CoV-2.
  • the invention provides extracellular vesicles that express on their surface (a.k.a. “display”) hemagglutinin protein or neuraminidase protein that are useful as a vaccine against multiple variants of influenza.
  • the invention provides a combination of vesicles, some of which display a SARS-CoV-2 protein and some of which display an influenza protein.
  • the spike protein may be a delta variant with any one or more of trimer stabilization mutation, a prefusion conformation stabilization mutation (e g., di-proline stabilization mutations), and a furin cleavage site mutation.
  • a prefusion conformation stabilization mutation e g., di-proline stabilization mutations
  • a furin cleavage site mutation e.g., a prefusion conformation stabilization mutation (e g., di-proline stabilization mutations), and a furin cleavage site mutation.
  • Walls et al. “Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein,” Cell 180, 281-292, April 16, 2020
  • Wrapp et al. “Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation,” Science367, 1260-1263 (2020)13 March 2020
  • Kirchdoerfer et al. “Stabilized coronavirus spikes are resistant to conform
  • nucleocapsid protein is a soluble protein that is not expressed at the virus surface, therefore the engineered fusion protein that includes tetraspanin and other transmembrane domains enables the placement of nucleocapsid protein at the vesicle surface to provide a readily accessible antigen for immunization (see Fig. 2B).
  • the exosomes that express spike protein or nucleocapsid protein on their surface were made from spike/nuclceocapsid protein-expressing 293F cells.
  • a packaging cell (300) was transfected with a plasmid that encodes the spike- or nucleocapsid- or hemagglutinin-tetraspanin fusion protein (301a) and plasmids that encode lentivirus structural proteins (302a and 303a).
  • the lentivirus proteins were produced (302b and 303b) and incorporated the fusion protein RNA to form a lentivirus vector containing the fusion protein RNA (304).
  • a host cell (311) was transduced with the fusion protein RNA-bearing lentivirus (304), enabling the production of the SARS-CoV-2 spike or nucleocapsid - tetraspanin fusion protein (307a), and its sorting to the plasma membrane (307b) (Fig. 4B) and the surface of exosomes that were produced by the transduced host cells (Figs. 4C, 5A, 5B, and 6C).
  • Figures 5A-C and 6A and 6B exosomes were isolated from 293F cells that harbored the spike-CD9 construct (Fig. 1 A and Fig. IB).
  • Figure 5A shows the size distribution of those exosomes from about 50 nm to about 270 nm, with a median of about 100 - 150 nm.
  • Figure 5B shows expression of spike-containing fusion protein in the transduced 293F cells (lane 4), and enriched expres si on/di splay of the spike-containing fusion protein in the exosomes that were derived from the transduced 293F cells (lane 5).
  • Figure 5C demonstrates significant expression of spike-containing fusion protein on those exosomes as determined by spike flow cytometry.
  • Figures 7A-C exosomes were isolated from 293F cells that harbored the nucleocapsid-CD9 construct (Fig. 2A and Fig. 2B).
  • Figure 7A shows the size distribution of those exosomes from about 50 nm to about 270 nm, with a median of about 100 - 150 nm.
  • Figure 7B shows expression of nucleocapsid-containing fusion protein in the exosomes that were derived from the transduced 293F cells (lane 5).
  • Figure 7C demonstrates significant expression of spike-containing fusion protein on those exosomes as determined by spike flow cytometry.
  • expression of the nucleocapsid-CD9 fusion protein was put under control of the tet-inducible promotor.
  • the exosomes derived from the uninduced, but transduced host cells did not express nucleocapsid (see second curve from left at Fig. 7C).
  • FIG. 8A - 8D 293F cells were transfected or transduced with constructs or virions encoding a hemagglutinin (e.g., H3)-CD9 construct (Fig. 3A and Fig. 3B).
  • Figures 8A and 8B demonstrate significant expression ( ⁇ 90%-96% in this example) of influenza H3- containing fusion protein on/in adherent 293F cells and 293F cells in suspension, respectively, as determined by anti-H3 flow cytometry.
  • Exosomes produced by the 293F cells expressing the H3 fusion protein were purified and assessed for H3 and H3-CD9 by western blot (Fig. 8C, panel 1 and panel 2 respectively).
  • subject mice were administered by intramuscular injection IX dose (i.e., 3E10 vesicles) or 10X dose i.e., 3E11 vesicles) or vesicles containing about 0.3 ng, about 3 ng, or about 10 ng of fusion protein, including spike-CD9, nucleocapsid-CD9, or influenza H3-CD9 fusion protein (710).
  • IX dose i.e., 3E10 vesicles
  • 10X dose i.e., 3E11 vesicles
  • vesicles containing about 0.3 ng, about 3 ng, or about 10 ng of fusion protein, including spike-CD9, nucleocapsid-CD9, or influenza H3-CD9 fusion protein (710).
  • blood was collected from the administered mice and assessed for early humoral immune-responses (720) (see Figs. 10A and 12A-F).
  • the subject mice received a second dose of the subject
  • splenocytes were collected from the administered mice and assessed via ELISpot assay for cellular immune-responses (750) (see Figs. 10C, 10D, 13C, and 13D).
  • FIG. 11A-C the administration of nanogram quantities of STX-S to subjects elicited potent neutralization of both delta and omicron variants of SARS-CoV-2 induced by STX-S exosome injection.
  • day-40 sera from subjects administered about 3.2 ng STX-S per injection (dose 2, Fig. 11A) and day-14 and day-40 sera from subjects administered about 9.8 ng STX-S per injection (dose 4, Fig. 11 A) were tested for neutralizing antibodies against SARS-CoV-2 Delta variant.
  • potent neutralizing activity was elicited by STX-S in each sample tested (Fig. 11 A) comparable to SARS-CoV-2 delta positive vaccine sera.
  • the STX-S engineered exosome vaccine induced in subject(s) neutralizing antibodies against delta spike by day 14 (after a single i.m. injection).
  • day 40 ⁇ 4 weeks post STX-S boost, robust neutralization was observed in all subjects regardless of dose (Fig. 11A).
  • one injection with about 9 ng of spike by STX-S dose 4, Fig. 11A
  • delivered about 65-75% neutralization against a delta variant whereas full immunization (i.e., initial injection and at least one booster, or two i.m. injections) resulted in about 80-85% of neutralization against delta variant at a dose of about 3-9 ng of spike delivered by STX-S exosomes.
  • an immunogenic composition containing single variant spike STX-S exosomes provides some level of protective immunity against other SARS-CoV-2 variants.
  • FIGS. 12A-F vaccine combinations containing both STX-S and exosomes that express influenza hemagglutinin 3 antigen on their surface (STX-H3) were administered to murine subjects.
  • Sera obtained from the mice 14 days after injection with low doses of STX-H3 alone shows significant levels of anti-H3 IgGs.
  • low doses (3E10 to 3E11 exosomes representing low nanogram amounts of H3 antigen) elicited significant IgG responses in the mice.
  • a low-dose immunogenic composition or vaccine contains an immunogenic dose inclusively between 1 ng and 1 pg viral antigen (e.g., SARS-CoV-2 spike, SARS-CoV-2 nucleocapsid, or influenza hemagglutinin, or combinations thereof), 1-900 ng, 1-800 ng, 1-700 ng, 1-600 ng, 1-500 ng, 1-400 ng, 1-300 ng, 1- 200 ng, 1-100 ng, 1-90 ng, 1-80 ng, 1-70 ng, 1-60 ng, 1-50 ng, 1-40 ng, 5-100 ng, 5-90 ng, 5-80 ng, 5-70 ng, 5-60 ng, 5-50 ng, 5-40 ng, ⁇ 100 pg, ⁇ 50 pg, ⁇ 25 pg, ⁇ 20 pg, ⁇ 10 pg, ⁇ 1 pg, ⁇ 900 ng, ⁇ 800 ng, ⁇ 700 ng
  • viral antigen e.g., SARS
  • a complete immunization cycle (two i.m. injections) induced a significant 3-fold increase of IgG against SARS-CoV-2 nucleocapsid protein (N) over PBS for the dose of about 3 ng/inj ection (dose 2, Fig. 9A) up to a 10-fold at 10 ng/injection (dose 3, Fig. 13B).
  • FIGs. 13C and 13D to characterize the T cell response to STX-N, antigenspecific T cell responses to nucleocapsid protein were measured by ELISpot assays performed on splenocytes obtained at day 40.
  • vaccination of subjects with STX-N elicited multifunctional, antigen-specific T cell responses against SARS-CoV-2 nucleocapsid protein at day 40 (after boost (2nd) injection).
  • STX-N administration resulted in a potent IFNy response (Figs. 13C and 13D).
  • the significant CD8+/IFNy response to nucleocapsid protein in subjects was effected with administered doses of STX-N in the 3 to 10 ng range without adjuvant, which is on the order of three orders of magnitude lower than prior art protein subunit vaccines.
  • the spike-exosome vaccine, the nucleocapsid-exosome vaccine, the hemagglutinin-exosome vaccine, and the RSV-exosome vaccine each presents several other advantages over currently available vaccines.
  • the subject exosome-based vaccines deliver antigen through an entirely endogenous, autologous lipid bilayer, that can be easily integrated into the host cell membrane and facilitate engineered antigen presentation to immune cells.
  • Membrane bound antigen can easily be presented to the circulating immune cells to quicky activate a response, while free antigen contained inside the exosome can additionally be processed by the lysosomal system and activate the cytotoxic T- lymphocyte response.
  • utilization of a natural delivery system promotes efficient delivery and response compared with synthetic lipid nanoparticle technology.
  • the spike, nucleocapsid, RSV, and hemagglutinin exosome vaccines are proteinbased vaccines, making the antigen readily available to the subject immune system without translation in a host cell as is required for RNA-based vaccines. Incomplete translation, and/or incorrect folding of mRNA encoding Spike, nucleocapsid, or hemagglutinin protein limits the amount of available antigen after vaccination, making the immune response extremely variable and with reduced efficacy.
  • the subject exosome-based spike, nucleocapsid, hemagglutinin, and RSV fusion protein vaccines do not require an adjuvant or synthetic lipid nanoparticle (LNP) for delivery and immune response.
  • Traditional protein-based vaccines require adjuvants (such as, e.g, aluminum salt and the squalene oil-in-water emulsion systems MF59 (Novartis) and AS03 (GlaxoSmithKline)) (see, e.g., Wong, S.S. and R.J. Webby, “Traditional and new influenza vaccines,” Clin Microbiol Rev, 2013. 26(3): p.
  • the subject exosomal spike fusion protein, nucleocapsid fusion protein, hemagglutinin fusion protein, and RSV-fusion protein immunogenic compositions exhibit greater immunogenic efficacy at a significantly lower, i.e., three orders of magnitude lower, dose than currently available LNP mRNA and protein vaccines.
  • clinical approved protein candidate vaccines use approximately 5ug to 25ug of antigen in conjunction with an adjuvant to induce immunization (see, e.g., Formica, et al., “Different dose regimens of a SARS- CoV-2 recombinant spike protein vaccine (NVX-CoV2373) in younger and older adults: A phase 2 randomized placebo-controlled trial,” PLoS Med, 2021. 18(10): p. el003769; Sun et al., “Development of a Recombinant RBD Subunit Vaccine for SARS-CoV-2,” Viruses, 2021.
  • exosomes including display exosomes
  • the spike fusion or nucleocapsid fusion-expressing exosome composition can also be lyophilized for long term storage at convenient temperature (see, e.g., U.S. Pat. App. No. 2017/0360842 Al).
  • the subject fusion protein-expressing exosomes may be engineered to express antigens of interest to target new CO VID variants and/or problematic influenza strains.
  • the antigen of interest could easily be swapped and adapted to the needs.
  • Antigens could be expressed as fusion proteins (e.g., chimeras) with, e.g., exosome expression or display domains fused to antigen domains to enable display of antigens on the exosome for delivery to the host subject immune system.
  • exosomes could be engineered to selectively target organs or tissues of interest and allow safe and targeted delivery of antigens to specific immune subsystems to elicit specific types of responses in the subject.
  • FIG 14B enrichment of Spike protein in exosomes was confirmed by Jess- automated Western Blot (Lane 5: STX-S exo).
  • lane 1 marker
  • lane 2 non-engineered 293F cells
  • lane 3 non-engineered 293F exosomes
  • lane 4 STX-S cells
  • lane 6 Spike protein.
  • FIG 15A high expression of influenza hemagglutinin 3 (H3) was detected on cell surfaces (right curve) by flow cytometry.
  • Parent non-engineered 293F cells (left curve) do not express Influenza H3 protein, as expected.
  • CD81 was detected on STX exosomes (right curve) by flowcytometry using a bead-assay compared with no signal from isotype control antibody (left curve).
  • FIG. 17-19 demonstrate the immunoresponse to intramuscular injection of a combination of spike expressing exosomes (STX-S) and H3 expressing exosomes (STX-H3) (the combination as STX-S+H3) vaccine in a mouse model.
  • STX-S spike expressing exosomes
  • STX-H3 H3 expressing exosomes
  • mice were injected on day 1, blood was collected on day 14, mice received a booster injection (IM) on day 21, blood was collected on day 35, and splenocytes were procured on day 40.
  • the STX-S+H3 vaccine induced robust expression of Influenza H3 and SARS-CoV-2 spike antibodies in mice after 1 (day 14) and 2 (day 35) IM injections as analyzed by ELISA.
  • PBS was used as a vehicle control in all studies.
  • FIG. 20 depicts five construct versions of RSV F.
  • RSV F version 1 (“VI”) is a DS-Cavl engineered form as described in McLellan et al., “Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus,” Science, 1 Nov 2013, Vol 342, Issue 6158, pp. 592-598, doi:
  • RSV F version 2 (“V2”) starts with the VI DS-Cavl engineered form with the polyadenylation signals removed across the F gene.
  • RSV F version 3 (“V3”) starts with the V2 engineered form with both furin cleavage sites mutated as described in Brakel et al., “Coexpression of respiratory syncytial virus (RSV) fusion (F) protein and attachment glycoprotein (G) in a vesicular stomatitis virus (VSV) vector system provides synergistic effects against RSV infection in a cotton rat model,” Vaccine, 2021 Nov 16;39(47):6817-6828. doi: 10.1016/j. vaccine.2021.10.042.
  • RSV respiratory syncytial virus
  • F respiratory syncytial virus
  • G attachment glycoprotein
  • V4 RSV F version 4
  • SEQ ID NO:20 A specific version of a V4 construct is exemplified in SEQ ID NO:20.
  • the V3 construct in which both furin cleavage sites were removed, showed a high percentage (about 96-97%) and high expression level of full-length RSV F protein in 293F transduced cells. However, the V3 expression was observed to be unstable.
  • RSV F V3-CD9 chimera transfected cells with top 10% RSV F expression were FACS sorted. CD9 and RSV F protein expression was measured 1 week and 2 weeks after FACS sorting. Both non-sorted pool and sorted pool showed a loss of RSV F protein expression (using an anti-AM22), however, CD9 expression remained at high level, indicating loss of V3 expression. 293F-generated exosomes produced from the sorted pool showed very low to no RSV F on the exosomes.
  • the RSV F V3-CD9-expressing exosomes were tested for a humoral immune response in vivo and the mice showed low antibody responses against RSV F protein on day 35 after two injections (Fig. 22, panels A and B).
  • two lots of exosomes expressing the V3 RSV F-CD9 chimera fusion protein with lot #1 producing 2.0E12 exosomes per mL at a protein concentration of about 43 ng/mL and lot #2 producing 1.8E12 exosomes per mL at a protein concentration of about 26 ng/mL.
  • Lot #1 (2 mL) and lot #2 (1.3 mL) were combined to obtain a combined lot #1.2 of 3.3mLl for a final concentration of 25.27 ng/mL.
  • 10 mice received two injections at ⁇ 3ng/inj ection (using lOOul of the stock).
  • Antibodies were detected after injection at days-14 (Fig. 22A) and day-35 (Fig. 22B), but the immune response was not sufficient for eliciting good humoral immunity.
  • V4 construct was observed to have improved (relative to V3) expression levels and stability in transduced 293F cells, as determined by anti-RSV F flow cytometry using 0 antigen site antibodies (AM22, D25) and two RSV-neutralizing site II monoclonal antibodies (palivizumab, motavizumab) and JESS western blotting.
  • RSV F V4-CD9-expressing exosomes were tested for a humoral immune response in vivo and observed to elicit a strong immune response at days 14 and 35 (Fig. 23, panels A and B) with a clear boost effect at day 35 (Fig. 23B).
  • the RSV concentration on exosomes was about 250 ug/mL and the total protein was 990ug/mL per 1.9E12 exosome/mL.
  • the RSV F protein is fused to a tetraspanin protein.
  • the RSV F protein is fused, i.e., as part of an engineered chimeric protein, to CD9.
  • the engineered chimeric protein contains at the N-terminal an RSV F polypeptide, preferably the RSV F V4 construct (SEQ ID NO: 18), followed by a linker, then the CD9 protein at the C-terminal end (Fig. 21 A), which permits the RSV F protein to be expressed on the outer surface of a vesicle (Fig. 21B).
  • the RSV F-CD9 chimeric construct has an amino acid sequence of SEQ ID NO:20.
  • the subject vaccine or immunogenic composition contains a combination of SARS-CoV2 spike-expressing vesicles and influenza hemagglutinin-expressing vesicles (Fig. 24). In another embodiment, the subject vaccine or immunogenic composition contains a combination of SARS-CoV2 spike-expressing vesicles and respiratory syncytial virus fusion protein (RSV F)-expressing vesicles (Fig. 25). In another embodiment, the subject vaccine or immunogenic composition contains a combination of influenza hemagglutinin-expressing vesicles and respiratory syncytial virus fusion protein (RSV F)-expressing vesicles (Fig. 26). In another embodiment, the subject vaccine or immunogenic composition contains a combination of SARS-CoV2 spike-expressing vesicles, RSV F-expressing vesicles, and influenza hemagglutinin-expressing vesicles (Fig. 27).
  • a combination of SARS-CoV2 spike-expressing exosomes (STX-S), RSV F-expressing exosomes (STX-RSV), and influenza hemagglutinin-expressing exosomes (STX-H3) were injected in mice in single formulation or in combination with each other, to verify the induction of an antibody response.
  • STX-S SARS-CoV2 spike-expressing exosomes
  • STX-RSV RSV F-expressing exosomes
  • STX-H3 influenza hemagglutinin-expressing exosomes

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Abstract

The present disclosure relates to compositions and methods for vaccinating a subject against multiple SARS-CoV-2 variants and other respiratory viruses that involves the making and delivery of extracellular vesicles expressing on their surface engineered spike protein, engineered nucleocapsid protein, engineered hemagglutinin protein, and/or engineered respiratory syncytial virus prefusion or fusion (RSV F) protein to the subject. The present invention also relates to compositions and methods for the design, preparation, manufacture, formulation, and/or use of spike-display, nucleocapsid-display, hemagglutinin-display, and/or RSV F-display vesicular vaccines designed to elicit strong humoral and cellular immune responses against multiple respiratory viruses and variants.

Description

COMBINATION EXOSOMAL IMMUNOGENIC COMPOSITIONS AND METHODS
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority dates of U.S. Provisional Application No. 63/412,226, filed September 30, 2022, and U.S. Provisional Application No. 63/455,902, filed March 30, 2023, the contents of which are incorporated herein by reference in their entireties.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 27, 2023, is named 516PCTseqlst and is 49,339 bytes in size.
BACKGROUND
[0003] The emergence, reemergence, and mutation of severe respiratory viral infections, such as acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza, and respiratory syncytial virus (RSV) have created an urgent need for vaccine development strategies to produce safe, effective, readily available and accessible vaccines that can be quickly and efficiently produced to combat the emergence of evolving variants. In the example of SARS-CoV-2, many vaccines have been developed, which try to achieve immunity to the virus, mainly directing the immune response to surface spike protein, which binds to the host cell receptor angiotensin-converting enzyme 2 (ACE2), mediating viral cell entry and infection. The spike (S) protein is a class I fusion glycoprotein, the major surface protein on the SARS-CoV-2 virus and the primary target for neutralizing antibodies. Spike is also the primary site of mutations identified in SARS-CoV-2 viruses, reducing the efficacy of the immune-response induced by vaccines. Since the beginning of the CO VID-19 pandemic, five variants of concern (VOC) and 8 variants of interest (VOI) have been reported, which poses challenges to current vaccines and creating the need for more effective vaccines. Vaccines directed against SARS-CoV-2 nucleocapsid protein, which is an internal soluble coronavirus protein, have heretofore been largely unsuccessful. [0004] Current leading vaccines have employed mRNA-lipid nanoparticle or virus vector technology, with more recent vaccines utilizing recombinant proteins (see, e.g., Krammer, F., SARS-CoV-2 vaccines in development. Nature, 2020. 586(7830): p. 516-527). Compared to the leading mRNA vaccines, recombinant or inactivated protein vaccines are a safe and reliable approach for immunization but generally suffer from weak immunogenicity, thus requiring formulation with an appropriate adjuvant. While all current mRNA vaccine candidates achieve a strong initial immune response to the virus with consequent reduction of hospitalization rates, they all lack long-term protection. It’s been reported that antibody levels drastically decline over time and therefore the efficacy to protect against infection drastically declines as well (see, e.g., Xiang, T., et al., Declining Levels of Neutralizing Antibodies Against SARS-CoV-2 in Convalescent COVID-19 Patients One Year Post Symptom Onset. Front Immunol, 2021. 12: p. 708523). Moreover, mRNA vaccines have thus far lacked cross-reactivity against new variants of concern, requiring multiple booster injections to maintain protection against the virus. Thus, there remains a significant medical and public health need for a better, more fast and efficient manufacturing and globally deployable vaccine for influenza, RSV, and SARS-CoV-2 to provide improved, broader, longer lasting neutralization of these viruses, a more robust T cell response, and an enhanced safety profile.
[0005] Briefly, a problem to which the present invention may be directed includes the production of SARS-CoV-2 variants of concern having more infectivity and less immunogenicity than some variants of lesser concern. This creates an ongoing public health and medical problem in which vaccines are no longer effective against new strains, and new strains that are less immunogenic (long term and short-term immunogenicity).
[0006] This problem is similar to that of influenza, where new variants develop each year or different variants become problematic from season to season. Thus a problem exists for the development and delivery of a combination vaccine, e.g., a seasonal vaccine, to both SARS- CoV-2 variants and influenza variants.
[0007] Disclosed is a solution to this problem, which includes a safe (LNP-free, adjuvant-free) and effective combination vaccine that confers long term humoral and cell-mediated immunity against multiple, emerging, and recalcitrant variants of SARS-CoV-2, influenza, and/or RSV which includes an extracellular vesicle that displays a spike protein or nucleocapsid protein from a single variant of SARS-CoV-2, which confers robust humoral and/or cellular immunity against several SARS-CoV-2 variants of concern or interest, combined with an extracellular vesicle that displays a hemagglutinin protein from a single variant of influenza, which also confers immunity against an influenza variant of interest, and or combined with an extracellular vesicle that displays an RSV protein from a single variant of RSV, which also confers immunity against an RSV variant of interest.
SUMMARY
[0008] In one aspect, an immunogenic composition is provided that contains a combination of two or more vesicle-expressing virus antigen substances. In one embodiment, the immunogenic composition contains (i) a plurality of vesicles that express on their surface a SARS-CoV-2 protein polypeptide or antigen, and (ii) a plurality of vesicles that express on their surface an influenza protein polypeptide or antigen.
[0009] In another aspect, an immunogenic composition is provided that contains a combination of two or more vesicle-expressing virus antigen substances. In one embodiment, the immunogenic composition contains (i) a plurality of vesicles that express on their surface a SARS-CoV-2 protein polypeptide or antigen, and (ii) a plurality of vesicles that express on their surface an RSV protein polypeptide or antigen.
[0010] In another aspect, an immunogenic composition is provided that contains a combination of two or more vesicle-expressing virus antigen substances. In one embodiment, the immunogenic composition contains (i) a plurality of vesicles that express on their surface an influenza protein polypeptide or antigen, and (ii) a plurality of vesicles that express on their surface an RSV protein polypeptide or antigen.
[0011] In another aspect, an immunogenic composition is provided that contains a combination of two or more vesicle-expressing virus antigen substances. In one embodiment, the immunogenic composition contains (i) a plurality of vesicles that express on their surface a SARS-CoV-2 protein polypeptide or antigen, (ii) a plurality of vesicles that express on their surface an influenza protein polypeptide or antigen, and (iii) a plurality of vesicles that express on their surface an RSV protein polypeptide or antigen. [0012] In one embodiment of the various immunogenic composition aspects, the SARS-CoV-2 protein is a spike glycoprotein, such as, for example, a SARS-CoV-2 delta spike protein or engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is a nucleocapsid protein or engineered variant thereof. In one embodiment, the influenza protein is a hemagglutinin protein or engineered variant of a hemagglutinin protein, such as, for example, an H3 glycoprotein or engineered variant thereof. In another embodiment, the influenza protein is a neuraminidase protein or engineered variant thereof. In one embodiment, the RSV protein is an RSV fusion (F) protein, such as, for example, RSV F protein or prefusion F protein (see, e.g., Simoes et al., N Engl J Med 2022; 386: 1615-1626). In one embodiment, the vesicle is an exosome. In one aspect, the virus protein polypeptide or antigen is fused to an exosomal protein, such as, for example, a tetraspanin, or more specifically, a CD9 protein or engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.
[0013] In some embodiments of the aforementioned immunogenic compositions, the immunogenic composition contains a plurality of vesicles containing the synthetic fusion protein at a concentration of about 2E9 vesicles/mL - 3E13 vesicles/mL. In those embodiments in which the immunogenic composition contains two or more types of vesicles, i.e., one type containing a first synthetic fusion protein and another type containing a second synthetic fusion protein, et cetera, each vesicle type may be provided at a concentration of about 1E9 vesicles/mL - 2E13 vesicles/mL or 2E9 vesicles/mL - 3E13 vesicles/mL.
[0014] In some embodiments, the immunogenic composition contains a plurality of vesicles containing the synthetic fusion protein at a concentration of about 0.3 ng/mL - 3 pg/mL of the synthetic fusion protein. In those embodiments in which the immunogenic composition contains two or more types of vesicles, i.e., one type containing a first synthetic fusion protein and another type containing a second synthetic fusion protein, et cetera, each vesicle type may be provided at a fusion protein concentration of about 0.1 ng/mL - 1.5 pg/mL or about 0.3 ng/mL - 3 pg/mL.
[0015] In some embodiments, the immunogenic composition further contains one or more pharmaceutically acceptable excipients. In one embodiment, the immunogenic composition does not contain an adjuvant. [0016] In some embodiments, a vesicle of the plurality of vesicles of the immunogenic composition has an average diameter of about 50-500 nm. In one embodiment, the vesicle is a synthetic vesicle. In one embodiment, the vesicle is produced by a cell. In one embodiment, the vesicle is an extracellular vesicle. In one embodiment, the vesicle is a microvesicle. In one embodiment, the vesicle is an exosome. In one embodiment, the vesicle is an apoptotic body. In one embodiment, the vesicle expresses a CD81 protein on its surface.
[0017] In one aspect, a method of eliciting an immune response in a subject is provided by administering to the subject a dose of an immunogenic composition described in the preceding aspects and embodiments. In some embodiments, the subject is administered more than one dose, such as a second dose administered a period of time after the first dose, and/or subsequent booster doses. In some embodiments, the period of time (e.g., between doses) is 14 days - 1 year.
[0018] In one embodiment, a dose contains about 100 pL - 1 mL of the immunogenic composition. In some embodiments, the immunogenic composition contains about 0.1 ng/mL - 3 pg/mL of synthetic fusion protein in a form expressed on the surface of vesicles. In some embodiments, the immunogenic composition contains about 2.81E9 vesicles/mL - 2.81E13 vesicles/mL that express synthetic fusion protein on their surfaces.
[0019] In some embodiments, the immune response that is elicited in the subject is the production of neutralizing antibodies against a virus, such as SARS-CoV-2, influenza, RSV, and the like. In some embodiments, the immune response that is elicited in the subject is the production of neutralizing antibodies against two or more SARS-CoV-2 variants, such as for example a delta variant, an omicron variant, or other variant of interest or concern now known or yet to be discovered.
[0020] In some embodiments, the immune response that is elicited in the subject is the production of anti-spike antibodies. In some embodiments, the immune response that is elicited in the subject is the production of anti-nucleocapsid antibodies. In some embodiments, the immune response that is elicited in the subject is a spike-specific T cell response, such as CD4+ and/or CD8+ response. In some embodiments, the immune response that is elicited in the subject is a nucleocapsid-specific T cell response, such as CD4+ and/or CD8+ response. [0021 ] In some embodiments, the immune response that is elicited in the subject is the production of anti-hemagglutinin antibodies. In some embodiments, the immune response that is elicited in the subject is a spike-specific T cell response, such as CD4+ and/or CD8+ response. In some embodiments, the immune response that is elicited in the subject is a hemagglutininspecific T cell response, such as CD4+ and/or CD8+ response.
[0022] In some embodiments, the immune response that is elicited in the subject is the production of anti-RSV F antibodies. In some embodiments, the immune response that is elicited in the subject is a spike-specific T cell response, such as CD4+ and/or CD8+ response. In some embodiments, the immune response that is elicited in the subject is a RSV F-specific T cell response, such as CD4+ and/or CD8+ response.
[0023] In another aspect, a method for vaccinating a subject against influenza and SARS-CoV-2 is provided. In one embodiment, a subject is administered a composition containing (i) a plurality of vesicles that express on their surface a SARS-CoV-2 protein polypeptide or antigen, and (ii) a plurality of vesicles that express on their surface an influenza protein polypeptide or antigen. In one embodiment, the SARS-CoV-2 protein is a spike glycoprotein, such as, for example, a SARS-CoV-2 delta variant spike protein or an engineered variant thereof. In another aspect, the SARS-CoV-2 protein is a nucleocapsid protein or an engineered variant thereof. In one embodiment, the influenza protein is a hemagglutinin protein, such as, for example, an H3 glycoprotein or an engineered variant thereof. In another embodiment, the influenza protein is a neuraminidase protein or an engineered variant thereof. In one embodiment, the vesicle is an exosome. In one embodiment, the virus protein polypeptide or antigen is fused to an exosomal protein, such as, for example, a tetraspanin, or more specifically, a CD9 protein or engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.
[0024] In another aspect, a method for vaccinating a subject against RSV and SARS-CoV-2 is provided. In one embodiment, a subject is administered a composition containing (i) a plurality of vesicles that express on their surface a SARS-CoV-2 protein polypeptide or antigen, and (ii) a plurality of vesicles that express on their surface an RSV protein polypeptide or antigen. In one embodiment, the SARS-CoV-2 protein is a spike glycoprotein, such as, for example, a SARS- CoV-2 delta variant spike protein or an engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is a nucleocapsid protein or an engineered variant thereof. In one embodiment, the RSV protein is a F-protein or prefusion F-protein or an engineered variant thereof. In one embodiment, the vesicle is an exosome. In one embodiment, the virus protein polypeptide or antigen is fused to an exosomal protein, such as, for example, a tetraspanin, or more specifically, a CD9 protein or engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.
[0025] In another aspect, a method for vaccinating a subject against influenza and RSV is provided. In one embodiment, a subject is administered a composition containing (i) a plurality of vesicles that express on their surface an RSV protein polypeptide or antigen, and (ii) a plurality of vesicles that express on their surface an influenza protein polypeptide or antigen. In one embodiment, the RSV protein is a F-protein or prefusion F-protein or an engineered variant thereof. In one embodiment, the influenza protein is a hemagglutinin protein, such as, for example, an H3 glycoprotein or an engineered variant thereof. In another embodiment, the influenza protein is a neuraminidase protein or an engineered variant thereof. In one aspect, the vesicle is an exosome. In one aspect, the virus protein polypeptide or antigen is fused to an exosomal protein, such as, for example, a tetraspanin, or more specifically, a CD9 protein or engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.
[0026] In another aspect, a method for vaccinating a subject against influenza and RSV is provided. In one aspect, a subject is administered a composition containing (i) a plurality of vesicles that express on their surface a SARS-CoV-2 protein polypeptide or antigen, (ii) a plurality of vesicles that express on their surface an influenza protein polypeptide or antigen, and (iii) a plurality of vesicles that express on their surface an RSV protein polypeptide or antigen. In one embodiment, the SARS-CoV-2 protein is a spike glycoprotein, such as, for example, a SARS-CoV-2 delta variant spike protein or an engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is a nucleocapsid protein or an engineered variant thereof. In one embodiment, the influenza protein is a hemagglutinin protein, such as, for example, an H3 glycoprotein or an engineered variant thereof. In another embodiment, the influenza protein is a neuraminidase protein or an engineered variant thereof. In one embodiment, the RSV protein is a F-protein or prefusion F-protein or an engineered variant thereof. In one embodiment, the vesicle is an exosome. In one embodiment, the virus protein polypeptide or antigen is fused to an exosomal protein, such as, for example, a tetraspanin, or more specifically, a CD9 protein or engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.
[0027] In one aspect, a synthetic fusion protein is provided that contains polypeptide sequences of a SARS-CoV-2 protein fused to polypeptide sequences of an exosomal tetraspanin protein. The fusion protein is designed and made such that a SARS-CoV-2 protein antigen can be expressed on the surface of an exosome to enable the eliciting of an immune response when administered to a subject.
[0028] In one embodiment, the SARS-CoV-2 protein is a spike protein or an engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is a nucleocapsid protein or an engineered variant thereof.
[0029] In some embodiments in which the SARS-CoV-2 protein is a spike protein and the exosomal tetraspanin protein is a CD9 protein, the spike protein polypeptide is positioned N- terminal to the CD9 protein polypeptide. In some cases, a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide, as depicted in Fig. 1A.
[0030] In some embodiments in which the SARS-CoV-2 protein is a nucleocapsid protein and the exosomal tetraspanin protein is a CD9 protein, the nucleocapsid protein polypeptide is positioned N-terminal to the CD9 protein polypeptide. In some cases, a signal peptide is positioned at the N-terminal end of the nucleocapsid protein polypeptide. In some cases, a hinge peptide, a transmembrane domain peptide, and a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide, as depicted in Fig. 2A.
[0031] In one aspect, a synthetic fusion protein is provided that contains polypeptide sequences of a SARS-CoV-2 protein fused to polypeptide sequences of an exosomal tetraspanin protein. The fusion protein is designed and made such that a SARS-CoV-2 protein antigen can be expressed on the surface of an exosome to enable the eliciting of an immune response when administered to a subject.
[0032] In one embodiment, the SARS-CoV-2 protein is a spike protein or an engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is a nucleocapsid protein or an engineered variant thereof. [0033] In some embodiments in which the SARS-CoV-2 protein is a spike protein (e.g., SEQ ID NO:1) and the exosomal tetraspanin protein is a CD9 protein (SEQ ID NO: 10), the spike protein polypeptide is positioned N-terminal to the CD9 protein polypeptide. In some cases, a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO:2), as depicted in Fig. 1A.
[0034] In some embodiments in which the SARS-CoV-2 protein is a spike protein, the SARS- CoV-2 spike protein polypeptide contains one or more mutations, such as, e.g., a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and/or a di-proline substitution (2P [986KV987-to-986PP987]). In a specific embodiment, the spike-containing fusion protein has an amino acid sequence as set forth in SEQ ID NO:3.
[0035] In one aspect, a nucleic acid encoding a spike-CD9 fusion protein is provided. In one embodiment, the nucleic acid encoding a spike-CD9 fusion protein has a nucleic acid sequence of SEQ ID NO:4.
[0036] In some embodiments in which the SARS-CoV-2 protein is a nucleocapsid protein (e.g., SEQ ID NO:5) and the exosomal tetraspanin protein is a CD9 protein (e.g., SEQ ID NO: 10), the nucleocapsid protein polypeptide is positioned N-terminal to the CD9 protein polypeptide. In some cases, a signal peptide is positioned at the N-terminal end of the nucleocapsid protein polypeptide. In some cases, a hinge peptide, a transmembrane domain peptide (e.g., SEQ ID NO:9), and a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO:6), depicted in Fig. 2A. In a specific embodiment, the nucleocapsid-containing fusion protein has an amino acid sequence as set forth in SEQ ID NO:7.
[0037] In one aspect, a nucleic acid encoding a nucleocapsid-CD9 fusion protein is provided. In one embodiment, the nucleic acid encoding a nucleocapsid-CD9 fusion protein has a nucleic acid sequence of SEQ ID NO: 8.
[0038] In another aspect, a synthetic fusion protein is provided that contains polypeptide sequences of an influenza protein fused to polypeptide sequences of an exosomal tetraspanin protein. The fusion protein is designed and made such that influenza protein antigen can be expressed on the surface of an exosome to enable the eliciting of an immune response when administered to a subject. [0039] In one embodiment, the influenza protein is a hemagglutinin protein or an engineered variant thereof. In a specific embodiment, the influenza protein is a hemagglutinin 3 (H3) protein or an engineered variant thereof (e.g., SEQ ID NO: 14).
[0040] In some embodiments in which the influenza protein is an H3 protein and the exosomal tetraspanin protein is a CD9 protein (e.g., SEQ ID NO: 10), the H3 protein polypeptide is positioned N-terminal to the CD9 protein polypeptide. In some cases, a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO: 15), as depicted in Fig. 3 A. In a specific embodiment, the H3-containing fusion protein has an amino acid sequence of SEQ ID NO: 16.
[0041] In one aspect, a nucleic acid encoding an H3-CD9 fusion protein is provided. In one embodiment, the nucleic acid encoding an H3-CD9 fusion protein has a nucleic acid sequence of SEQ ID NO: 17.
[0042] In one aspect, a synthetic fusion protein is provided that contains polypeptide sequences of a respiratory syncytial virus (RSV) protein fused to polypeptide sequences of an exosomal tetraspanin protein. The fusion protein is designed and made such that an RSV protein antigen can be expressed on the surface of an exosome to enable the eliciting of an immune response when administered to a subject.
[0043] In one embodiment, the RSV protein is a prefusion (F) protein or an engineered variant thereof (e.g., SEQ ID NO: 18).
[0044] In some embodiments in which the RSV protein is a prefusion protein (RSV F) and the exosomal tetraspanin protein is a CD9 protein (e.g., SEQ ID NO: 10), the RSV F protein polypeptide is positioned N-terminal to the CD9 protein polypeptide. In some cases, a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO: 19), as depicted in Fig. 21 A. In a specific embodiment, the RSV F-containing fusion protein has an amino acid sequence of SEQ ID NO:20.
[0045] In one aspect, a nucleic acid encoding an RSV F-CD9 fusion protein is provided. In one embodiment, the nucleic acid encoding an RSV F-CD9 fusion protein has a nucleic acid sequence of SEQ ID NO:21. [0046] In one aspect, a polynucleotide is provided that encodes a synthetic fusion protein that contains polypeptide sequences of a SARS-CoV-2 protein fused to polypeptide sequences of an exosomal tetraspanin protein. The encoded fusion protein is designed and made such that a SARS-CoV-2 protein antigen can be expressed in a cell and sorted onto the surface of an exosome to enable the eliciting of an immune response when that exosome is administered to a subject.
[0047] In one embodiment, the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide. In some embodiments, the encoded SARS-CoV-2 spike protein polypeptide contains one or more mutations, such as, e.g., a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and/or a di-proline substitution (2P [986KV987-to- 986PP987]). In a specific embodiment, the spike protein has an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 or is essentially identical to SEQ ID NOT. In a specific embodiment, the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 11 or is essentially identical to SEQ ID NOT E
[0048] In some embodiments in which the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded spike protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some cases, a linker peptide is encoded by the polynucleotide to be positioned between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 1A. In a specific embodiment, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NOTO. In a specific embodiment, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13. In a specific embodiment the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NOT or is essentially identical to SEQ ID NOT. In a specific embodiment, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:4 or is essentially identical to SEQ ID NO:4. [0049] In another embodiment, the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide. In a specific embodiment, the nucleocapsid protein has an amino acid sequence that is at least 80% identical to SEQ ID NO: 5 or is essentially identical to SEQ ID NO:4. In a specific embodiment, the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 12 or is essentially identical to SEQ ID NO: 12.
[0050] In some embodiments in which the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide, and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded nucleocapsid protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide. In some cases, a signal peptide is positioned at the N- terminal end of the nucleocapsid protein polypeptide. In some cases, a hinge peptide, a transmembrane domain peptide, and a linker peptide are encoded by the polynucleotide to be positioned between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 2A. In a specific embodiment, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10. In a specific embodiment, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13. In a specific embodiment the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:7 or is essentially identical to SEQ ID NO:7. In a specific embodiment, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 8 or is essentially identical to SEQ ID NO:8.
[0051] In another aspect, a polynucleotide is provided that encodes a synthetic fusion protein that contains polypeptide sequences of an influenza protein fused to polypeptide sequences of an exosomal tetraspanin protein. The encoded fusion protein is designed and made such that an influenza protein antigen can be expressed in a cell and sorted onto the surface of an exosome to enable the eliciting of an immune response when that exosome is administered to a subject.
[0052] In one embodiment, the encoded influenza protein polypeptide is a hemagglutinin protein polypeptide. In one embodiment, the encoded hemagglutinin protein polypeptide is an H3 protein polypeptide. In a specific embodiment, the encoded H3 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 14 or is essentially identical to SEQ ID NO: 14. In a specific embodiment, the polynucleotide encoding the H3 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:22 or is essentially identical to SEQ ID NO:22. In one embodiment, the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide. In a specific embodiment, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10 or is essentially identical to SEQ ID NO: 10. In a specific embodiment, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13.
[0053] In some embodiments in which the encoded influenza protein polypeptide is a hemagglutinin 3 (H3) protein polypeptide and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded hemagglutinin protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some embodiments, a linker peptide is encoded by the polynucleotide to be positioned between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 3A. In a specific embodiment the H3-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO: 16 or is essentially identical to SEQ ID NO: 16. In a specific embodiment, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 17 or is essentially identical to SEQ ID NO: 17.
[0054] In another aspect, a polynucleotide is provided that encodes a synthetic fusion protein that contains polypeptide sequences of a respiratory syncytial virus (RSV) protein fused to polypeptide sequences of an exosomal tetraspanin protein. The encoded fusion protein is designed and made such that an RSV protein antigen can be expressed in a cell and sorted onto the surface of an exosome to enable the eliciting of an immune response when that exosome is administered to a subject.
[0055] In one embodiment, the encoded RSV protein polypeptide is a prefusion protein polypeptide (RSV F). In a specific embodiment, the encoded RSV F protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 18 or is essentially identical to SEQ ID NO: 18. In a specific embodiment, the polynucleotide encoding the RSV F protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:23 or is essentially identical to SEQ ID NO:23. In one embodiment, the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide. In a specific embodiment, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10. In a specific embodiment, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13.
[0056] In some embodiments, the encoded RSV F protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some embodiments, a linker peptide is encoded by the polynucleotide to be positioned between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 21 A. In a specific embodiment the RSV F-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:20 or is essentially identical to SEQ ID NO:20. In a specific embodiment, the polynucleotide encoding the spike- CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:21 or is essentially identical to SEQ ID NO:21.
[0057] In one aspect, a cell is provided that contains a polynucleotide that encodes a synthetic fusion protein that contains polypeptide sequences of a SARS-CoV-2 fused to polypeptide sequences of an exosomal tetraspanin protein. The encoded fusion protein is designed and made such that a SARS-CoV-2 protein antigen can be expressed in the cell and sorted onto the surface of an exosome to enable the eliciting of an immune response when that exosome is administered to a subject.
[0058] In some embodiments of the cell aspects, the cell is a metazoan cell. In some embodiments, the cell is a vertebrate cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a primate cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is of an established cell line. In some embodiments, the cell is a human embryonic kidney cell. In some embodiments, the cell is a HEK293 cell. In some embodiments, the cell is a 293F cell.
[0059] In one embodiment, the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide. In some embodiments, the encoded SARS-CoV-2 spike protein polypeptide contains one or more mutations, such as, e.g., a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and/or a di-proline substitution (2P [986KV987-to- 986PP987]). In a specific embodiment, the spike protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:1 or is essentially identical to SEQ ID NO: 1. In a specific embodiment, the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 11 or is essentially identical to SEQ ID NO:11.
[0060] In some embodiments in which the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded spike protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some cases, a linker peptide is encoded by the polynucleotide to be positioned between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 1A. In a specific embodiment, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10. In a specific embodiment, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13. In a specific embodiment the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:3 or is essentially identical to SEQ ID NO:3. In a specific embodiment, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:4 or is essentially identical to SEQ ID NO:4.
[0061] In another embodiment, the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide. In a specific embodiment, the nucleocapsid protein has an amino acid sequence that is at least 80% identical to SEQ ID NO: 5 or is essentially identical to SEQ ID NO:4. In a specific embodiment, the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 12 or is essentially identical to SEQ ID NO: 12.
[0062] In some embodiments in which the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide, and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded nucleocapsid protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide. Tn some cases, a signal peptide is positioned at the N- terminal end of the nucleocapsid protein polypeptide. In some cases, a hinge peptide, a transmembrane domain peptide, and a linker peptide are encoded by the polynucleotide to be positioned between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 2A. In a specific embodiment, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10. In a specific embodiment, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13. In a specific embodiment the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:7 or is essentially identical to SEQ ID NO:7. In a specific embodiment, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80%> identical to SEQ ID NO:8 or is essentially identical to SEQ ID NO:8.
[0063] In another aspect, a cell is provided that contains a polynucleotide that encodes a synthetic fusion protein that contains polypeptide sequences of an influenza virus fused to polypeptide sequences of an exosomal tetraspanin protein. The encoded fusion protein is designed and made such that an influenza SARS-CoV-2 protein antigen can be expressed in the cell and sorted onto the surface of an exosome to enable the eliciting of an immune response when that exosome is administered to a subject.
[0064] In some embodiments of the cell aspects, the cell is a metazoan cell. In some embodiments, the cell is a vertebrate cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a primate cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is of an established cell line. In some embodiments, the cell is a human embryonic kidney cell. In some embodiments, the cell is a HEK293 cell. In some embodiments, the cell is a 293F cell.
[0065] In one embodiment, the encoded influenza protein polypeptide is a hemagglutinin protein polypeptide. In one embodiment, the encoded hemagglutinin protein polypeptide is an H3 protein polypeptide. In a specific embodiment, the encoded H3 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 14 or is essentially identical to SEQ ID NO: 14. In a specific embodiment, the polynucleotide encoding the H3 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:22 or is essentially identical to SEQ ID NO:22. In one embodiment, the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide. In a specific embodiment, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10 or is essentially identical to SEQ ID NO: 10. In a specific embodiment, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13.
[0066] In some embodiments in which the encoded influenza protein polypeptide is a hemagglutinin 3 (H3) protein polypeptide and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded hemagglutinin protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some embodiments, a linker peptide is encoded by the polynucleotide to be positioned between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 3A. In a specific embodiment the H3-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO: 16 or is essentially identical to SEQ ID NO: 16. In a specific embodiment, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:17 or is essentially identical to SEQ ID N0: 17.
[0067] In another aspect, a cell is provided that contains a polynucleotide that encodes a synthetic fusion protein that contains polypeptide sequences of a respiratory syncytial virus (RSV) fused to polypeptide sequences of an exosomal tetraspanin protein. The encoded fusion protein is designed and made such that an RSV protein antigen can be expressed in the cell and sorted onto the surface of an exosome to enable the eliciting of an immune response when that exosome is administered to a subject.
[0068] In some embodiments of the cell aspects, the cell is a metazoan cell. In some embodiments, the cell is a vertebrate cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a primate cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is of an established cell line. In some embodiments, the cell is a human embryonic kidney cell. In some embodiments, the cell is a HEK293 cell. In some embodiments, the cell is a 293F cell. [0069] In one embodiment, the encoded RSV protein polypeptide is a prefusion protein polypeptide (RSV F). In a specific embodiment, the encoded RSV F protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 18 or is essentially identical to SEQ ID NO: 18. In a specific embodiment, the polynucleotide encoding the RSV F protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:23 or is essentially identical to SEQ ID NO:23. In one embodiment, the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide. In a specific embodiment, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO: 10. In a specific embodiment, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13 or is essentially identical to SEQ ID NO: 13.
[0070] In some embodiments, the encoded RSV F protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some embodiments, a linker peptide is encoded by the polynucleotide to be positioned between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as depicted in Fig. 21 A. In a specific embodiment the RSV F-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:20 or is essentially identical to SEQ ID NO:20. In a specific embodiment, the polynucleotide encoding the spike- CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:21 or is essentially identical to SEQ ID NO:21.
DRAWINGS
[0071] Figure 1A depicts a linear cartoon of a SARS-CoV-2 spike CD9 fusion protein oriented from left-to-right amino-terminus-to-carboxy -terminus with a linker sequence positioned between the spike protein polypeptide and the CD9 protein polypeptide.
[0072] Figure IB depicts a cartoon of a SARS-CoV-2 spike CD9 fusion protein of Fig. 1A relative to a vesicle membrane oriented from left-to-right amino-terminus-to-carboxy-terminus. The spike protein polypeptide spans the membrane once with its amino terminus oriented on the outside of the vesicle. The linker is positioned on the luminal side of the membrane. The CD9 protein polypeptide spans the membrane 4 times with its carboxy terminus oriented in the lumen of the vesicle.
[0073] Figure 2A depicts a linear cartoon of a SARS-CoV-2 nucleocapsid CD9 fusion protein oriented from left-to-right amino-terminus-to-carboxy -terminus with an amino terminal signal peptide fused to a nucleocapsid protein polypeptide fused to a hinge region peptide fused to a transmembrane domain peptide fused to a linker peptide fused to a CD9 protein polypeptide.
[0074] Figure 2B depicts a cartoon of a SARS-CoV-2 nucleocapsid CD9 fusion protein of Fig. 2A relative to a vesicle membrane oriented from left-to-right amino-terminus-to-carboxy- terminus. The nucleocapsid protein polypeptide with the amino terminal signal sequence is positioned on the outside (cytoplasmic or external side) with a linker sequence connecting the nucleocapsid protein polypeptide to the transmembrane domain peptide, which spans the membrane and which in turn connects to the linker positioned in the lumen and which connects to the CD9 protein polypeptide which spans the membrane 4 times with its carboxy terminus oriented in the lumen of the vesicle.
[0075] Figure 3A depicts a linear cartoon of an influenza hemagglutinin CD9 fusion protein oriented from left-to-right amino-terminus-to-carboxy-terminus with a linker sequence positioned between the spike protein polypeptide and the CD9 protein polypeptide.
[0076] Figure 3B depicts a cartoon of an influenza hemagglutinin CD9 fusion protein of Fig. 3 A relative to a vesicle membrane oriented from left-to-right amino-terminus-to-carboxy-terminus. The hemagglutinin protein polypeptide spans the membrane once with its amino terminus oriented on the outside of the vesicle. The linker is positioned on the luminal side of the membrane. The CD9 protein polypeptide spans the membrane 4 times with its carboxy terminus oriented in the lumen of the vesicle.
[0077] Figure 4A is a flow chart depicting the elements and steps for producing cells expressing a spike, nucleocapsid, influenza hemagglutinin, or other antigen protein polypeptide fusion protein using a packaging cell (301) and a host cell (311) mediated by a lentivirus vector (304).
[0078] Figure 4B is a histogram depicting relative fluorescent intensity flow analysis of host cells expressing spike protein on their surface. [0079] Figure 5A is a graph depicting the concentration of spike-expressing exosomes per milliliter as a function of exosome diameter in nanometers.
[0080] Figure 5B is a western blot stained for SARS-CoV-2 spike protein. The first column from left to right depicts lane loaded with size markers, second column represents lane loaded with non-transduced 293F (host cell) protein, third column represents lane loaded with protein from exosomes derived from non-transduced 293F cells, fourth column represents lane loaded with protein from 293F cells constitutively expressing spike fusion protein, fifth column represents lane loaded with protein from exosomes derived from 293F cells constitutively expressing spike fusion protein, and sixth column represents lane loaded with spike fusion protein.
[0081] Figure 5C is a histogram depicting relative fluorescent intensity flow analysis of exosomes with spike expressed on the surface. The left curve represents exosomes derived from 293F cells that do not express a spike-CD9 fusion protein. The right curve represents exosomes derived from 293F cells expressing a spike-CD9 fusion protein.
[0082] Figure 6A depicts a transmission electron micrograph of exosomes expressing SARS- CoV-2 spike protein. Inset is a blow up showing an exosome decorated with spike protein. Arrows point to SARS-CoV-2 spikes around the circumference of exosomes.
[0083] Figure 6B depicts a higher magnification transmission electron micrograph of exosomes expressing SARS-CoV-2 spike protein. Arrows point to SARS-CoV-2 spikes around the circumference of exosomes.
[0084] Figure 7A is a graph depicting the concentration of nucleocapsid-expressing exosomes per milliliter as a function of exosome diameter in nanometers.
[0085] Figure 7B is a western blot stained for SARS-CoV-2 nucleocapsid protein. The first column from left to right depicts lane loaded with size markers, second column represents lane loaded with nucleocapsid protein, third column represents lane loaded with protein from exosomes derived from non-transduced 293F cells, fourth column represents lane loaded with protein from non-transduced 293F cells, and fifth column represents lane loaded with protein from exosomes derived from 293F cells expressing nucleocapsid fusion protein. [0086] Figure 7C is a histogram depicting relative fluorescent intensity flow analysis of exosomes with or without nucleocapsid protein expressed on the surface. Moving from left to right, the first curve represents exosomes derived from unmodified 293F cells, the second curve represents exosomes derived from uninduced 293F cells harboring a nucleocapsid-CD9 fusion protein-encoding polynucleotide, the third curve represents exosomes derived from 293F cells expressing nucleocapsid-CD9 fusion protein, the smaller third curve represents exosomes derived from 293F cells expressing nucleocapsid-CD9 fusion protein under tet induction, the larger third curve represents exosomes derived from 293F cells harboring a CD9 knock-out and expressing nucleocapsid-CD9 fusion protein.
[0087] Figures 8A and 8B are histograms depicting relative fluorescent intensity flow analysis of 293F cells with or without influenza H3 hemagglutinin protein expressed on the surface. Moving from left to right on each panel, the first curve represents unmodified 293F cells, the second curve represents 293F cells harboring and expressing an influenza H3-CD9 fusion proteinencoding polynucleotide. Panel A represents adherent 293F cells. Panel B represents 293F cells in suspension.
[0088] Figure 8C is a western blot stained for influenza H3 protein (panel 1) or CD9 (panel 2). For each panel, the first column from left to right depicts lane loaded with size markers, second column represents lane loaded with unmodified 293F (host cell) protein, third column represents lane loaded with protein from exosomes derived from unmodified 293F cells, fourth column represents lane loaded with protein from 293F cells expressing H3-CD9 fusion protein, and fifth column represents lane loaded with protein from exosomes derived from 293F cells expressing H3-CD9 fusion protein. Each sample was loaded at 0.6mg/ml total protein.
[0089] Figure 8D is a flow cytometry dot plot of H3 -expressing exosomes depicting CD81 surface expression as a function of H3 surface expression. In this example, about 54.2% of the flowed exosomes express both CD81 and H3, thus demonstrating that the expression of a hemagglutinin polypeptide or antigen at the surface of a vesicle when fused to an exosomal protein such as a tetraspanin like CD9.
[0090] Figure 9 is a timeline depicting a process for generating am immune response from administering an immunogenic composition containing exosomes expressing SARS-CoV-2 fusion proteins on their surface. [0091 ] Figures 10A-10D. STX-S exosome elicited a robust immune response using significantly lower antigen than recombinant protein vaccines. Data are shown as mean ± SEM. **** p<0.0005, ***p<0.001, **p<0.01, *p<0.05, ns= not significant, *l-way ANOVA; ##p<0.01, #p<0.05, # - 2 tail-t test.
[0092] Figure 10A is a histogram depicting fold change in murine day-14 l : 100-diluted serum antibody titer against spike protein as a function of exosome-expressing spike dosage in nanograms. The left-most bar represents PBS control; the second bar represents a 10 ng dose of spike expressing exosomes (STX-S); the third bar represents a 32 ng dose of STX-S; the right most bar represents sera from mice injected with 32 ng spike protein dose combined with adjuvant.
[0093] Figure 10B is a histogram depicting fold change in murine day-35 l : 100-diluted serum antibody titer against spike protein as a function of exosome-expressing spike dosage in nanograms. The left-most bar represents PBS control; the second bar represents a 10-ng dose of spike expressing exosomes (STX-S); the third bar represents a 32 ng dose of STX-S; the right most bar represents sera from mice injected with 32 ng spike protein dose combined with adjuvant.
[0094] Figure 10C is a bar graph depicting an ELISpot assay showing the number of IL4 producing spots responding to spike protein as a function of antigen dose. Moving from left to right on the x-axis, the first bar represents non-spike-containing wells from PBS control splenocytes; the second bar represents spike protein reactivity from PBS control splenocytes; the third bar represents non-spike-containing wells from splenocytes of 10 ng STX-S dosed subject; the fourth bar represents spike protein reactivity from splenocytes of 10 ng STX-S dosed subject; the fifth bar represents non-spike-containing wells from splenocytes of 32 ng STX-S dosed subject; the sixth bar represents spike protein reactivity from splenocytes of 32 ng STX-S dosed subject; the seventh bar represents non-spike-containing wells from splenocytes of 32 ng spike protein with adjuvant dosed subject; the eighth bar represents spike protein reactivity from splenocytes of 32 ng spike protein with adjuvant dosed subject.
[0095] Figure 10D is a bar graph depicting an ELISpot assay showing the number of IFNy producing spots responding to spike protein as a function of antigen dose. Moving from left to right on the x-axis, the first bar represents non-spike-containing wells from PBS control splenocytes; the second bar represents spike protein reactivity from PBS control splenocytes; the third bar represents non-spike-containing wells from splenocytes of 10 ng STX-S dosed subject; the fourth bar represents spike protein reactivity from splenocytes of 10 ng STX-S dosed subject; the fifth bar represents non-spike-containing wells from splenocytes of 32 ng STX-S dosed subject; the sixth bar represents spike protein reactivity from splenocytes of 32 ng STX-S dosed subject; the seventh bar represents non-spike-containing wells from splenocytes of 32 ng spike protein with adjuvant dosed subject; the eighth bar represents spike protein reactivity from splenocytes of 32 ng spike protein with adjuvant dosed subject.
[0096] Figures 11, panels A-C: Line graphs depicting percent SARS-CoV-2 neutralization as a function of immune sera at escalating serum dilution. The graphs depict the production of neutralizing antibodies after STX-S injection. Data are shown as mean ± SEM. COV-02-Delta = plasma from a patient immunized with Modema’s mRNA vaccine with a breakthrough SARS- CoV-2 delta spike infection. Dose 4 = 9.8 ng S per injection; dose 2 = 3.2 ng S per injection.
[0097] Panel 11 A. STX-S vaccine generated strong neutralization against SARS-CoV-2 delta spike (B.1.617.2)
[0098] Panel 11B. STX-S vaccine resulted in neutralization of SARS-CoV-2 spike Omicron BA. l
[0099] Panel 11C. STX-S vaccine resulted in neutralization of SARS-CoV-2 spike Omicron BA.5.2.1.
[0100] Figure 12, panels A-F are histograms depicting anti-H3 (panels A-C) and anti-spike (panels D-F) antibody quantities (OD [panels A and D], fold-change [panels B and E], or loglO titer [panels C and F) in day-14 post-injection sera from subjects as a function of immunogenic composition (STX-H3 or STX-H3 plus STX-S). Data are shown as mean±SEM. *p<0.05, ***p<0.005, ****p<0.001, ANOVA, corrected multiple comparisons; ns= not significant.
[0101] Figure 12A is a histogram depicting OD of day-14 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3), and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S). [0102] Figure 12B is a histogram depicting fold change in levels of day-14 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3), and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).
[0103] Figure 12C is a histogram depicting the loglO titer of day-14 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3), and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).
[0104] Figure 12D is a histogram depicting OD of day-14 serum anti-spike IgG as a function of injected material dose, which is, from left to right, PBS control, and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).
[0105] Figure 12E is a histogram depicting fold change in levels of day-14 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3), and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).
[0106] Figure 12FE is a histogram depicting the loglO titer of day-14 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3), and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).
[0107] Figure 13, panels A-D. STX-N vaccine elicits a multivalent immune-response. Data are shown as mean±SEM. *p<0.05, ***p<0.005, ****p<0.001, ANOVA, corrected multiple comparisons; ns= not significant. Dose 1 = 0.32 ng N per injection; dose 2 = 3.2 ng N per injection; dose 3 = 10 ng N per injection
[0108] Panels 13A and 13B: Histograms depicting anti-nucleocapsid antibody titers of day-35 post immunization sera as a function of dose. STX-N vaccine induced modest expression of SARS-CoV-2 Nucleocapsid antibodies in two sample bins (N 1 and N 2). PBS was used as a vehicle control. [0109] Panels 13C and 13D: Histograms depicting anti-nucleocapsid IFNy ELISpot positive wells of day-40 post immunization splenocytes as a function of dose.
[0110] Figure 14 depicts the expression of SARS-CoV-2 spike protein on 293 cells.
[0111] Panel 14A is a histogram depicting relative fluorescent intensity flow analysis of 293F cells with SARS-CoV2 spike expressed on the surface. The left curve represents 293F cells that do not express a spike-CD9 fusion protein. The right curve represents 293F cells expressing a spike-CD9 fusion protein.
[0112] Panel 14B is a western blot stained for SARS-CoV-2 spike protein. The first column from left to right depicts lane loaded with size markers, second column represents lane loaded with non-transduced 293F (host cell) protein, third column represents lane loaded with protein from exosomes derived from non-transduced 293F cells, fourth column represents lane loaded with protein from 293F cells expressing spike fusion protein, fifth column represents lane loaded with protein from exosomes derived from 293F cells expressing spike fusion protein, and sixth column represents lane loaded with spike fusion protein.
[0113] Panel 15A is a histogram depicting relative fluorescent intensity flow analysis of 293F cells with influenza hemagglutinin 3 (H3) expressed on the surface. The left curve represents 293F cells that do not express an H3-CD9 fusion protein. The right curve represents 293F cells expressing an H3-CD9 fusion protein.
[0114] Panel 15B is a western blot stained for influenza hemagglutinin 3 (H3) protein. The first column from left to right depicts lane loaded with size markers, second column represents lane loaded with non-transduced 293F (host cell) protein, third column represents lane loaded with protein from exosomes derived from non-transduced 293F cells, fourth column represents lane loaded with protein from 293F cells expressing H3 fusion protein, and fifth column represents lane loaded with protein from exosomes derived from 293F cells expressing H3 fusion protein.
[0115] Figure 16 are histograms depicting expression of surface markers on 293F-derived exosomes.
[0116] Panel 16A is a histogram depicting relative fluorescent intensity flow analysis of exosomes derived from 293F cells with naturally-occurring CD81 expressed on the surface. The left curve represents 293F-derived exosomes decorated with isotype control antibody. The right curve represents 293F-derived exosomes decorated with anti-CD81 antibody.
[0117] Panel 16B is a histogram depicting relative fluorescent intensity flow analysis of exosomes derived from 293F cells with spike expressed on the surface. The left curve represents exosomes derived from 293F cells that do not express a spike-CD9 fusion protein. The right curve represents exosomes derived from 293F cells expressing a spike-CD9 fusion protein.
[0118] Panel 16C is a histogram depicting relative fluorescent intensity flow analysis of exosomes derived from 293F cells transfected with hemagglutinin 3 (H3) expressed on the surface. The left curve represents exosomes derived from 293F cells that do not express an H3- CD9 fusion protein. The right curve represents exosomes derived from 293F cells expressing an H3-CD9 fusion protein.
[0119] Figure 17 presents histograms depicting fold-change in antigen-specific IgG production.
[0120] Panel 17A is a histogram depicting fold change in levels of day-14 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, and a combination of exosomes expressing hemagglutinin 3 (STX-H3) and exosomes expressing SARS-CoV-2 spike (STX-S).
[0121] Panel 17B is a histogram depicting fold change in levels of day-14 serum anti-spike IgG as a function of injected material dose, which is, from left to right, PBS control, and a combination of exosomes expressing hemagglutinin 3 (STX-H3) and exosomes expressing SARS-CoV-2 spike (STX-S).
[0122] Figure 18 presents histograms depicting fold-change in antigen-specific IgG production.
[0123] Panel 18A is a histogram depicting fold change in levels of day-35 serum anti-H3 IgG as a function of injected material dose, which is, from left to right, PBS control, and a combination of exosomes expressing hemagglutinin 3 (STX-H3) and exosomes expressing SARS-CoV-2 spike (STX-S).
[0124] Panel 18B is a histogram depicting fold change in levels of day-35 serum anti-spike IgG as a function of injected material dose, which is, from left to right, PBS control, and a combination of exosomes expressing hemagglutinin 3 (STX-H3) and exosomes expressing SARS-CoV-2 spike (STX-S).
[0125] Figure 19 presents ELISpot histograms of IFNg production.
[0126] Panel 19A is a histogram depicting IFNg production by splenocytes as a function of immunogen and antigen. From left to right, the first histogram represents splenocytes from animals immunized with PBS and not exposed to antigen, the second histogram represents splenocytes from animals immunized with PBS and exposed to H3 antigen, the third histogram represents splenocytes from animals immunized with a combination of exosomes that express spike and exosomes that express H3 and not exposed to antigen, and the fourth histogram represents splenocytes from animals immunized with a combination of exosomes that express spike and exosomes that express H3 and exposed to H3 antigen.
[0127] Panel 19A is a histogram depicting IFNg production by splenocytes as a function of immunogen and antigen. From left to right, the first histogram represents splenocytes from animals immunized with PBS and not exposed to antigen, the second histogram represents splenocytes from animals immunized with PBS and exposed to H3 antigen, the third histogram represents splenocytes from animals immunized with a combination of exosomes that express spike and exosomes that express H3 and not exposed to antigen, and the fourth histogram represents splenocytes from animals immunized with a combination of exosomes that express spike and exosomes that express H3 and exposed to H3 antigen.
[0128] Panel 19B is a histogram depicting IFNg production by splenocytes as a function of immunogen and antigen. From left to right, the first histogram represents splenocytes from animals immunized with PBS and not exposed to antigen, the second histogram represents splenocytes from animals immunized with PBS and exposed to spike antigen, the third histogram represents splenocytes from animals immunized with a combination of exosomes that express spike and exosomes that express H3 and not exposed to antigen, and the fourth histogram represents splenocytes from animals immunized with a combination of exosomes that express spike and exosomes that express H3 and exposed to spike antigen.
[0129] Figure 20 depicts graphic representations of respiratory syncytial virus fusion protein (RSFV F) coding sequence. Bar A represents non-engineered RSV F. Bar B represents engineered RSV F version 1 (VI) which is DS-Cavl . Bar C represents engineered RSV F version 2 (V2) which is DS-Cavl with polyA signals deleted. Bar D represents engineered RSV F version 3 (V3) which is DS-Cavl with polyA signals and two furin cleavage sites deleted. Bar D represents engineered RSV F version 3 (V4) which is DS-Cavl with polyA signals and the N- terminal most furin cleavage site deleted. SP = signal peptide; F2 = RSV fusion protein subunit 2; p27 = RSV fusion protein p27 subunit; FP = hydrophobic fusion peptide (FP); Fl = RSV fusion protein subunit 1; TM = transmembrane domain.
[0130] Figures 22 are histograms depicting anti-RSV antibody titer fold change as a function of antigen (RSV version 3). The Y-axes represent antibody titer fold change, and the X-axes represent antigen, the left bars are PBS control, the right bars are RSV F antigen. Panel 22A represents antibody fold change at 14 days post injection. Panel 22B represents antibody fold change at 35 days post injection and after a booster at day -21.
[0131] Figures 23 are histograms depicting anti-RSV antibody titer fold change as a function of antigen (RSV version 4). The Y-axes represent antibody titer fold change, and the X-axes represent antigen. Panel 23 A represents antibody fold change at 14 days post injection. Panel 23B represents antibody fold change at 35 days post injection and after a booster at day-21. X- axes labels for both panels are: 1 = PBS, 2 = 1E9 exosomes expressing RSV F V4-CD9, 3 = 1E10 exosomes expressing RSV F V4-CD9, 4 = 3E10 exosomes expressing RSV F V4-CD9, 5 = 1E11 exosomes expressing RSV F V4-CD9.
[0132] Figure 24 is a graphic representing a combination of exosomes expressing SARS-CoV2 spike-tetraspanin (STX-S) and exosomes expressing influenza H3 -tetraspanin (STX-H3).
[0133] Figure 25 is a graphic representing a combination of exosomes expressing SARS-CoV2 spike-tetraspanin (STX-S) and exosomes expressing respiratory syncytial virus fusion protein- tetraspanin (STX-F).
[0134] Figure 26 is a graphic representing a combination of exosomes expressing influenza H3- tetraspanin (STX-H3) and exosomes expressing respiratory syncytial virus fusion protein- tetraspanin (STX-F). [0135] Figure 27 is a graphic representing a combination of exosomes expressing SARS-CoV2 spike-tetraspanin (STX-S), exosomes expressing influenza H3 -tetraspanin (STX-H3), and exosomes expressing respiratory syncytial virus fusion protein-tetraspanin (STX-F).
[0136] Figures 28 are histograms depicting antibody titer fold change as a function of antigen. The Y-axes represent antibody titer fold change, and the X-axes represent antigen. Panel 28A represents anti-spike antibody fold change at 14 days post injection. Panel 28B represents anti- 143 antibody fold change at 14 days post injection. Panel 28C represents anti-RSV antibody fold change at 14 days post injection. X-axes labels for each panels are: 1 = PBS, 2 = exosomes expressing spike (STX-S), 3 = exosomes expressing H3 (STX-H3), 4 = exosomes expressing RSV F (STX-RSV), 5 = combination of exosomes expressing H3 and exosomes expressing spike (STX-H3 + STX-S), 6 = combination of exosomes expressing RSV F and exosomes expressing spike (STX-RSV + STX-S), 7 = combination of exosomes expressing RSV F and exosomes expressing H3 (STX-RSV + STX-H3), 8 = combination of exosomes expressing RSV F, exosomes expressing H3, and exosomes expressing spike (STX-RSV + STX-H3 +STX-S).
[0137] Figures 29 are histograms depicting antibody titer fold change as a function of antigen. The Y-axes represent antibody titer fold change, and the X-axes represent antigen. Panel 29 A represents anti-spike antibody fold change at 35 days post injection and after booster at day-21. Panel 28B represents anti-H3 antibody fold change at 35 days post injection and after booster at day-21. Panel 28C represents anti-RSV antibody fold change at 35 days post injection and after booster at day-21. X-axes labels for each panels are: 1 = PBS, 2 = exosomes expressing spike (STX-S), 3 = exosomes expressing H3 (STX-H3), 4 = exosomes expressing RSV F (STX-RSV), 5 = combination of exosomes expressing H3 and exosomes expressing spike (STX-H3 + STX- S), 6 = combination of exosomes expressing RSV F and exosomes expressing spike (STX-RSV + STX-S), 7 = combination of exosomes expressing RSV F and exosomes expressing H3 (STX- RSV + STX-H3), 8 = combination of exosomes expressing RSV F, exosomes expressing H3, and exosomes expressing spike (STX-RSV + STX-H3 +STX-S). DETAILED DESCRIPTION OF EMBODIMENTS
Definitions
[0138] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
[0139] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).
[0140] The terms “about” and “approximate”, as used herein when referring to a measurable value such as an amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length and the like, is meant to encompass variations of ± 15%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length and the like. In instances in which the terms “about” and “approximate” are used in connection with the location or position of regions within a reference polypeptide, these terms encompass variations of ± up to 20 amino acid residues, ± up to 15 amino acid residues, ± up to 10 amino acid residues, ± up to 5 amino acid residues, ± up to 4 amino acid residues, ± up to 3 amino acid residues, ± up to 2 amino acid residues, or even ± 1 amino acid residue.
[0141] The term “derived from” as in “A is derived from B” means that A is obtained from B in such a manner that A is not identical to B.
[0142] The terms “treat”, “therapeutic”, “prophylactic” and “prevent” are not intended to be absolute terms. Treatment, prevention and prophylaxis can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, increase in survival time or rate, etc. Treatment, prevention, and prophylaxis can be complete or partial. The term “prophylactic” means not only “prevent”, but also minimize illness and disease. For example, a “prophylactic” agent can be administered to a subject, e.g., a human subject, to prevent infection, or to minimize the extent of illness and disease caused by such infection. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. In some aspects, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects, the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques.
[0143] A treatment can be considered “effective,” as used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 2%, 3%, 4%, 5%, 10%, or more, following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (e.g., progression of the disease is halted). Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. One skilled in the art can monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters.
[0144] The term “effective amount” as used herein refers to the amount of a composition or an agent needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of therapeutic composition to provide the desired effect. The term “therapeutically effective amount” refers to an amount of a composition or therapeutic agent that is sufficient to provide a particular effect when administered to a typical subject. An effective amount as used herein, in various contexts, can include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of therapeutic effect at least any of 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. For example, a therapeutically effective amount can have at least any of a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. The therapeutically effective amount may be administered in one or more doses of the therapeutic agent. The therapeutically effective amount may be administered in a single administration, or over a period of time in a plurality of doses.
[0145] “Administering” as used herein can include any suitable routes of administering a therapeutic agent or composition as disclosed herein. Suitable routes of administration include, without limitation, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection or topical administration. Administration can be local or systemic.
[0146] As used herein, the term “pharmaceutically acceptable” refers to a carrier that is compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The term is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0147] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. For the present invention, the dose can refer to the concentration of the extracellular vesicles or associated components, e.g., the amount of therapeutic agent or dosage of radiolabel. The dose will vary depending on a number of factors, including frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; the route of administration; and the imaging modality of the detectable moiety (if present). One of skill in the art will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical and depends on the route of administration. For example, a dosage form can be in a liquid, e.g., a saline solution for injection.
[0148] “Subject,” “patient,” “individual” and like terms are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision. A patient can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc.
[0149] As used herein, the following meanings apply unless otherwise specified. The word “may” is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). The words “include”, “including”, and “includes” and the like mean including, but not limited to. The singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “an element” includes a combination of two or more elements, notwithstanding use of other terms and phrases for one or more elements, such as “one or more.” The term “or” is, unless indicated otherwise, non-exclusive, i.e., encompassing both “and” and “or.” The term “any of’ between a modifier and a sequence means that the modifier modifies each member of the sequence. So, for example, the phrase “at least any of 1 , 2 or 3” means “at least 1, at least 2 or at least 3”. The phrase “at least one” includes “a plurality”.
[0150] Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-91 1910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9);
Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds. [0151 ] The term “native form” corresponds to the polypeptide as it is understood to be encoded by the infectious agent’s genome. The term “exosomal form” corresponds to any derivative of the protein that, in whole or in part, is fused to an exosome-associated protein. The term “cytoplasmic form” corresponds to any derivative of the protein that, in whole or in part, is configured, or designed, to be expressed within the cytoplasm of the cell, rather than entering the canonical secretory pathway.
[0152] The expression that a certain protein is “configured, or designed, to be expressed” in a certain way means that its nucleotide sequence encodes certain a particular amino acid sequence such that when that protein is expressed in a cell, that protein will be in its native form, exosomal form, or cytoplasmic form by virtue of that particular amino acid sequence. For instance, if a spike protein (S) is expressed in its native form, it is configured, or designed, to induce a humoral or cellular immune response by virtue of the fact that it is a transmembrane protein with an extracellular domain.
[0153] The term “extracellular vesicle” (EV) refers to lipid bilayer-delimited particles that are naturally released from cells. EVs range in diameter from around 20-30 nanometers to about 10 microns or more. EVs can comprise proteins, nucleic acids, lipids and metabolites from the cells that produced them. EVs include exosomes (about 50 to about 200 nm), microvesicles (about 100 to about 300 nm), ectosomes (about 50 to about 1000 nm), apoptotic bodies (about 50 to about 5000 nm) and lipid-protein aggregates of the same dimensions.
[0154] The term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), as well as chemically modified nucleic acids such as morpholino (PMO), peptide nucleic acid (PNA), 2'O-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate, and phosphorothioate. Nucleic acids may be of any size. Nucleic acids include but are not limited to genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules such as aptamers, plasmids, anti-sense DNA strands, shRNA, ribozymes, nucleic acids conjugated and oligonucleotides. According to the invention, a nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid might be employed for introduction into, e.g., transfection of, cells, e g., in the form of RNA which can be prepared by in vitro transcription from a DNA template. The RNA can moreover be modified before application by stabilizing sequences, capping, and polyadenylation. Generally, nucleic acid can be extracted, isolated, amplified, or analyzed by a variety of techniques such as those described by, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY 2,028 pages (2012).
[0155] The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein and refer to any chain of at least two amino acids, linked by a covalent chemical bound. As used herein a peptide can refer to the complete amino acid sequence coding for an entire protein or to a portion thereof. A “protein coding sequence” or a sequence that “encodes” a particular polypeptide or peptide, is a nucleic acid sequence that is transcribed (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence.
[0156] As used herein, the phrase “protein polypeptide” means a polypeptide sequence of or derived from a protein. For example, a CD9 protein polypeptide may be any polypeptide of the CD9 protein, such as, e.g., a full length CD9 protein, a transmembrane domain polypeptide of a CD9 protein, a C-terminal stretch of a CD9 protein, an extracellular loop region of a CD9 protein, the intracellular (intralumenal) loop region of a CD9 protein, a C-terminal stretch of a CD9 protein, combinations thereof, and/or the like. Here, a protein polypeptide may be at least 10 amino acids long.
[0157] As used herein, the term “spike protein” includes any SARS-CoV-2 spike glycoprotein, fragment of a SARS-CoV-2 spike glycoprotein, monomer of a SARS-CoV-2 spike glycoprotein, trimer of SARS-CoV-2 spike glycoprotein monomers, variant of a SARS-CoV-2 spike glycoprotein, fusion protein or chimeral protein containing a SARS-CoV-2 spike glycoprotein sequence and another non-SARS-CoV-2 spike glycoprotein sequence, SARS-CoV-2 spike glycoproteins having one or more deletions, additions, or substitutions of one or more amino acids, and conservatively substituted variations of a SARS-CoV-2 spike glycoprotein having at least 80% amino acid sequence identity of e.g., at least the stem region of an S2 subunit, membrane-proximal stem helix region, or the receptor binding domain, or other like domains.
[0158] A fragment of a SARS-CoV-2 spike glycoprotein includes peptide or polypeptides the encompass, comprise, consist of, or overlap with e.g., antigenic epitopes, specific domains like receptor binding domain (RBD) in up or down conformational states, a receptor-binding fragment SI, fusion fragment S2, N-terminal domain (NTD), receptor-binding domain (RBD) C- terminal domain 1 (CTD1), C-terminal domain 2 (CTD2), fusion peptide (FP), fusion-peptide proximal region (FPPR), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), heptad repeat 2 (HR2), transmembrane segment (TM), the cytoplasmic tail (CT), and the like.
[0159] A variant of a SARS-CoV-2 spike glycoprotein includes any known or yet to be discovered, including alpha, beta, gamma, delta, epsilon, eta, iota, kappa, 1.617.3, mu, zeta, omicron, or their subvariants, lineages, and conservatively substituted spike protein sequence.
[0160] SARS-CoV-2 spike glycoprotein may have additions, deletions, substitution, point mutations. For example, a spike protein may have a deletion of several (2-20) amino acids from its C-terminus (see Johnson et al., 2020 and Xiong et al., 2020), or furin cleavage site change (see e.g., Johnson et al., 2020).
[0161] As used herein, the term “nucleocapsid protein” means a soluble coronavirus structural protein that binds and forms complexes with RNA and viral membrane protein (M) and is critical for viral genome pacjkaging. Nucleocapsid protein contains (from amino terminus to carboxy terminus) an intrinsically disordered region (IDR), and N terminal domain containing an RNA binding domain (NTD), a serine-arginine rich linker region (LKR), a C terminal domain containing an RNA binding and dimerization domain and a nuclear localization signal, followed by a C terminal IDR. Nucleocapsid protein is generally described, e.g., in McBride et al., “The Coronavirus Nucleocapsid Is a Multifunctional Protein,” Viruses. 2014 Aug; 6(8): 2991-3018; Cubuk et al., “The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA,” Nature Communications volume 12, Article number: 1936 (2021); and references cited therein.
[0162] Fusion proteins or chimeral proteins of SARS-CoV-2 spike glycoprotein or SARS-CoV-2 nucleocapsid protein includes fusions of spike or nucleocapsid protein sequences and sequences of another protein to effect a particular outcome, such as improved sorting or targeting to endosomes and resultant extracellular vesicles such as exosomes. For example, spike protein may be a fusion of spike or nucleocapsid protein sequences and other glycoproteins known to sort to exosomes or useful in the production of pseudovirions, such as VSV glycoproteins, lentivirus glycoprotein, and the like. Spike or nucleocapsid protein may be a fusion of spike protein sequences and other proteins known to sort to exosomes such as various tetraspanins (CD9, CD63, and CD81). Fusion proteins may contain mostly SARS-CoV-2 sequences with short (i.e., dipeptides to peptides of 100 amino acids) sequences of the other protein.
[0163] As used herein, the term “tetraspanin” or “tetraspanin protein” means any member (or chimera thereof) of a family of proteins having four transmembrane domains and in some cases present in exosome membranes. Tetraspanin proteins are known to regulate trafficking and cell and membrane compartmentalization. Tetraspanins include inter alia CD9, CD37, CD63, CD81, CD82, CD151, TSPAN7, TSPAN8, TSPAN12, TSPAN33, peripherin, UPla/lb, TSP-15, TSP- 12, TSP3A, TSP86D, TSP26D, TSP-2, and analogs, orthologs, and homologs thereof. It is envisioned that useful tetraspanins may include chimeras of any one or more canonical tetraspanins, such as, e.g., a CD9/CD81 chimera or the like. Tetraspanins are generally described in Charrin et al., “Tetraspanins at a glance,” J Cell Sci (2014) 127 (17): 3641-3648; Kummer et al., “Tetraspanins: integrating cell surface receptors to functional microdomains in homeostasis and disease,” Med Microbiol Immunol. 2020; 209(4): 397-405; and references cited therein. The CD9 member of the tetraspanin superfamily is generally described in Umeda et al., “Structural insights into tetraspanin CD9 function,” Nature Communications volume 11, Article number: 1606 (2020), and references cited therein.
[0164] The term “sequence identity” as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions times 100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present application. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website).
[0165] Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4: 11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
[0166] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
[0167] For antibodies, percentage sequence identities can be determined when antibody sequences are maximally aligned by IMGT. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, multiplied by 100 to convert to percentage.
[0168] Percent amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be obtained from the National Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length= 15/5, multi-pass e-value=0.01, constant for multi-pass=25, dropoff for final gapped alignment=25 and scoring matrix=BLOSUM62.
[0169] In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y,
[0170] where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. The term “nucleic acid sequence” as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages and includes cDNA. The term also includes modified or substituted sequences comprising non- naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. It is understood that polynucleotides comprising non-transcribable nucleotide bases may be useful as probes in, for example, hybridization assays. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid" includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents.
[0171] As used herein the term “antibody” refers to immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds an antigen. Antibodies are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. The light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (X), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, 8, s, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The antibody may have one or more effector functions which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region or any other modified Fc region) of an antibody. Non-limiting examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor (BCR); and cross-presentation of antigens by antigen presenting cells or dendritic cells).
[0172] The term “neutralizing antibody” (Nab) refers an antibody that defends a cell from a pathogen or infectious particle by neutralizing any effect it has biologically. Neutralization renders the particle no longer infectious or pathogenic. Neutralizing antibodies are part of the humoral response of the adaptive immune system against viruses, intracellular bacteria and microbial toxin. By binding specifically to surface antigen on an infectious particle, neutralizing antibodies prevent the particle from interacting with its host cells it might infect and destroy.
[0173] Immunity due to neutralizing antibodies is also known as sterilizing immunity, as the immune system eliminates the infectious particle before any infection took place.
[0174] The term “antigen” refers to any substance that will elicit an immune response. For instance, an antigen relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T-lymphocytes (T cells). As used herein, the term “antigen” comprises any molecule which comprises at least one epitope. For instance, an antigen is a molecule which, optionally after processing, induces an immune reaction. For instance, any suitable antigen may be used, which is a candidate for an immune reaction, wherein the immune reaction may be a cellular immune reaction. For instance, the antigen may be presented by a cell, which results in an immune reaction against the antigen. For example, an antigen is a product which corresponds to or is derived from a naturally occurring antigen. Such antigens include, but are not limited to, SARS-CoV-2 structural proteins S, N, M, and E, and any variants or mutants thereof.
[0175] The term “pharmaceutical composition” refers to a formulation comprising an active ingredient, and optionally a pharmaceutically acceptable carrier, diluent or excipient. The term “active ingredient” can interchangeably refer to an “effective ingredient,” and is meant to refer to any agent that is capable of inducing a sought-after effect upon administration. By “pharmaceutically acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof, nor to the activity of the active ingredient of the formulation. Pharmaceutically acceptable carriers, excipients or stabilizers are well known in the art, for example Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (for example, Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Examples of carrier include, but are not limited to, liposome, nanoparticles, ointment, micelles, microsphere, microparticle, cream, emulsion, and gel.
[0176] Examples of excipient include, but are not limited to, anti-adherents such as magnesium stearate, binders such as saccharides and their derivatives (sucrose, lactose, starches, cellulose, sugar alcohols and the like) protein like gelatin and synthetic polymers, lubricants such as talc and silica, and preservatives such as antioxidants, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate and parabens. Examples of diluent include, but are not limited to, water, alcohol, saline solution, glycol, mineral oil and dimethyl sulfoxide (DMSO).
[0177] The term “vaccine” or “immunogenic composition” relates to a pharmaceutical preparation (pharmaceutical composition) or product that upon administration induces an immune response, e.g., a cellular immune response, which recognizes and attacks a pathogen or a diseased cell. The term “immune response” refers to an integrated bodily response to an antigen and refers to a cellular immune response and/or a humoral immune response. The immune response may be protective/preventive/prophy lactic and/or therapeutic.
[0178] The term “cellular immune response” or “cell-mediated immune response” describes any adaptive immune response in which antigen-specific T cells have the main role. It is defined operationally as all adaptive immunity that cannot be transferred to a native recipient by serum antibody. In contrast, the term “humoral immune response” describes immunity due to antibodies.
[0179] The cellular response relates to cells called T cells or T-lymphocytes which act as either “helpers” or “killers”. The helper T cells (also termed CD4+ T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill diseased cells such as cancer cells, preventing the production of more diseased cells.
[0180] The terms “immunoreactive cell” “immune cells” or “immune effector cells” relate to a cell which exerts effector functions during an immune reaction. An “immunoreactive cell” preferably is capable of binding an antigen or a cell characterized by presentation of an antigen, or an antigen peptide derived from an antigen and mediating an immune response. For example, such cells secrete cytokines and/or chemokines, secrete antibodies, recognize cancerous cells, and optionally eliminate such cells. For example, immunoreactive cells comprise T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells.
[0181] The term “adjuvant” refers to a pharmacological or immunological agent that modifies the effect of other agents. An adjuvant may be added to the vaccine composition of the invention to boost the immune response to produce more antibodies and longer-lasting immunity, thus minimizing the dose of antigen needed. Adjuvants may also be used to enhance the efficacy of a vaccine by helping to modify the immune response to particular types of immune system cells: for example, by activating T cells instead of antibody-secreting B cells depending on the purpose of the vaccine. Immunologic adjuvants are added to vaccines to stimulate the immune system's response to the target antigen, but do not provide immunity themselves. Examples of adjuvants include, but are not limited to analgesic adjuvants; inorganic compounds such as alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide; mineral oil such as paraffin oil; bacterial products such as killed bacteria (Bordetella pertussis, Mycobacterium bovis, toxoids); nonbacterial organics such as squalene; delivery systems such as detergents (Quil A); plant saponins from Quillaja, soybean, or Polygala senega; cytokines such as IL-1, IL- 2, IL- 12; combination such as Freund’s complete adjuvant, Freund’s incomplete adjuvant; foodbased oil such as Adjuvant 65, which is based on peanut oil.
Embodiments
[0182] Embodiments provided include:
[0183] Embodiment 1. An immunogenic composition comprising a first vesicle and a first fusion protein, and a second vesicle and a second fusion protein, wherein said first fusion protein comprises a first virus polypeptide and an exosomal polypeptide and said second fusion protein comprises a second virus polypeptide and an exosomal polypeptide.
[0184] Embodiment 2. An immunogenic composition comprising a first vesicle and a first fusion protein, a second vesicle and a second fusion protein, and a third vesicle and a third fusion protein, wherein said first fusion protein comprises a first virus polypeptide and an exosomal polypeptide, said second fusion protein comprises a second virus polypeptide and an exosomal polypeptide, and said third fusion protein comprises a third virus polypeptide and an exosomal polypeptide.
[0185] Embodiment 3. An immunogenic composition of embodiment 1 or embodiment 2 further comprising an excipient.
[0186] Embodiment 4. An immunogenic composition of embodiment 3, wherein the excipient comprises a buffer.
[0187] Embodiment 5. An immunogenic composition of embodiment 3 or 4, wherein the excipient comprises a cryoprotectant.
[0188] Embodiment 6. An immunogenic composition of any one of embodiments 1-5 not comprising an adjuvant.
[0189] Embodiment 7. An immunogenic composition of any one of embodiments 1-4, wherein said first fusion protein and said second fusion protein are present in a membrane of the each respective vesicle.
[0190] Embodiment 8. An immunogenic composition of any one of embodiments 1-7, wherein a part or all of each virus polypeptide is present at or on an outer surface of the vesicle.
[0191] Embodiment 9. An immunogenic composition of any one of embodiments 1-8, wherein each fusion protein is present in the composition at a concentration of about 1 ng/100 pL to about 50 ng/100 pL.
[0192] Embodiment 10. An immunogenic composition of any one of embodiments 1-9, wherein the first virus polypeptide is a SARS-CoV-2 polypeptide and the second virus polypeptide is an influenza polypeptide. [0193] Embodiment 1 1. An immunogenic composition of any one of embodiments 1-10, wherein the first virus polypeptide is a SARS-CoV-2 spike protein polypeptide.
[0194] Embodiment 12. An immunogenic composition of any one of embodiments 1-11, wherein the first virus polypeptide is a SARS-CoV-2 delta variant spike protein polypeptide.
[0195] Embodiment 13. An immunogenic composition of any one of embodiments 1-12, wherein the first virus polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO SEQ ID NO: 1, or identical to SEQ ID NO:1.
[0196] Embodiment 14. An immunogenic composition of any one of embodiments 1-10, wherein the first virus polypeptide is a SARS-CoV-2 nucleocapsid protein polypeptide.
[0197] Embodiment 15. An immunogenic composition of embodiment 14, wherein the first virus polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO SEQ ID NO:5, or identical to SEQ ID NO:5.
[0198] Embodiment 16. An immunogenic composition of any one of embodiments 1-15, wherein the second virus polypeptide is an influenza hemagglutinin protein polypeptide.
[0199] Embodiment 17. An immunogenic composition of any one of embodiments 1-16, wherein the second virus polypeptide is an influenza hemagglutinin 3 (H3) protein polypeptide.
[0200] Embodiment 18. An immunogenic composition of any one of embodiments 1-17, wherein the second virus polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO: 14, or identical to SEQ ID NO: 14.
[0201] Embodiment 19. An immunogenic composition of any one of embodiments 2-18, wherein the third virus polypeptide is a respiratory syncytial virus (RSV) protein polypeptide.
[0202] Embodiment 20. An immunogenic composition of any one of embodiments 2-19, wherein the third virus polypeptide is an RSV fusion (RSV F) protein polypeptide.
[0203] Embodiment 21. An immunogenic composition of any one of embodiments 2-20, wherein the third virus polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO: 18, or identical to SEQ ID NO: 18. [0204] Embodiment 22. An immunogenic composition of any one of embodiments 1-21, wherein the exosomal polypeptide is a tetraspanin protein polypeptide.
[0205] Embodiment 23. An immunogenic composition of any one of embodiments 1-22, wherein the exosomal polypeptide is a CD9 protein polypeptide.
[0206] Embodiment 24. An immunogenic composition of any one of embodiments 1-23, wherein the exosomal polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO SEQ ID NO:10, or identical to SEQ ID NO:10.
[0207] Embodiment 25. An immunogenic composition of any one of embodiments 1-13 and 17-24, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO:2, or identical to SEQ ID NO:2.
[0208] Embodiment 26. An immunogenic composition of any one of embodiments 1-13 and 17-24, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO:3, or identical to SEQ ID NO:3.
[0209] Embodiment 27. An immunogenic composition of any one of embodiments 1-10 and 14-23, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO:6, or identical to SEQ ID NO:6.
[0210] Embodiment 28. An immunogenic composition of any one of embodiments 1-10, 14-23 and 27, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO:7, or identical to SEQ ID NO:7.
[0211] Embodiment 29. An immunogenic composition of any one of embodiments 1-28, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO: 15, or identical to SEQ ID NO: 15.
[0212] Embodiment 30. An immunogenic composition of any one of embodiments 1-29, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO: 16, or identical to SEQ ID NO:16.
[0213] Embodiment 31. An immunogenic composition of any one of embodiments 2-30, wherein the third fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO:19, or identical to SEQ ID NO:19. [0214] Embodiment 32. An immunogenic composition of any one of embodiments 2-31, wherein the third fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO:20, or identical to SEQ ID NO:20.
[0215] Embodiment 33. An immunogenic composition of any one of embodiments 1-32, wherein an immunogenic dose of said composition comprises about 1 ng to about 50 ng of the first fusion protein or first virus polypeptide, and about 1 ng to about 50 ng of the second fusion protein or second virus polypeptide.
[0216] Embodiment 34. An immunogenic composition of any one of embodiments 2-33, wherein an immunogenic dose of said composition comprises about 1 ng to about 50 ng of the third fusion protein or third virus polypeptide.
[0217] Embodiment 35. A method of immunizing a subject against a virus infection comprising administering to the subject an immunogenically effective dose of an immunogenic composition of any one of embodiments 1-34.
[0218] Embodiment 36. A method of embodiment 35, wherein said immunogenically effective dose elicits protective immunity in said subject against a first virus and a second virus.
[0219] Embodiment 37. A method of embodiment 35 or 36, wherein said immunogenically effective dose elicits protective immunity in said subject against a first virus, a second virus, and a third virus.
[0220] Embodiment 38. A method of embodiment 35 or 36, wherein the immunogenic composition comprises a SARS-CoV-2 spike protein polypeptide and an influenza hemagglutinin polypeptide, and the first virus is a SARS-CoV2 and the second virus is an influenza virus.
[0221] Embodiment 39. A method of any one of embodiments 35-38, wherein the immunogenic composition comprises a SARS-CoV-2 spike protein polypeptide, an influenza hemagglutinin polypeptide, and a respiratory syncytial virus (RSV) fusion protein polypeptide, and the first virus is a SARS-CoV2, the second virus is an influenza virus, and the third virus is an RSV. [0222] Embodiment 40. A method of any one of embodiments 35-39, wherein said immunogenically effective dose elicits protective immunity in said subject against a SARS-CoV- 2 delta variant and a SARS-CoV-2 omicron variant.
[0223] Embodiment 41. A method of any one of embodiments 35-40, wherein the immunogenically effective dose comprises about 1 ng to about 50 ng of said first fusion protein, about 1 ng to about 50 ng of said second fusion protein, and about 1 ng to about 50 ng of said third fusion protein.
[0224] Embodiment 42. A method of any one of embodiments 35-41 further comprising administering to the subject a second effective dose of the immunogenic composition of any one of embodiments 1-34.
[0225] Embodiment 43. A method of any one of embodiments 35-42, wherein the elicited protective immunity comprises (a) a high antibody titer directed against the first, second, and third virus, (b) a CD4+ T-cell response against the first, second, and third virus, and (c) a CD8+ cytotoxic T-cell response against the first, second, and third virus.
[0226] Embodiment 44. A synthetic fusion protein comprising a virus polypeptide and an exosomal polypeptide.
[0227] Embodiment 45. A synthetic fusion protein of embodiment 44 further comprising a linker polypeptide positioned between the virus polypeptide and the exosomal polypeptide.
[0228] Embodiment 46. A synthetic fusion protein of embodiment 44 or 45 further comprising a hinge polypeptide positioned between the virus polypeptide and the exosomal polypeptide.
[0229] Embodiment 47. A synthetic fusion protein of any one of embodiments 44-46 further comprising a transmembrane domain polypeptide positioned between the virus polypeptide and the exosomal polypeptide.
[0230] Embodiment 48. A synthetic fusion protein of any one of embodiments 44-47, wherein the exosomal polypeptide is a tetraspanin polypeptide.
[0231] Embodiment 49. A synthetic fusion protein of any one of embodiments 44-48, wherein the exosomal polypeptide is a CD9 polypeptide. [0232] Embodiment 50. A synthetic fusion protein of any one of embodiments 44-49, wherein the virus polypeptide is a SARS-CoV-2 structural protein polynucleotide.
[0233] Embodiment 51. A synthetic fusion protein of any one of embodiments 44-50, wherein the virus polypeptide is a SARS-CoV-2 spike protein polypeptide.
[0234] Embodiment 52. A synthetic fusion protein of embodiment 51, wherein said SARS- CoV-2 spike protein polypeptide comprises a one of more of a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and a di-proline substitution (2P [986KV987-to- 986PP987]).
[0235] Embodiment 53. A synthetic fusion protein of any one of embodiments 44-52, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, a SARS-CoV-2 spike protein polypeptide, a linker polypeptide, and a CD9 polypeptide.
[0236] Embodiment 54. A synthetic fusion protein of any one of embodiments 44-53, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:3.
[0237] Embodiment 55. A synthetic fusion protein of any one of embodiments 44-54, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:3.
[0238] Embodiment 56. A synthetic fusion protein of any one of embodiments 44-50, wherein the virus polypeptide in a SARS-CoV-2 nucleocapsid protein polypeptide.
[0239] Embodiment 57. A synthetic fusion protein of any one of embodiments 44-50 and 56, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, a signal peptide, a SARS-CoV-2 nucleocapsid protein polypeptide, a hinge region, a transmembrane domain polypeptide, a linker polypeptide, and a CD9 polypeptide.
[0240] Embodiment 58. A synthetic fusion protein of any one of embodiments 44-50, 56 and 57, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:7.
[0241] Embodiment 59. A synthetic fusion protein of any one of embodiments 44-50 and 56-58, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:7. [0242] Embodiment 60. A synthetic fusion protein of any one of embodiments 44-49, wherein the virus polypeptide is an influenza protein polypeptide.
[0243] Embodiment 61. A synthetic fusion protein of embodiment 60, wherein said influenza protein polypeptide comprises a hemagglutinin.
[0244] Embodiment 62. A synthetic fusion protein of embodiment 60 or 61, wherein said influenza protein polypeptide comprises a hemagglutinin 3 (H3).
[0245] Embodiment 63. A synthetic fusion protein of any one of embodiments 60-62, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, a hemagglutinin protein polypeptide, a linker polypeptide, and a CD9 polypeptide.
[0246] Embodiment 64. A synthetic fusion protein of any one of embodiments 60-63, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16.
[0247] Embodiment 65. A synthetic fusion protein of any one of embodiments 60-64, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 16.
[0248] Embodiment 66. A synthetic fusion protein of any one of embodiments 44-49, wherein the virus polypeptide is a respiratory syncytial virus (RSV) protein polypeptide.
[0249] Embodiment 67. A synthetic fusion protein of embodiment 66, wherein said RSV protein polypeptide comprises an RSV fusion (RSV F) protein.
[0250] Embodiment 68. A synthetic fusion protein of embodiment 66 or 67, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, an RSV F protein polypeptide, a linker polypeptide, and a CD9 polypeptide.
[0251] Embodiment 69. A synthetic fusion protein of any one of embodiments 66-68, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 20.
[0252] Embodiment 70. A synthetic fusion protein of any one of embodiments 60-64, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:20. [0253] Embodiment 71 . A synthetic polynucleotide encoding a synthetic fusion protein of any one of embodiments 44-70.
[0254] Embodiment 72. A synthetic polynucleotide of embodiment 71 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 13.
[0255] Embodiment 73. A synthetic polynucleotide of embodiment 71 or 72 comprising a nucleic acid sequence set forth in SEQ ID NO: 13.
[0256] Embodiment 74. A synthetic polynucleotide of any one of embodiments 71-73 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 11.
[0257] Embodiment 75. A synthetic polynucleotide of any one of embodiments 71-74 comprising a nucleic acid sequence set forth in SEQ ID NO: 11.
[0258] Embodiment 76. A synthetic polynucleotide of any one of embodiments 71-75 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:4.
[0259] Embodiment 77. A synthetic polynucleotide of any one of embodiments 71-76 comprising a nucleic acid sequence set forth in SEQ ID NO:4.
[0260] Embodiment 78. A synthetic polynucleotide of any one of embodiments 71-73 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 12.
[0261] Embodiment 79. A synthetic polynucleotide of any one of embodiments 71-73 and 78 comprising a nucleic acid sequence set forth in SEQ ID NO: 12.
[0262] Embodiment 80. A synthetic polynucleotide of any one of embodiments 71-73, 78 and 79 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:8.
[0263] Embodiment 81. A synthetic polynucleotide of any one of embodiments 71-73 and 78-80 comprising a nucleic acid sequence set forth in SEQ ID NO:8.
[0264] Embodiment 82. A synthetic polynucleotide of any one of embodiments 71-73 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:22.
[0265] Embodiment 83. A synthetic polynucleotide of any one of embodiments 71-73 and 82 comprising a nucleic acid sequence set forth in SEQ ID NO:22. [0266] Embodiment 84. A synthetic polynucleotide of any one of embodiments 71-73, 82 and 83 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 17.
[0267] Embodiment 85. A synthetic polynucleotide of any one of embodiments 71-73 and 82-84 comprising a nucleic acid sequence set forth in SEQ ID NO: 17.
[0268] Embodiment 86. A synthetic polynucleotide of any one of embodiments 71-73 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:23.
[0269] Embodiment 87. A synthetic polynucleotide of any one of embodiments 71-73 and 86 comprising a nucleic acid sequence set forth in SEQ ID NO:23.
[0270] Embodiment 88. A synthetic polynucleotide of any one of embodiments 71-73, 86 and 87 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:21.
[0271] Embodiment 89. A synthetic polynucleotide of any one of embodiments 71-73 and 86-88 comprising a nucleic acid sequence set forth in SEQ ID NO:21.
[0272] Embodiment 90. A cell comprising a synthetic polynucleotide of any one of embodiments 71-89.
[0273] Embodiment 91. A cell of embodiment 90, wherein the cell is a metazoan cell.
[0274] Embodiment 92. A cell of embodiment 90 or 91 wherein the cell is a vertebrate cell.
[0275] Embodiment 93. A cell of any one of embodiments 90-92, wherein the cell is a mammalian cell.
[0276] Embodiment 94. A cell of any one of embodiments 90-93, wherein the cell is a primate cell.
[0277] Embodiment 95. A cell of any one of embodiments 90-94, wherein the cell is a human cell.
[0278] Embodiment 96. A cell of any one of embodiments 90-95, wherein the cell is a primary cell.
[0279] Embodiment 97. A cell of any one of embodiments 90-95, wherein the cell is a human embryonic kidney cell. [0280] Embodiment 98. A cell of embodiment 97, wherein the cell is a 293 cell.
[0281] Embodiment 99. A cell of any one of embodiments 90-98, wherein said cell is produced by transducing the cell with a lentivirus comprising the synthetic polynucleotide.
[0282] Embodiment 100. A cell of any one of embodiments 90-99, wherein said cell comprises a synthetic fusion protein of any one of embodiments 44-70.
[0283] Embodiment 101. A vesicle comprising a synthetic fusion protein of any one of embodiments 44-70.
[0284] Embodiment 102. A vesicle of embodiment 101, wherein said vesicle is an exosome.
[0285] Embodiment 103. A vesicle of embodiment 101 or 102, wherein said vesicle has a diameter of about 50-500 nm.
[0286] Embodiment 104. A vesicle of any one of embodiments 101-103, wherein a SARS- CoV-2 spike protein polypeptide is expressed on an outer surface of said vesicle.
[0287] Embodiment 105. A vesicle of any one of embodiments 101-103, wherein said vesicle expresses a SARS-CoV-2 nucleocapsid protein polypeptide on its surface.
[0288] Embodiment 106. A vesicle of any one of embodiments 101-103, wherein said vesicle expresses an influenza hemagglutinin protein polypeptide on its surface.
[0289] Embodiment 107. A vesicle of any one of embodiments 101-103, wherein said vesicle expresses a respiratory syncytial virus fusion protein polypeptide on its surface.
[0290] Embodiment 108. A method of making a vesicle of any one of embodiments 101-107 comprising culturing a cell of any one of embodiments 90-100 in a cell culture medium, collecting the cell culture medium, and purifying a plurality of vesicles comprising said vesicle from the cell culture medium.
[0291] Embodiment 109. A method of embodiment 108 further comprising inducing expression of a synthetic polynucleotide of any one of embodiments 71-89 to produce a synthetic fusion protein of any one of embodiments 44-70.
[0292] Embodiment 110. A method of embodiment 105, wherein said inducing comprises contacting the cell with tetracycline, doxycycline, or analogs thereof. [0293] Embodiment 1 11. A method of embodiment 105, wherein said inducing comprises removing from the cell tetracycline, doxycycline, or analogs thereof.
[0294] Embodiment 112. A method of eliciting an immune response in a subject comprising administering to said subject a first dose of an immunogenic composition comprising a plurality of vesicles comprising a vesicle of any one of embodiments 101-107 or a vesicle produced according to a method of any one of embodiments 108-111, wherein a synthetic fusion protein of any one of embodiments 44-70 is expressed on an outer surface of said vesicle.
[0295] Embodiment 113. A method of embodiment 112 further comprising administering to said subject a second dose of the immunogenic composition a period of time after administering the first dose.
[0296] Embodiment 114. A method of embodiment 113, wherein the period of time is 14 days - 1 year.
[0297] Embodiment 115. A method of any one of embodiments 112-114, wherein the first dose or the second dose comprises about 100 pL - 1 mL of the immunogenic composition, wherein said immunogenic composition comprises about 0.3 ng/mL - 3 pg/mL of said synthetic fusion protein.
[0298] Embodiment 116. A method of any one of embodiments 112-115, wherein the first dose or the second dose comprises about 100 pL - 1 mL of the immunogenic composition which comprises about 2E9 vesicles/mL - 3E13 vesicles/mL.
[0299] Embodiment 117. A method of any one of embodiments 112-115, wherein said synthetic fusion protein comprises a SARS-CoV-2 spike protein polypeptide.
[0300] Embodiment 118. A method of any one of embodiments 112-117, wherein said synthetic fusion protein comprises a SARS-CoV-2 nucleocapsid protein polypeptide.
[0301] Embodiment 119. A method of any one of embodiments 112-117, wherein said synthetic fusion protein comprises an influenza hemagglutinin protein polypeptide.
[0302] Embodiment 120. A method of any one of embodiments 112-117, wherein said synthetic fusion protein comprises a respiratory syncytial virus fusion (RSV F) protein polypeptide. [0303] Embodiment 121 . A method of any one of embodiments 1 12-120, wherein said immunogenic composition comprises vesicles that express on the outer surface SARS-CoV-2 spike protein polypeptides, vesicles that express on the outer surface influenza hemagglutinin protein polypeptides, and vesicles that express on the outer surface RSV F protein polypeptides.
[0304] Embodiment 122. A method of any one of embodiments 112-121, wherein the elicited immune response comprises producing neutralizing antibodies against an antigen present in the synthetic fusion protein.
[0305] Embodiment 123. A method of any one of embodiments 112-123, wherein the elicited immune response comprises producing anti-spike antibodies.
[0306] Embodiment 124. A method of any one of embodiments 112-123, wherein the elicited immune response comprises a spike-specific T cell response.
[0307] Embodiment 125. A method of any one of embodiments 112-124, wherein the elicited immune response comprises producing anti-nucleocapsid antibodies.
[0308] Embodiment 126. A method of any one of embodiments 112-125, wherein the elicited immune response comprises a nucleocapsid-specific T cell response.
[0309] Embodiment 127. A method of any one of embodiments 112-126, wherein the elicited immune response comprises producing anti-hemagglutinin antibodies.
[0310] Embodiment 128. A method of any one of embodiments 112-127, wherein the elicited immune response comprises a hemagglutinin -specific T cell response.
[0311] Embodiment 129. A method of any one of embodiments 112-128, wherein the elicited immune response comprises producing anti-RSV F antibodies.
[0312] Embodiment 130. A method of any one of embodiments 112-129, wherein the elicited immune response comprises a RSV F -specific T cell response.
[0313] Embodiment 131. A method of any one of embodiments 112-130, wherein the immune response persists in the subject for up to nine months.
[0314] Embodiment 132. A method of any one of embodiments 112-130, wherein the immune response persists in the subject for at least nine months. Extracellular Vesicles and Exosomes
[0315] A variety of host cells are known in the art and suitable for proteins expression and extracellular vesicles production. Non-limiting examples of typical cell used for transfection include, but are not limited to, a bacterial cell, a eukaryotic cell, a yeast cell, an insect cell, or a plant cell. For example, human embryonic kidney 293 (HEK293), E. coli, Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SJ9, CHO, COS (e g. COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H, or 293F. See, e.g., Portolano et al., “Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach,” Journal of Visualized Experiments, October 2014, 92, e51897, pp. 1-8 for a description of the recombinant proteins in 293 cells in suspension culture.
[0316] Extracellular vesicles (EVs) are lipid bound vesicles secreted by cells into the extracellular space. The three main subtypes of EVs are microvesicles (MVs), exosomes, and apoptotic bodies, which are differentiated based upon their biogenesis, release pathways, size, content, and function. For a review of extracellular vesicles, see, e.g., Doyle and Wang, “Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis,” Cells, v.8(7), 2019 Jul., and references therein.
[0317] Exosomes include small, secreted vesicles of about 20-200 nm in diameter that are released by inter alia mammalian cells, and made either by budding into endosomes or by budding from the plasma membrane of a cell. In some cases, exosomes have a characteristic buoyant density of approximately 1.1 -1.2 g/mL, and a characteristic lipid composition. Their lipid membrane is typically rich in cholesterol and contains sphingomyelin, ceramide, lipid rafts and exposed phosphatidylserine. Exosomes express certain marker proteins, such as integrins and cell adhesion molecules, but generally lack markers of lysosomes, mitochondria, or caveolae. In some embodiments, the exosomes contain cell-derived components, such as, but not limited to, proteins, DNA and RNA (e.g., microRNA [miR] and noncoding RNA). In some embodiments, exosomes can be obtained from cells obtained from a source that is allogeneic, autologous, xenogeneic, or syngeneic with respect to the recipient of the exosomes. [0318] Certain types of RNA, e.g., microRNA (miRNA), are known to be carried by exosomes. miRNAs function as post-transcriptional regulators, often through binding to complementary sequences on target messenger RNA transcripts (mRNAs), thereby resulting in translational repression, target mRNA degradation and/or gene silencing.
[0319] Useful exosomes can be obtained from any cell source, including prokaryotes, plants, fungi, metazoans, vertebrate, mammalian, primate, human, autologous cells and allogeneic cells. See, e.g., Kim et al., “Platform technologies and human cell lines for the production of therapeutic exosomes,” Extracell Vesicles Circ Nucleic Acids 2021;2:3-17. For example, exosomes may be derived from mesenchymal stem cells, embryonic stem cells, iPS cells, immune cells, PBMCs, neural stem cells, HEK293 cells, which are described in e.g., Dumont et al., “Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives,” Crit Rev Biotechnol 2016;36: 1110-22, HEK293T cells, which are described in e.g., Li et al., “Identification and characterization of 293T cell-derived exosomes by profiling the protein, mRNA and MicroRNA components,” PLoS One 2016; 1 l:e0163043, 293F cells, Stenkamp et al., “Exosomes represent a novel mechanism of regulatory T cell suppression (P1079),” J Immunol May 1, 2013, 190 (1 Supplement) 121.11, amniotic cells, CAR-T cells, cardiospheres and cardiosphere-derived cells (CDCs), which are described in, e.g., WO2014028493, W02022006178A1, US20210032598A1, US9828603B2, EP2914273A1, US20200316226A1, US20120315252A1, US20170360842A1, and references therein., and the like.
[0320] Briefly, methods for preparing exosomes can include the steps of: culturing cells in media, isolating the cells from the media, purifying the exosome by, e.g., sequential centrifugation, and optionally, clarifying the exosomes on a density gradient, e.g., sucrose density gradient. In some instances, the isolated and purified exosomes are essentially free of non-exosome components, such as cellular components or whole cells. Exosomes can be resuspended in a buffer such as a sterile PBS buffer containing 0.01-1% human serum albumin. The exosomes may be frozen and stored for future use.
[0321] Exosomes can be collected, concentrated and/or purified using methods known in the art.
For example, differential centrifugation has become a leading technique wherein secreted exosomes are isolated from the supernatants of cultured cells. This approach allows for separation of exosomes from larger extracellular vesicles and from most non-particulate contaminants by exploiting their size. Exosomes can be prepared as described in a wide array of papers, including but not limited to, Fordjour et al., “A shared pathway of exosome biogenesis operates at plasma and endosome membranes”, bioRxiv , preprint posted February 11, 2019, at https://www.biorxiv.org/content/10.1101/545228vl; Booth et ah, “Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane”, J Cell BioL, 172:923-935 (2006); and, Fang et ah, “Higher-order oligomerization targets plasma membrane proteins and HIV gag to exosomes”, PLoS Biol., 5 :el58 (2007). Exosomes using a commercial kit such as, but not limited to the ExoSpin™ Exosome Purification Kit, Invitrogen® Total Exosome Purification Kit, PureExo® Exosome Isolation Kit, and ExoCap™ Exosome Isolation kit. Methods for isolating exosome from stem cells are found in, e.g., Tan et ah, Journal of Extracellular Vesicles, 2:22614 (2013); Ono et ah, Sci Signal, 7(332):ra63 (2014) and U.S. Application Publication Nos. 2012/0093885 and 2014/0004601. Methods for isolating exosome from cardiosphere-derived cells are found in, e.g., Ibrahim et al., “Exosomes as critical agents of cardiac regeneration triggered by cell therapy,” Stem Cell Reports, 2014. Specific methodologies include ultracentrifugation, density gradient, HPLC, adherence to substrate based on affinity, or filtration based on size exclusion.
[0322] Size exclusion allows for their separation from biochemically similar, but biophysically different microvesicles, which possess larger diameters of up to 1,000 nm. Differences in flotation velocity further allows for separation of differentially sized exosomes. In general, exosome sizes will possess a diameter ranging from 30-200 nm, including sizes of 40-100 nm. Further purification may rely on specific properties of the particular exosomes of interest. This includes, e.g., use of immunoadsorption with a protein of interest to select specific vesicles with exoplasmic or outward orientations.
[0323] Among current methods, e.g., differential centrifugation, discontinuous density gradients, immunoaffmity, ultrafiltration and high-performance liquid chromatography (HPLC), differential ultracentrifugation is the most commonly used for exosome isolation. This technique utilizes increasing centrifugal force from 2,000xg to 10,000xg to separate the medium- and larger-sized particles and cell debris from the exosome pellet at 100,000xg. Centrifugation alone allows for significant separation/collection of exosomes from a conditioned medium, although it may be insufficient to remove various protein aggregates, genetic materials, particulates from media and cell debris that are common contaminants. Enhanced specificity of exosome purification may deploy sequential centrifugation in combination with ultrafiltration, or equilibrium density gradient centrifugation in a sucrose density gradient, to provide for the greater purity of the exosome preparation (flotation density 1.1 - 1.2 g/mL) or application of a discrete sugar cushion in preparation.
[0324] Ultrafiltration can be used to purify exosomes without compromising their biological activity. Membranes with different pore sizes - such as 100 kDa molecular weight cutoff (MWCO) and gel filtration to eliminate smaller particles - have been used to avoid the use of a nonneutral pH or non-physiological salt concentration. Currently available tangential flow filtration (TFF) systems are scalable (to >10,000L), allowing one to not only purify, but concentrate the exosome fractions, and such approaches are less time consuming than differential centrifugation. HPLC can also be used to purify exosomes to more uniformly sized particle preparations and preserve their biological activity as the preparation is maintained at a physiological pH and salt concentration. Other chemical methods have exploit differential solubility of exosomes for precipitation techniques, addition to volume-excluding polymers (e.g., polyethylene glycols (PEGs)), possibly combined additional rounds of centrifugation or filtration. For example, a precipitation reagent, ExoQuick®, can be added to conditioned cell media to quickly and rapidly precipitate a population of exosomes, although re-suspension of pellets prepared via this technique may be difficult. Flow field-flow fractionation (F1FFF) is an elution-based technique that is used to separate and characterize macromolecules (e.g., proteins) and nano-to micro-sized particles (e.g., organelles and cells) and which has been successfully applied to fractionate exosomes from culture media.
[0325] Beyond these techniques relying on general biochemical and biophysical features, focused techniques may be applied to isolate specific exosomes of interest. This includes relying on antibody immunoaffinity to recognizing certain exosome-associated antigens. As described, exosomes further express the extracellular domain of membrane-bound receptors at the surface of the membrane. This presents an opportunity for isolating and segregating exosomes in connection with their parental cellular origin, based on a shared antigenic profile. Conjugation to magnetic beads (e.g., such as anti-CD81 magnetic beads), chromatography matrices, plates or microfluidic devices allows isolating of specific exosome populations of interest as may be related to their production from a parent cell of interest or associated cellular regulatory state. Other affinity-capture methods use lectins which bind to specific saccharide residues on the exosome surface.
[0326] For example, exosomes (and other extracellular vesicles) may be produced via 293F cells. The 293F cells may be transfected with (or transduced with a lentivirus bearing) a polynucleotide that encodes a spike protein or a nucleocapsid protein or an influenza hemagglutinin protein, or chimeral fusions thereof, as described herein (see Fig. 4A), and expressing the spike protein or nucleocapsid protein, such that the spike protein or nucleocapsid is sorted into and displayed in or on the exosomes isolated therefrom. An example procedure for making exosomes from 293F cells may include steps as follows: 293F cells (Gibco™, Cat.# 51- 0029, ThermoFisher Scientific, Waltham, MA) may be tested for pathogens and found to be free of viral (cytomegalovirus, human immunodeficiency virus I and II, Epstein Barr virus, hepatitis B virus, and parvovirus Bl 9) and bacterial ( Mycoplasma ) contaminants. Cells may be maintained in FreeStyle™ 293 Expression Medium (Gibco, Cat.# 12338-018, ThermoFisher Scientific, Waltham, MA) and incubated at 37°C in 8% CO2. For exosome production, 293F cells may be seeded at a density of 1.5E6 cells/ml in shaker flasks in a volume of about 1/4 the flask volume and grown at a shaking speed of about 110 rpm. HEK293 cells may be grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum.
[0327] To purify exosomes, the 293F cells may be cultured in shaker flasks for a period of three days. Cells and large cell debris may be removed by centrifugation at 300 x g for 5 minutes followed by 3,000 x g for 15 minutes. The resulting supernatant may be passed through a 0.22 pm sterile filtration filter unit (Thermo Fisher, Cat.# 566-0020) to generate a clarified tissue culture supernatant (CTCS). The CTCS may be concentrated by centrifugal filtration (Centricon Plus-70, Ultracel-PL Membrane, 100 kDa size exclusion, Millipore Sigma, Cat.# UFC710008, St. Louis, MO), with aboutl20 mL CTCS concentrated to about 0.5 mL. Concentrated CTCS my then be purified by size exclusion chromatography (SEC) in lx PBS (qEV original columns/35 nm: Izon Science, Cat.# SP5), with the exosomes present in each 0.5 ml starting sample eluting in three 0.5 ml fractions. Purified exosomes may be reconcentrated using Amicon® Ultra-4 100 kDa cutoff spin columns (Cat.# UFC810024). This process may yield a population of exosomes/small EVs that have the expected ultrastructure and size distribution profile of human exosomes and contain the exosomal marker proteins CD9 and CD63, at a concentrating effect of about 500-fold, to a final concentration of 1E10 - 2E12 exosomes/ml. The concentration and size of the isolated extracellular vesicles may be measured using NANOSIGHT nanoparticle tracking analysis system (Malvern Panalytical, Malvern, UK).
SARS-CoV-2 Proteins
[0328] Disclosed are membrane-bound vesicles that contain one or more populations of SARS- CoV-2 structural proteins. By contain, what is meant is that the contained protein may be within the lumen of the vesicle, displayed on the surface of the vesicle, or both within the lumen and on the surface. Here, those fusion proteins containing a tetraspanin protein polypeptide sequence are mostly displayed on the surface of the vesicle. Those proteins that are displayed on the surface of the vesicle may have a portion of the protein inside the lumen, a portion of the protein spanning the membrane of the vesicle, i.e., a transmembrane spanning region or domain, and a portion of the protein extending outside the vesicle. In one embodiment, the SARS-CoV-2 structural protein is a spike glycoprotein (S), a nucleocapsid (N) protein, a membrane (M) protein, or an envelope (E) protein, or any combination thereof. See Satarker and Nampoothiri, “Structural Proteins in Severe Acute Respiratory Syndrome Coronavirus-2,” Arch Med Res. 2020 Aug;
51(6): 482-491. See, e.g., Figs. 1 A, IB, 2A, and 2C, which depict spike/CD9 and nucleocapsid/CD fusion proteins for expression of spike or nucleocapsid antigen on the surface of the exosomes.
[0329] In one embodiment, the antigenic protein is SARS-CoV-2 spike glycoprotein (a.k.a. spike protein or simply “spike”). The spike protein can be of any variant of SARS-CoV-2, such as, e.g., the Wuhan-1 strain, an omicron variant (e.g., BA.2 variant), a delta variant (e.g., B.1.617.2, AYA, AY.103, AY.44, AY.43 variant, or the like), and epsilon variant (e.g., B.1.427 or B.1.429 variant), or any variant now known or yet to be discovered. As used herein, the term spike refers to any SARS-CoV-2 spike glycoprotein, chimera, or fragment thereof unless otherwise specified.
[0330] In some embodiments, the SARS-CoV-2 spike protein is the Wuhan-1 strain SARS-CoV-
2 spike protein or a Delta variant SARS-CoV-2 spike protein; a furin-blocked, trimer-stabilized form of the Wuhan- 1 strain SARS-CoV-2 spike protein; the Wuhan- 1 strain SARS-CoV-2 spike protein with an amino acid change of D614G; the Wuhan-1 strain SARS-CoV-2 spike protein with di-proline substitutions of 986KV987-to-986PP987 (S-2P); and/or the Wuhan- 1 strain SARS-CoV-2 spike protein with cleavage site mutations of 682RRAR685-to-682GSAG685, or equivalent (S-CSM).
Extracellular Vesicles Displaying Viral Protein
[0331] In one embodiment, the invention provides extracellular vesicles that express on their surface (a.k.a. “display”) spike protein or nucleocapsid that are useful as a vaccine against multiple variants of SARS-CoV-2. In one embodiment, the invention provides extracellular vesicles that express on their surface (a.k.a. “display”) hemagglutinin protein or neuraminidase protein that are useful as a vaccine against multiple variants of influenza. In one embodiment, the invention provides a combination of vesicles, some of which display a SARS-CoV-2 protein and some of which display an influenza protein. The spike protein may be a delta variant with any one or more of trimer stabilization mutation, a prefusion conformation stabilization mutation (e g., di-proline stabilization mutations), and a furin cleavage site mutation. See, e.g., Walls et al., “Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein,” Cell 180, 281-292, April 16, 2020; Wrapp et al., “Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation,” Science367, 1260-1263 (2020)13 March 2020; Kirchdoerfer et al., “Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis,” Sci Rep 8, 15701 (2018), doi.org/10.1038/ s41598-018-34171-7; Pallesen et al., “Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen,” PNAS, E7348-E7357, published online August 14, 2017 pnas.org/cgi/doi/10.1073/pnas.1707304114; Juraszek, et al., “Stabilizing the closed SARS-CoV- 2 spike trimer,” Nat Commun 12, 244 (2021), doi.org/10.1038/s41467-020-20321-x; Johnson, 2020; and Xiong 2020; and references therein.
[0332] Here, synthetic fusion proteins containing a C-terminal tetraspanin protein and either a SARS-CoV-2 spike protein (Figs. 1A and IB), a SARS-CoV-2 nucleocapsid protein (Figs. 2A and 2B), or an influenza hemagglutinin protein were produced. It is important to note that nucleocapsid protein is a soluble protein that is not expressed at the virus surface, therefore the engineered fusion protein that includes tetraspanin and other transmembrane domains enables the placement of nucleocapsid protein at the vesicle surface to provide a readily accessible antigen for immunization (see Fig. 2B).
[0333] In one embodiment, the exosomes that express spike protein or nucleocapsid protein on their surface were made from spike/nuclceocapsid protein-expressing 293F cells. Turning to Figure 4A, in specific exemplar embodiment, a packaging cell (300) was transfected with a plasmid that encodes the spike- or nucleocapsid- or hemagglutinin-tetraspanin fusion protein (301a) and plasmids that encode lentivirus structural proteins (302a and 303a). The lentivirus proteins were produced (302b and 303b) and incorporated the fusion protein RNA to form a lentivirus vector containing the fusion protein RNA (304). A host cell (311) was transduced with the fusion protein RNA-bearing lentivirus (304), enabling the production of the SARS-CoV-2 spike or nucleocapsid - tetraspanin fusion protein (307a), and its sorting to the plasma membrane (307b) (Fig. 4B) and the surface of exosomes that were produced by the transduced host cells (Figs. 4C, 5A, 5B, and 6C).
[0334] Turning to Figures 5A-C and 6A and 6B, exosomes were isolated from 293F cells that harbored the spike-CD9 construct (Fig. 1 A and Fig. IB). Figure 5A shows the size distribution of those exosomes from about 50 nm to about 270 nm, with a median of about 100 - 150 nm.
Figure 5B shows expression of spike-containing fusion protein in the transduced 293F cells (lane 4), and enriched expres si on/di splay of the spike-containing fusion protein in the exosomes that were derived from the transduced 293F cells (lane 5). Figure 5C demonstrates significant expression of spike-containing fusion protein on those exosomes as determined by spike flow cytometry.
[0335] Transmission electron micrography confirmed that spike protein was expressed on the surface of the exosomes derived from the spike-CD9 transduced 293F cells (arrows at Figures 5A and 5B point to spikes at the surface of the vesicles).
[0336] Turning to Figures 7A-C, exosomes were isolated from 293F cells that harbored the nucleocapsid-CD9 construct (Fig. 2A and Fig. 2B). Figure 7A shows the size distribution of those exosomes from about 50 nm to about 270 nm, with a median of about 100 - 150 nm. Figure 7B shows expression of nucleocapsid-containing fusion protein in the exosomes that were derived from the transduced 293F cells (lane 5). Figure 7C demonstrates significant expression of spike-containing fusion protein on those exosomes as determined by spike flow cytometry. In one embodiment, expression of the nucleocapsid-CD9 fusion protein was put under control of the tet-inducible promotor. Here, the exosomes derived from the uninduced, but transduced host cells did not express nucleocapsid (see second curve from left at Fig. 7C).
[0337] Turning to Figures 8A - 8D, 293F cells were transfected or transduced with constructs or virions encoding a hemagglutinin (e.g., H3)-CD9 construct (Fig. 3A and Fig. 3B). Figures 8A and 8B demonstrate significant expression (~90%-96% in this example) of influenza H3- containing fusion protein on/in adherent 293F cells and 293F cells in suspension, respectively, as determined by anti-H3 flow cytometry. Exosomes produced by the 293F cells expressing the H3 fusion protein were purified and assessed for H3 and H3-CD9 by western blot (Fig. 8C, panel 1 and panel 2 respectively). The double detection of both H3 and CD90 at just below 180 kDa in lanes 6 of panels 1 and 2 demonstrates significant loading of H3-CD9 fusion protein in 293F- derived exosomes. Additionally, Figure 8D demonstrates significant expression of spikecontaining fusion protein on those exosomes as determined by spike flow cytometry. In one embodiment, exosomes produced from 293F cells expressing the influenza H3-CD9 fusion protein were combined with micron-sized magnetic beads coupled with anti -human CD81 (an exosome marker) and subjected to flow cytometry. Here, about 54% of those exosomes that express CD81 express influenza H3 antigen (Fig. 8D).
[0338] Turning to Figure 9, at day-1, subject mice were administered by intramuscular injection IX dose (i.e., 3E10 vesicles) or 10X dose i.e., 3E11 vesicles) or vesicles containing about 0.3 ng, about 3 ng, or about 10 ng of fusion protein, including spike-CD9, nucleocapsid-CD9, or influenza H3-CD9 fusion protein (710). At day-14, blood was collected from the administered mice and assessed for early humoral immune-responses (720) (see Figs. 10A and 12A-F). At day-21, the subject mice received a second dose of the subject spike or nucleocapsid expressing exosomes (730). At day-35, blood was collected from the administered mice and assessed for humoral immune-responses, including antibody production (see Figs. 10B and 11A-C) and neutralizing antibody production (740). At day-40, splenocytes were collected from the administered mice and assessed via ELISpot assay for cellular immune-responses (750) (see Figs. 10C, 10D, 13C, and 13D).
[0339] Turning to Figures 10A and 10B, immune responses to exosome-expressing COVID-19 spike antigen (STX-S) at the humoral level were induced in a subject as early as two weeks after the first injection (Fig. 10A) and persisting at least until day-35 post-injection (Fig. 10B). Furthermore, the robust and persistent anti-spike antibody response was elicited using nanogram amounts of spike protein expressed on exosomes and without the inclusion of an adjuvant. Here, for example, as little as 10 ng of exosome-expressed spike protein without adjuvant elicited a significant and useful antibody response as measured at day-14 and day-35. Also, 32 ng of exosome-expressed spike protein without adjuvant elicited a significant > 30-fold antibody response compared to 32 ng of purified spike protein (exosomeless) with adjuvant. It is generally known in the art that prior art spike protein vaccines are administered in microgram quantities and with adjuvant to generate an antibody response similar to that observed with nanogram quantities of the instant exosome-expressed spike protein antigen.
[0340] Turning to Figures 10C and 10D, immune responses to STX-S at the cellular level were induced in a subject as shown in ELISpot assays performed on splenocytes obtained at day 35- 40. Here, a dose of as little as 10 ng of adjuvantless exosome-expressed spike antigen elicited in a subject a significant CD4+ T-cell response against spike protein as demonstrated in an IL4 ELISpot assay of subject splenocytes (Figure 810C). Here also, doses of 10 ng and 32 ng of adjuvantless exosome-expressed spike antigen elicited in a subject a significant CD8+ cytotoxic T-cell response against spike protein as demonstrated in an IFNy ELISpot assay of subject splenocytes (Figure 10D).
[0341] Turning to Figures 11A-C, the administration of nanogram quantities of STX-S to subjects elicited potent neutralization of both delta and omicron variants of SARS-CoV-2 induced by STX-S exosome injection. In one embodiment, day-40 sera from subjects administered about 3.2 ng STX-S per injection (dose 2, Fig. 11A) and day-14 and day-40 sera from subjects administered about 9.8 ng STX-S per injection (dose 4, Fig. 11 A) were tested for neutralizing antibodies against SARS-CoV-2 Delta variant. Here, potent neutralizing activity was elicited by STX-S in each sample tested (Fig. 11 A) comparable to SARS-CoV-2 delta positive vaccine sera. STX-S displayed dose-dependent neutralization of the virus, estimated by the ability to protect infected cells from the virus-induced cytopathic effects (compare dose 4 to dose 2 at day-40 sera, Fig. HA).
[0342] Thus, according to one embodiment, the STX-S engineered exosome vaccine induced in subject(s) neutralizing antibodies against delta spike by day 14 (after a single i.m. injection). By day 40, ~4 weeks post STX-S boost, robust neutralization was observed in all subjects regardless of dose (Fig. 11A). Here, one injection with about 9 ng of spike by STX-S (dose 4, Fig. 11A) delivered about 65-75% neutralization against a delta variant. Moreover, full immunization (i.e., initial injection and at least one booster, or two i.m. injections) resulted in about 80-85% of neutralization against delta variant at a dose of about 3-9 ng of spike delivered by STX-S exosomes.
[0343] The delta-variant neutralization elicited by the administration of STX-S in subjects induced a response comparable to the human control plasma (CoV02-delta, plasma from a patient immunized with Moderna’s mRNA vaccine, with breakthrough delta infection), with a complete neutralizing response at higher dilutions (see, e.g., the 1 :320, STX-S dose 4 day-40) (Fig. HA).
[0344] Additionally, turning to Figures 1 IB and 11C, day-40 sera samples from subjects given the about 9-10 ng STX-S dose (here, the spike protein of the STX-S is of a delta variant - STX- S5) were also tested for neutralizing antibodies against SARS-CoV-2 Omicron variants (Omicron BA. l and BA.5.2.1). As shown in Figs. 1 IB and 11C, a strong cross neutralization was observed for the sera obtained from STX-S5-treated subjects, achieving, in this embodiment, about 84% neutralization of Omicron BAI variant (Fig. 1 IB) and a range between 16% to 97% for Omicron BA5 variant (Fig. 11C), after full immunization (i.e., at least 2 i.m. injections) with a dose of about 9-10 ng of delta spike delivered by STX-S exosomes. Thus, in some embodiments, the administration of an immunogenic composition containing single variant spike STX-S exosomes provides some level of protective immunity against other SARS-CoV-2 variants.
[0345] Since such low amounts of exosome expressing antigen (nanogram quantities, which are 1,000-fold less than standard protein or subunit vaccines) are required to elicit immunity as measured by neutralization, antibody titer and IL4/IFNY, specific different antigen-expressing exosomes can be combined to provide a multiplexed vaccine or immunogenic composition. For example, exosome-expressing SARS-CoV-2 antigens (e.g., STX-S) can be combined with exosome-expressing influenza antigens (e.g., hemagglutinin, e.g., H3).
[0346] Turning to Figures 12A-F, vaccine combinations containing both STX-S and exosomes that express influenza hemagglutinin 3 antigen on their surface (STX-H3) were administered to murine subjects. Sera obtained from the mice 14 days after injection with low doses of STX-H3 alone (Figs. 12A-C, second and third bars from left) shows significant levels of anti-H3 IgGs. As in the case of STX-S dose size and STX-N dose size (below and at Figs. 13A-D), low doses (3E10 to 3E11 exosomes representing low nanogram amounts of H3 antigen) elicited significant IgG responses in the mice.
[0347] Low nanogram amounts of STX-S and STX-H3 were combined and administered to murine subjects. Day- 14 sera obtained from those mice demonstrate significant eliciting of IgGs against both H3 antigen (Figs. 12A-C, fourth bars from the left) and against spike antigen (Figs, 12D-F, right bars). Thus, low nanogram doses of viral antigen-expressing exosomes can be combined to make multiplex vaccines. Here, for example, nanogram amounts of STX-S and STX-H3 were combined to make in immunogenic composition capable of eliciting strong antibody responses to both SARS-CoV-2 and influenza.
[0348] Similar nanogram doses of adjuvantless exosome-expressing SARS-CoV-2 nucleocapsid protein also elicit useful, robust, and significant humoral and cell-mediated immune responses (Figures 13A-D). The inventors envision that any or most viral antigens (such as, e.g., influenza hemagglutinin, influenza neuraminidase, respiratory syncytial virus, and/or other viral glycoproteins or other protein or fragments thereof) expressed on the surface of exosomes according to the present disclosure will elicit humoral and/or cell-mediated immune response in the recipient subject.
[0349] Thus, in some embodiments, a low-dose immunogenic composition or vaccine is provided that contains an immunogenic dose inclusively between 1 ng and 1 pg viral antigen (e.g., SARS-CoV-2 spike, SARS-CoV-2 nucleocapsid, or influenza hemagglutinin, or combinations thereof), 1-900 ng, 1-800 ng, 1-700 ng, 1-600 ng, 1-500 ng, 1-400 ng, 1-300 ng, 1- 200 ng, 1-100 ng, 1-90 ng, 1-80 ng, 1-70 ng, 1-60 ng, 1-50 ng, 1-40 ng, 5-100 ng, 5-90 ng, 5-80 ng, 5-70 ng, 5-60 ng, 5-50 ng, 5-40 ng, < 100 pg, < 50 pg, < 25 pg, < 20 pg, < 10 pg, < 1 pg, < 900 ng, < 800 ng, < 700 ng, < 600 ng, < 500 ng, < 400 ng, < 300 ng, < 200 ng, < 100 ng, < 90 ng, < 80 ng, < 70 ng, < 60 ng, < 50 ng, < 40 ng, about 1 ng, about 1.5 ng, about 2 ng, about 2.5 ng, about 3 ng, about 3.5 ng, about 4 ng, about 4.5 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about 18 ng, about 19 ng, about 20 ng, about 25 ng, about 30 ng, about 35 ng, about 40 ng, about 45 ng, or about 50 ng of viral antigen (e.g., SARS-CoV-2 spike or nucleocapsid).
[0350] Turning to Figures 13A and 13B, immune responses to exosome-expressing COVID-19 nucleocapsid antigen (STX-N) at the humoral level were induced in a subject and detected a day- 35 post-injection (Figs. 13A and 13B). Furthermore, the robust and persistent anti-spike antibody response was elicited using nanogram amounts of spike protein expressed on exosomes and without the inclusion of an adjuvant. Here, for example, as little as 3.2 ng of exosome-expressed nucleocapsid protein without adjuvant elicited a significant and useful antibody response as measured at day-35 (Fig. 13 A, STX-N dose 2). Thus, in one embodiment, a complete immunization cycle (two i.m. injections) induced a significant 3-fold increase of IgG against SARS-CoV-2 nucleocapsid protein (N) over PBS for the dose of about 3 ng/inj ection (dose 2, Fig. 9A) up to a 10-fold at 10 ng/injection (dose 3, Fig. 13B).
[0351] Turning to Figures 13C and 13D, to characterize the T cell response to STX-N, antigenspecific T cell responses to nucleocapsid protein were measured by ELISpot assays performed on splenocytes obtained at day 40. Here, vaccination of subjects with STX-N elicited multifunctional, antigen-specific T cell responses against SARS-CoV-2 nucleocapsid protein at day 40 (after boost (2nd) injection). STX-N administration resulted in a potent IFNy response (Figs. 13C and 13D). Here, evaluation of IFNy secreting cells in response to ex-vivo nucleocapsid protein stimulation showed a 6-fold increase in spleens immunized with STX-N (Figs. 13C and 13D), suggesting a Thl-biased CD8+ T cell response. This response is of invaluable importance for the development of exosome-expressing immunogenic compositions with the potential to protect against a wide range of SARS-CoV-2 variants, since nucleocapsid protein is not a surface protein and otherwise most likely will only been presented to the immune cells once the infecting viri are processed inside the infected cells.
[0352] As in the humoral antibody response to nucleocapsid protein (Figs. 13 A, 13B), in this particular embodiment, the significant CD8+/IFNy response to nucleocapsid protein in subjects was effected with administered doses of STX-N in the 3 to 10 ng range without adjuvant, which is on the order of three orders of magnitude lower than prior art protein subunit vaccines.
[0353] In addition to their superior immune-response elicitation, the spike-exosome vaccine, the nucleocapsid-exosome vaccine, the hemagglutinin-exosome vaccine, and the RSV-exosome vaccine each presents several other advantages over currently available vaccines. First, the subject exosome-based vaccines deliver antigen through an entirely endogenous, autologous lipid bilayer, that can be easily integrated into the host cell membrane and facilitate engineered antigen presentation to immune cells. Membrane bound antigen can easily be presented to the circulating immune cells to quicky activate a response, while free antigen contained inside the exosome can additionally be processed by the lysosomal system and activate the cytotoxic T- lymphocyte response. Thus, utilization of a natural delivery system promotes efficient delivery and response compared with synthetic lipid nanoparticle technology.
[0354] Second, the spike, nucleocapsid, RSV, and hemagglutinin exosome vaccines are proteinbased vaccines, making the antigen readily available to the subject immune system without translation in a host cell as is required for RNA-based vaccines. Incomplete translation, and/or incorrect folding of mRNA encoding Spike, nucleocapsid, or hemagglutinin protein limits the amount of available antigen after vaccination, making the immune response extremely variable and with reduced efficacy.
[0355] Third, the subject exosome-based spike, nucleocapsid, hemagglutinin, and RSV fusion protein vaccines do not require an adjuvant or synthetic lipid nanoparticle (LNP) for delivery and immune response. Traditional protein-based vaccines require adjuvants (such as, e.g, aluminum salt and the squalene oil-in-water emulsion systems MF59 (Novartis) and AS03 (GlaxoSmithKline)) (see, e.g., Wong, S.S. and R.J. Webby, “Traditional and new influenza vaccines,” Clin Microbiol Rev, 2013. 26(3): p. 476-92) to augment and orchestrate immune responses and influence affinity, specificity, magnitude and functional profile of B and T cell responses. Current mRNA vaccines do not use an adjuvant, which combined with the need for mRNA translation prior to antigen availability, may also reduce long term efficacy. Current approved COVID vaccines use LNPs to deliver the mRNA or adjuvants for the proteins and undesirable side effects have been reported.
[0356] Fourth, the subject exosomal spike fusion protein, nucleocapsid fusion protein, hemagglutinin fusion protein, and RSV-fusion protein immunogenic compositions exhibit greater immunogenic efficacy at a significantly lower, i.e., three orders of magnitude lower, dose than currently available LNP mRNA and protein vaccines. For example, clinical approved protein candidate vaccines use approximately 5ug to 25ug of antigen in conjunction with an adjuvant to induce immunization (see, e.g., Formica, et al., “Different dose regimens of a SARS- CoV-2 recombinant spike protein vaccine (NVX-CoV2373) in younger and older adults: A phase 2 randomized placebo-controlled trial,” PLoS Med, 2021. 18(10): p. el003769; Sun et al., “Development of a Recombinant RBD Subunit Vaccine for SARS-CoV-2,” Viruses, 2021. 13(10); Worzner et al., “Adjuvanted SARS-CoV-2 spike protein elicits neutralizing antibodies and CD4 T cell responses after a single immunization in mice. EBioMedicine, 2021. 63: p. 103197; and references therein. Here, the immunology data from subjects immunized with either the spike fusion-exosome or the nucleocapsid fusion-exosome elicits a complete immunization that is correlated with high antibody levels, strong viral neutralization, broad multi-variant activity, and both B and T cell memory with approximately 1/1000th of the protein antigen amount (i.e., nanogram amounts versus microgram amounts) currently being administrated in current clinically approved vaccines.
[0357] Fifth, an additional limitation of the available vaccines is the need of refrigeration and short shelf life of the products at higher temperatures. Because of their high stability at physiological pH and temperature, exosomes, including display exosomes, can be stored at 4°C for longer times. The spike fusion or nucleocapsid fusion-expressing exosome composition can also be lyophilized for long term storage at convenient temperature (see, e.g., U.S. Pat. App. No. 2017/0360842 Al).
[0358] Thus, here, a strong CD8+ T cell response as well as a strong B cell response, as demonstrated by IgG production and potentially neutralizing antibodies, was induced when spike protein, nucleocapsid protein, or hemagglutinin protein was delivered by exosomes. While not wishing to be bound by theory, this result could be explained by the role of extracellular vesicles in intercellular communication and antigen presentation. In particular, numerous copies of the spike protein (Figs. 6A and 6B), nucleocapsid protein, or hemagglutinin protein may be present on the vesicle surface, facilitating the crosslinking to B-cell receptors. Moreover, spike proteins in extracellular vesicle-based vaccines could indirectly activate B cells and CD8(+) T cells through the antigen cross-presentation.
[0359] It is further envisioned that the subject fusion protein-expressing exosomes may be engineered to express antigens of interest to target new CO VID variants and/or problematic influenza strains. The antigen of interest could easily be swapped and adapted to the needs. Antigens could be expressed as fusion proteins (e.g., chimeras) with, e.g., exosome expression or display domains fused to antigen domains to enable display of antigens on the exosome for delivery to the host subject immune system.
[0360] It is further envisioned that the exosomes could be engineered to selectively target organs or tissues of interest and allow safe and targeted delivery of antigens to specific immune subsystems to elicit specific types of responses in the subject.
Characterization of STX-S and STX-H3 cells and exosomes
[0361] Turning to Figure 14A, high expression of spike was detected on cell surface (right curve) by flow cytometry. Parent, non-engineered 293F cells do not express SARS-CoV-2 spike protein, as expected.
[0362] Turning to figure 14B, enrichment of Spike protein in exosomes was confirmed by Jess- automated Western Blot (Lane 5: STX-S exo). Here, lane 1 : marker, lane 2: non-engineered 293F cells, lane 3: non-engineered 293F exosomes, lane 4: STX-S cells, and lane 6: Spike protein.
[0363] Turning to figure 15A, high expression of influenza hemagglutinin 3 (H3) was detected on cell surfaces (right curve) by flow cytometry. Parent, non-engineered 293F cells (left curve) do not express Influenza H3 protein, as expected.
[0364] Turning to figure 15B, the enrichment of H3 protein in exosomes was confirmed by Jess- automated Western Blot (Lane 5: STX-H3 exo). Here, Lane L marker, Lane 2: non-engineered 293F cells, Lane 3: non-engineered 293F exosomes, Lane 4: STX-H3 cells, Lane 5: STX-H3 exo.
[0365] Turning to figure 16A, CD81 was detected on STX exosomes (right curve) by flowcytometry using a bead-assay compared with no signal from isotype control antibody (left curve).
[0366] Turning to figure 16B, spike protein was detected on STX-S exosomes (right curve) but not on 293F parental exosomes (left curve). [0367] Turning to figure 16C, H3 protein was detected on STX-H3 exosomes (right curve) but not on 293F parental exosomes (left curve).
[0368] Figures 17-19 demonstrate the immunoresponse to intramuscular injection of a combination of spike expressing exosomes (STX-S) and H3 expressing exosomes (STX-H3) (the combination as STX-S+H3) vaccine in a mouse model. Here, mice were injected on day 1, blood was collected on day 14, mice received a booster injection (IM) on day 21, blood was collected on day 35, and splenocytes were procured on day 40. The STX-S+H3 vaccine induced robust expression of Influenza H3 and SARS-CoV-2 spike antibodies in mice after 1 (day 14) and 2 (day 35) IM injections as analyzed by ELISA. PBS was used as a vehicle control in all studies. The STX-S+H3 vaccine was also observed to induce a T-cell response against both H3 and spike after in vitro stimulation, as shown by the IFNg ELISPOT on isolated splenocytes (panels 19A and 19B). All data presented in figures 17-19 are shown as mean ± SEM. **** p<0.0005, ***p<0.001, **p<0.01, *p<0.05, ns= not significant, 1-way ANOVA. Here, N= 10/experimental group.
[0369] Turning to figure 17A, IgG against H3 was observed at day 14 after a single IM injection.
[0370] Turning to figure 17B, IgG against Spike was observed at day 14 after a single IM injection.
[0371] Turning to figure 18A, IgG against H3 was observed at day 35 after complete immunization (two IM injections).
[0372] Turning to figure 18B, IgG against spike was observed at day 35 after complete immunization (two IM injections).
[0373] Turning to figure 19A, an IFNg response to H3 in vitro stimulation was observed.
[0374] Turning to figure 19B, an IFNg response to spike in vitro stimulation was observed.
Multivalent vaccine directed against covid 19, influenza, and/or respiratory syncytial virus
Vesicles Expressing Respiratory Syncytial Virus - F
[0375] Various constructs of the RSV F protein were constructed to discover a stable and native conformation of the F protein that confers robust and stable immunity in a subject, especially when presented on the surface of a vesicle such as an exosome. Figure 20 depicts five construct versions of RSV F. RSV F version 1 (“VI”) is a DS-Cavl engineered form as described in McLellan et al., “Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus,” Science, 1 Nov 2013, Vol 342, Issue 6158, pp. 592-598, doi:
10.1126/science.1243283. RSV F version 2 (“V2”) starts with the VI DS-Cavl engineered form with the polyadenylation signals removed across the F gene. RSV F version 3 (“V3”) starts with the V2 engineered form with both furin cleavage sites mutated as described in Brakel et al., “Coexpression of respiratory syncytial virus (RSV) fusion (F) protein and attachment glycoprotein (G) in a vesicular stomatitis virus (VSV) vector system provides synergistic effects against RSV infection in a cotton rat model,” Vaccine, 2021 Nov 16;39(47):6817-6828. doi: 10.1016/j. vaccine.2021.10.042. Epub 2021 Oct 23. RSV F version 4 (“V4”) starts with the V2 engineered form with the deletion of the N-terminal most furin cleavage site as described in Patel et al., “Flexible RSV Prefusogenic Fusion Glycoprotein Exposes Multiple Neutralizing Epitopes that May Collectively Contribute to Protective Immunity,” Vaccines 2020, 8(4), 607; doi. org/10.3390/ vaccines8040607. A specific version of a V4 construct is exemplified in SEQ ID NO:20.
[0376] The various RSV engineered constructs were expressed in cells and protein stability, expression levels, and exosome loading were determined. Here, it was observed that the VI construct showed low RSV F protein expression in multiple transduced 293F cell lines generated. Those cells that did show high RSV F expression (-10%) were sorted by FACS but the 50% of those cells lost expression within one week. Here, it was observed that the V2 construct, in which the polyadenylation signals across the F gene were removed, was also poorly expressed in the transduced 293F cell lines.
[0377] The V3 construct, in which both furin cleavage sites were removed, showed a high percentage (about 96-97%) and high expression level of full-length RSV F protein in 293F transduced cells. However, the V3 expression was observed to be unstable. RSV F V3-CD9 chimera transfected cells with top 10% RSV F expression were FACS sorted. CD9 and RSV F protein expression was measured 1 week and 2 weeks after FACS sorting. Both non-sorted pool and sorted pool showed a loss of RSV F protein expression (using an anti-AM22), however, CD9 expression remained at high level, indicating loss of V3 expression. 293F-generated exosomes produced from the sorted pool showed very low to no RSV F on the exosomes.
[0378] Furthermore, the RSV F V3-CD9-expressing exosomes were tested for a humoral immune response in vivo and the mice showed low antibody responses against RSV F protein on day 35 after two injections (Fig. 22, panels A and B). Here, two lots of exosomes expressing the V3 RSV F-CD9 chimera fusion protein, with lot #1 producing 2.0E12 exosomes per mL at a protein concentration of about 43 ng/mL and lot #2 producing 1.8E12 exosomes per mL at a protein concentration of about 26 ng/mL. Lot #1 (2 mL) and lot #2 (1.3 mL) were combined to obtain a combined lot #1.2 of 3.3mLl for a final concentration of 25.27 ng/mL. 10 mice received two injections at ~3ng/inj ection (using lOOul of the stock). Antibodies were detected after injection at days-14 (Fig. 22A) and day-35 (Fig. 22B), but the immune response was not sufficient for eliciting good humoral immunity.
[0379] The V4 construct was observed to have improved (relative to V3) expression levels and stability in transduced 293F cells, as determined by anti-RSV F flow cytometry using 0 antigen site antibodies (AM22, D25) and two RSV-neutralizing site II monoclonal antibodies (palivizumab, motavizumab) and JESS western blotting.
[0380] RSV F V4-CD9-expressing exosomes were tested for a humoral immune response in vivo and observed to elicit a strong immune response at days 14 and 35 (Fig. 23, panels A and B) with a clear boost effect at day 35 (Fig. 23B). Here, the RSV concentration on exosomes was about 250 ug/mL and the total protein was 990ug/mL per 1.9E12 exosome/mL.
[0381] Table 1
Figure imgf000076_0001
[0382] In some embodiments, the RSV F protein is fused to a tetraspanin protein. In a specific embodiment, the RSV F protein is fused, i.e., as part of an engineered chimeric protein, to CD9. Here the engineered chimeric protein contains at the N-terminal an RSV F polypeptide, preferably the RSV F V4 construct (SEQ ID NO: 18), followed by a linker, then the CD9 protein at the C-terminal end (Fig. 21 A), which permits the RSV F protein to be expressed on the outer surface of a vesicle (Fig. 21B). In a specific embodiment, the RSV F-CD9 chimeric construct has an amino acid sequence of SEQ ID NO:20.
Combination Vaccines
[0383] In one embodiment, the subject vaccine or immunogenic composition contains a combination of SARS-CoV2 spike-expressing vesicles and influenza hemagglutinin-expressing vesicles (Fig. 24). In another embodiment, the subject vaccine or immunogenic composition contains a combination of SARS-CoV2 spike-expressing vesicles and respiratory syncytial virus fusion protein (RSV F)-expressing vesicles (Fig. 25). In another embodiment, the subject vaccine or immunogenic composition contains a combination of influenza hemagglutinin-expressing vesicles and respiratory syncytial virus fusion protein (RSV F)-expressing vesicles (Fig. 26). In another embodiment, the subject vaccine or immunogenic composition contains a combination of SARS-CoV2 spike-expressing vesicles, RSV F-expressing vesicles, and influenza hemagglutinin-expressing vesicles (Fig. 27).
[0384] In one embodiment, a combination of SARS-CoV2 spike-expressing exosomes (STX-S), RSV F-expressing exosomes (STX-RSV), and influenza hemagglutinin-expressing exosomes (STX-H3) were injected in mice in single formulation or in combination with each other, to verify the induction of an antibody response. For Spike and H3, 10-15 ng of protein was used for immunization. For RSV, 130ng of RSV protein was used for immunization. Antibody response was analyzed for all groups at day 14 (after 1 injection) (Fig. 28) and day 35 (after booster injections at day-21, full immunization) (Fig. 29).
[0385] Here, it was observed that injection of STX-S, STX-H3 and STX-RSV induced production of virus-type-specific antibodies, when injected as exosomes expressing a single virus-antigen type, and in combination with one or more other exosomes each expressing another virus antigen. A clear booster effect was also observed. Also, no immune interference was observed when two or more exosome-virus species were injected. [0386] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
[0387] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
[0388] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

Claims

WHAT IS CLAIMED IS:
1. An immunogenic composition comprising a first vesicle and a first fusion protein, and a second vesicle and a second fusion protein, wherein said first fusion protein comprises a first virus polypeptide and an exosomal polypeptide and said second fusion protein comprises a second virus polypeptide and an exosomal polypeptide.
2. An immunogenic composition comprising a first vesicle and a first fusion protein, a second vesicle and a second fusion protein, and a third vesicle and a third fusion protein, wherein said first fusion protein comprises a first virus polypeptide and an exosomal polypeptide, said second fusion protein comprises a second virus polypeptide and an exosomal polypeptide, and said third fusion protein comprises a third virus polypeptide and an exosomal polypeptide.
3. The immunogenic composition of claim 1 or claim 2 further comprising an excipient.
4. The immunogenic composition of claim 3, wherein the excipient comprises a buffer.
5. The immunogenic composition of claim 3, wherein the excipient comprises a cryoprotectant.
6. The immunogenic composition of claim 1 or claim 2 not comprising an adjuvant.
7. The immunogenic composition of claim 1 or claim 2, wherein said first fusion protein and said second fusion protein are present in a membrane of each respective vesicle.
8. The immunogenic composition of claim 7, wherein a part or all of each virus polypeptide is present at or on an outer surface of the vesicle.
9. The immunogenic composition of claim 1 or claim 2, wherein each fusion protein is present in the composition at a concentration of about 1 ng/100 pL to about 50 ng/100 pL.
10. The immunogenic composition of claim 1 or claim 2, wherein the first virus polypeptide is a SARS-CoV-2 polypeptide and the second virus polypeptide is an influenza polypeptide.
11. The immunogenic composition of claim 10, wherein the first virus polypeptide is a SARS-CoV-2 spike protein polypeptide.
12. The immunogenic composition of claim 10, wherein the first virus polypeptide is a SARS-CoV-2 delta variant spike protein polypeptide.
13. The immunogenic composition of claim 11, wherein the first virus polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO: 1, or identical to SEQ ID NO:1.
14. The immunogenic composition of claim 10, wherein the first virus polypeptide is a SARS-CoV-2 nucleocapsid protein polypeptide.
15. The immunogenic composition of claim 14, wherein the first virus polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO:5, or identical to SEQ ID N0:5.
16. The immunogenic composition of any one of claims 10, wherein the second virus polypeptide is an influenza hemagglutinin protein polypeptide.
17. The immunogenic composition of claim 10, wherein the second virus polypeptide is an influenza hemagglutinin 3 (H3) protein polypeptide.
18. The immunogenic composition of claim 17, wherein the second virus polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO: 14, or identical to SEQ ID NO: 14.
19. The immunogenic composition of claim 2, wherein the third virus polypeptide is a respiratory syncytial virus (RSV) protein polypeptide.
20. The immunogenic composition of any one of claim 2 or claim 19, wherein the third virus polypeptide is an RSV fusion (RSV F) protein polypeptide.
21. The immunogenic composition of claim 20, wherein the third virus polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO: 18, or identical to SEQ ID NO: 18.
22. The immunogenic composition of claim 1 or claim 2, wherein the exosomal polypeptide is a tetraspanin protein polypeptide.
23. The immunogenic composition of claim 22, wherein the exosomal polypeptide is a CD9 protein polypeptide.
24. The immunogenic composition of claim 22, wherein the exosomal polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO: 10, or identical to SEQ ID NO: 10.
25. The immunogenic composition of any one of claims 1-13 and 17-24, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO:2, or identical to SEQ ID NO:2.
26. The immunogenic composition of claim 22, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO:3, or identical to SEQ ID NO:3.
27. The immunogenic composition of claim 10, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO:6, or identical to SEQ ID NO:6.
28. The immunogenic composition of claim 10, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO:7, or identical to SEQ ID NO:7.
29. The immunogenic composition of claim 10, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO: 15, or identical to SEQ ID NO: 15.
30. The immunogenic composition of claim 10, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO: 16, or identical to SEQ ID NO: 16.
31. The immunogenic composition of claim 20, wherein the third fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:SEQ ID NO: 19, or identical to SEQ ID NO:19.
32. The immunogenic composition of claim 20, wherein the third fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: SEQ ID NO:20, or identical to SEQ ID NO:20.
33. The immunogenic composition of claim 1 or claim 2, wherein an immunogenic dose of said composition comprises about 1 ng to about 50 ng of the first fusion protein or first virus polypeptide, and about 1 ng to about 50 ng of the second fusion protein or second virus polypeptide.
34. The immunogenic composition of claim 2, wherein an immunogenic dose of said composition comprises about 1 ng to about 50 ng of the third fusion protein or third virus polypeptide.
35. A method of immunizing a subject against a virus infection comprising administering to the subject an immunogenically effective dose of an immunogenic composition of claim 1 or claim 2.
36. The method of claim 35, wherein said immunogenically effective dose elicits protective immunity in said subject against a first virus and a second virus.
37. The method of claim 35, wherein said immunogenically effective dose elicits protective immunity in said subject against a first virus, a second virus, and a third virus.
38. The method of claim 35, wherein the immunogenic composition comprises a SARS- CoV-2 spike protein polypeptide and an influenza hemagglutinin polypeptide, and the first virus is a SARS-CoV2 and the second virus is an influenza virus.
39. The method of claim 35, wherein the immunogenic composition comprises a SARS- CoV-2 spike protein polypeptide, an influenza hemagglutinin polypeptide, and a respiratory syncytial virus (RSV) fusion protein polypeptide, and the first virus is a SARS-CoV2, the second virus is an influenza virus, and the third virus is an RSV.
40. The method of claim 35, wherein said immunogenically effective dose elicits protective immunity in said subject against a SARS-CoV-2 delta variant and a SARS-CoV-2 omicron variant.
41. The method of claim 35, wherein the immunogenically effective dose comprises about 1 ng to about 50 ng of said first fusion protein, about 1 ng to about 50 ng of said second fusion protein, and about 1 ng to about 50 ng of said third fusion protein.
42. The method of claim 35 further comprising administering to the subject a second effective dose of the immunogenic composition of claim 1 or claim 2.
43. The method of claim 35, wherein the elicited protective immunity comprises (a) a high antibody titer directed against the first, second, and third virus, (b) a CD4+ T-cell response against the first, second, and third virus, and (c) a CD8+ cytotoxic T-cell response against the first, second, and third virus.
44. A synthetic fusion protein comprising a virus polypeptide and an exosomal polypeptide.
45. The synthetic fusion protein of claim 44 further comprising a linker polypeptide positioned between the virus polypeptide and the exosomal polypeptide.
46. The synthetic fusion protein of claim 45 further comprising a hinge polypeptide positioned between the virus polypeptide and the exosomal polypeptide.
47. The synthetic fusion protein of claim 46 further comprising a transmembrane domain polypeptide positioned between the virus polypeptide and the exosomal polypeptide.
48. The synthetic fusion protein of claim 44, wherein the exosomal polypeptide is a tetraspanin polypeptide.
49. The synthetic fusion protein of any one of claim 44 or 48, wherein the exosomal polypeptide is a CD9 polypeptide.
50. The synthetic fusion protein of claim 44, wherein the virus polypeptide is a SARS-CoV-2 structural protein polynucleotide.
51. The synthetic fusion protein of claim 44 or 50, wherein the virus polypeptide is a SARS- CoV-2 spike protein polypeptide.
52. The synthetic fusion protein of claim 51, wherein said SARS-CoV-2 spike protein polypeptide comprises a one of more of a furin cleavage site mutation (CSM [682RRAR685-to- 682GSAG685]) and a di-proline substitution (2P [986KV987-to-986PP987]).
53. The synthetic fusion protein of claim 51, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, a SARS-CoV-2 spike protein polypeptide, a linker polypeptide, and a CD9 polypeptide.
54. The synthetic fusion protein of claim 53, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:3.
55. The synthetic fusion protein of claim 53 or 54, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:3.
56. The synthetic fusion protein of claim 44 or 50, wherein the virus polypeptide in a SARS- CoV-2 nucleocapsid protein polypeptide.
57. The synthetic fusion protein of claim 56, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, a signal peptide, a SARS-CoV-2 nucleocapsid protein polypeptide, a hinge region, a transmembrane domain polypeptide, a linker polypeptide, and a CD9 polypeptide.
58. The synthetic fusion protein of claim 57, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:7.
59. The synthetic fusion protein of claim 57 or 58, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:7.
60. The synthetic fusion protein of claim 44 or 45, wherein the virus polypeptide is an influenza protein polypeptide.
61 . The synthetic fusion protein of claim 60, wherein said influenza protein polypeptide comprises a hemagglutinin.
62. The synthetic fusion protein of claim 61, wherein said influenza protein polypeptide comprises a hemagglutinin 3 (H3).
63. The synthetic fusion protein of claim 61 or 62, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, a hemagglutinin protein polypeptide, a linker polypeptide, and a CD9 polypeptide.
64. The synthetic fusion protein of claim 63, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16.
65. The synthetic fusion protein of claim 63, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 16.
66. The synthetic fusion protein of claim 44 or 45, wherein the virus polypeptide is a respiratory syncytial virus (RSV) protein polypeptide.
67. The synthetic fusion protein of claim 66, wherein said RSV protein polypeptide comprises an RSV fusion (RSV F) protein.
68. The synthetic fusion protein of claim 66, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, an RSV F protein polypeptide, a linker polypeptide, and a CD9 polypeptide.
69. The synthetic fusion protein of claim 68, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:20.
70. The synthetic fusion protein of claim 68, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:20.
71. A synthetic polynucleotide encoding a synthetic fusion protein of claim 44.
72. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 13.
73. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence set forth in SEQ ID NO: 13.
74. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 11.
75. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence set forth in SEQ ID NO: 11.
76. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:4.
77. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence set forth in SEQ ID NO:4.
78. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 12.
79. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence set forth in SEQ ID NO: 12.
80. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 8.
81. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence set forth in SEQ ID NO:8.
82. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:22.
83. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence set forth in SEQ ID NO: 22.
84. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 17.
85. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence set forth in SEQ ID NO: 17.
86. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:23.
87. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence set forth in SEQ ID NO: 23.
88. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:21.
89. The synthetic polynucleotide of claim 71 comprising a nucleic acid sequence set forth in SEQ ID NO:21.
90. A cell comprising a synthetic polynucleotide of claim 71.
91. The cell of claim 90, wherein the cell is a metazoan cell.
92. The cell of claim 91 wherein the cell is a vertebrate cell.
93. The cell of claim 92, wherein the cell is a mammalian cell.
94. The cell of claim 93, wherein the cell is a primate cell.
95. The cell of claim 94, wherein the cell is a human cell.
96. The cell of claim 90 or 95, wherein the cell is a primary cell.
97. The cell of claim 90 or 95, wherein the cell is a human embryonic kidney cell.
98. The cell of claim 97, wherein the cell is a 293 cell.
99. The cell of claim 90 or 95, wherein said cell is produced by transducing the cell with a lentivirus comprising the synthetic polynucleotide.
100. The cell of claim 90, wherein said cell comprises a synthetic fusion protein of claim 44.
101. A vesicle comprising a synthetic fusion protein of claim 44.
102. The vesicle of claim 101, wherein said vesicle is an exosome.
103. The vesicle of claim 101 or 102, wherein said vesicle has a diameter of about 50-500 nm.
104. The vesicle of claim 101 or 102, wherein a SARS-CoV-2 spike protein polypeptide is expressed on an outer surface of said vesicle.
105. The vesicle of claim 101 or 102, wherein said vesicle expresses a SARS-CoV-2 nucleocapsid protein polypeptide on its surface.
106. The vesicle of claim 101 or 102, wherein said vesicle expresses an influenza hemagglutinin protein polypeptide on its surface.
107. The vesicle of claim 101 or 102, wherein said vesicle expresses a respiratory syncytial virus fusion protein polypeptide on its surface.
108. A method of making a vesicle of claim 101 comprising culturing a cell of claim 90 in a cell culture medium, collecting the cell culture medium, and purifying a plurality of vesicles comprising said vesicle from the cell culture medium.
109. The method of claim 108 further comprising inducing expression of a synthetic polynucleotide of claim 71 to produce a synthetic fusion protein of claim 44.
110. The method of claim 105, wherein said inducing comprises contacting the cell with tetracycline, doxycycline, or analogs thereof.
111. The method of claim 105, wherein said inducing comprises removing from the cell tetracycline, doxycycline, or analogs thereof.
112. A method of eliciting an immune response in a subject comprising administering to said subject a first dose of an immunogenic composition comprising a plurality of vesicles comprising a vesicle of claim 101 or a vesicle produced according to a method of claim 108, wherein a synthetic fusion protein of claim 44 is expressed on an outer surface of said vesicle.
113. The method of claim 112 further comprising administering to said subject a second dose of the immunogenic composition a period of time after administering the first dose.
114. The method of claim 113, wherein the period of time is 14 days - 1 year.
115. The method of claim 113 or 114, wherein the first dose or the second dose comprises about 100 pL - 1 mL of the immunogenic composition, wherein said immunogenic composition comprises about 0.3 ng/mL - 3 pg/mL of said synthetic fusion protein.
116. The method of any one of claims 113 or 114, wherein the first dose or the second dose comprises about 100 pL - 1 mL of the immunogenic composition which comprises about 2E9 vesicles/mL - 3E13 vesicles/mL.
117. The method of claim 112, wherein said synthetic fusion protein comprises a SARS-CoV- 2 spike protein polypeptide.
118. The method of claim 112, wherein said synthetic fusion protein comprises a SARS-CoV- 2 nucleocapsid protein polypeptide.
119. The method of claim 112, wherein said synthetic fusion protein comprises an influenza hemagglutinin protein polypeptide.
120. The method of claim 112, wherein said synthetic fusion protein comprises a respiratory syncytial virus fusion (RSV F) protein polypeptide.
121. The method of claim 112, wherein said immunogenic composition comprises vesicles that express on the outer surface SARS-CoV-2 spike protein polypeptides, vesicles that express on the outer surface influenza hemagglutinin protein polypeptides, and vesicles that express on the outer surface RSV F protein polypeptides.
122. The method of claim 112 or 121, wherein the elicited immune response comprises producing neutralizing antibodies against an antigen present in the synthetic fusion protein.
123. The method of claim 112 or 121, wherein the elicited immune response comprises producing anti-spike antibodies.
124. The method of claim 112 or 121, wherein the elicited immune response comprises a spike-specific T cell response.
125. The method of claim 112 or 121, wherein the elicited immune response comprises producing anti-nucleocapsid antibodies.
126. The method of claim 112 or 121, wherein the elicited immune response comprises a nucleocapsid-specific T cell response.
127. The method of claim 112 or 121, wherein the elicited immune response comprises producing anti-hemagglutinin antibodies.
128. The method of claim 112 or 121, wherein the elicited immune response comprises a hemagglutinin -specific T cell response.
129. The method of claim 112 or 121, wherein the elicited immune response comprises producing anti-RSV F antibodies.
130. The method of claim 112 or 121, wherein the elicited immune response comprises a RSV F -specific T cell response.
131. The method of claim 112 or 121, wherein the immune response persists in the subject for up to nine months.
132. The method of claim 112 or 121, wherein the immune response persists in the subject for at least nine months.
PCT/US2023/075343 2022-09-30 2023-09-28 Combination exosomal immunogenic compositions and methods WO2024073552A2 (en)

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