Combination Exosome Immunogenic Compositions and Methods

Abstract

The present disclosure relates to compositions and methods for vaccinating a subject against multiple SARS-CoV-2 variants and other respiratory viruses, including the creation and delivery to a subject of extracellular vesicles that express an engineered spike protein, an engineered nucleocapsid protein, an engineered hemagglutinin protein, and / or an engineered respiratory syncytial virus (RSV) pre-fusion fusion (F) protein or RSV F protein on their surface. The present invention also relates to compositions and methods for the design, preparation, manufacturing, formulation, and / or use of spike-, nucleocapsid-, hemagglutinin-, and / or RSV-F-displaying vesicle vaccines designed to elicit strong humoral and cellular immune responses against multiple respiratory viruses and variants. TIFF2025532934000004.tif59170

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the 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.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy created on September 27, 2023 is named 516PCTseqlst and is 49,339 bytes in size. [Background technology]

[0003] background The emergence, re-emergence, 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 create safe, effective, readily available, and accessible vaccines that can be rapidly and efficiently produced to combat the emergence of evolved variants. In the case of SARS-CoV-2, many vaccines have been developed in an attempt to achieve immunity against the virus, primarily directing the immune response against the surface spike protein, which binds to the host cell receptor angiotensin-converting enzyme 2 (ACE2), which mediates viral entry and infection into cells. The spike (S) protein, a class I fusion glycoprotein, is the major surface protein of the SARS-CoV-2 virus and the primary target for neutralizing antibodies. The spike is also the primary site of mutations identified in the SARS-CoV-2 virus that reduce the effectiveness of vaccine-induced immune responses. Since the start of the COVID-19 pandemic, five variants of concern (VOC) and eight variants of interest (VOI) have been reported, posing challenges to current vaccines and creating a need for more effective vaccines. Vaccines against the SARS-CoV-2 nucleocapsid protein, an intraparticle soluble coronavirus protein, have so far been largely unsuccessful.

[0004] Current major vaccines utilize mRNA-lipid nanoparticle or viral vector technology, while more recent vaccines utilize recombinant proteins (see, e.g., Krammer, F., SARS-CoV-2 vaccines in development. Nature, 2020. 586(7830): pp. 516-527). Compared with the major mRNA vaccines, recombinant protein vaccines or inactivated protein vaccines are safe and reliable approaches for immunization, but generally suffer from weak immunogenicity and therefore require formulation with appropriate adjuvants. All current mRNA vaccine candidates achieve strong initial immune responses against the virus, resulting in reduced hospitalization rates, but all lack long-term protection. It has been reported that antibody levels decline significantly over time, and therefore their effectiveness in protecting against infection also declines significantly (see, for example, 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). Furthermore, mRNA vaccines to date lack cross-reactivity to emerging variants of concern and require multiple booster injections to maintain protection against the virus. Therefore, there remains a significant medical and public health need for better, more rapidly and efficiently produced, and globally accessible vaccines against influenza, RSV, and SARS-CoV-2 to provide improved, broader, and more durable neutralization of these viruses, more robust T cell responses, and enhanced safety profiles.

[0005] Briefly, the problem to which the present invention may be directed includes the emergence of SARS-CoV-2 variants of concern that have greater infectivity and lower immunogenicity than some variants of less concern, creating an ongoing public health and medical problem of vaccine ineffectiveness against new strains and new strains with reduced immunogenicity (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 a problem each season. Thus, problems exist in developing and delivering combination vaccines, e.g., seasonal vaccines, against both SARS-CoV-2 and influenza variants.

[0007] Solutions to this problem are disclosed, including safe (LNP-free, adjuvant-free) and effective combination vaccines that confer long-lasting humoral and cell-mediated immunity against multiple variants, emerging variants, and refractory variants of SARS-CoV-2, influenza, and / or RSV, comprising extracellular vesicles displaying spike or nucleocapsid proteins from a single variant of SARS-CoV-2 that confer robust humoral and / or cellular immunity against several variants of concern or variants of note of SARS-CoV-2, combined with extracellular vesicles displaying hemagglutinin proteins from a single variant of influenza that also confer immunity against variants of note of influenza, and / or combined with extracellular vesicles displaying RSV proteins from a single variant of RSV that also confer immunity against variants of note of RSV. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Krammer, F., SARS-CoV-2 vaccines in development. Nature, 2020.586(7830):p.516-527 [Non-patent document 2] 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 Summary of the Invention

[0009] overview In one aspect, an immunogenic composition is provided that contains a combination of two or more viral antigenic substance-expressing vesicles. In one embodiment, the immunogenic composition contains (i) a plurality of vesicles that express a SARS-CoV-2 protein polypeptide or antigen on their surface, and (ii) a plurality of vesicles that express an influenza protein polypeptide or antigen on their surface.

[0010] In another aspect, an immunogenic composition is provided that contains a combination of two or more viral antigenic substance-expressing vesicles. In one embodiment, the immunogenic composition contains (i) a plurality of vesicles expressing SARS-CoV-2 protein polypeptides or antigens on their surface, and (ii) a plurality of vesicles expressing RSV protein polypeptides or antigens on their surface.

[0011] In another aspect, an immunogenic composition is provided that contains a combination of two or more viral antigen-expressing vesicles. In one embodiment, the immunogenic composition contains (i) a plurality of vesicles expressing influenza protein polypeptides or antigens on their surface, and (ii) a plurality of vesicles expressing RSV protein polypeptides or antigens on their surface.

[0012] In another aspect, an immunogenic composition containing a combination of two or more viral antigenic substance-expressing vesicles is provided. In one embodiment, the immunogenic composition contains (i) a plurality of vesicles expressing a SARS-CoV-2 protein polypeptide or antigen on their surface, (ii) a plurality of vesicles expressing an influenza protein polypeptide or antigen on their surface, and (iii) a plurality of vesicles expressing an RSV protein polypeptide or antigen on their surface.

[0013] In one embodiment of various immunogenic composition aspects, the SARS-CoV-2 protein is a spike glycoprotein, e.g., a SARS-CoV-2 delta spike protein, or a genetically engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is a nucleocapsid protein, or a genetically engineered variant thereof. In one embodiment, the influenza protein is a hemagglutinin protein or a genetically engineered variant thereof, e.g., an H3 glycoprotein, or a genetically engineered variant thereof. In another embodiment, the influenza protein is a neuraminidase protein, or a genetically engineered variant thereof. In one embodiment, the RSV protein is a RSV fusion (F) protein, e.g., a RSV F protein or a pre-fusion F protein (see, e.g., Simoes et al., N Engl J Med 2022;386:1615-1626). In one embodiment, the vesicles are exosomes. In one aspect, the viral protein polypeptide or antigen is fused to an exosomal protein, such as a tetraspanin, more specifically, the CD9 protein, or a genetically engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.

[0014] In some embodiments of the immunogenic compositions described above, the immunogenic composition contains a plurality of vesicles containing a synthetic fusion protein at a concentration of about 2E9 to 3E13 vesicles / mL. In 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, etc., each vesicle type can be provided at a concentration of about 1E9 to 2E13 vesicles / mL or 2E9 to 3E13 vesicles / mL.

[0015] In some embodiments, the immunogenic composition contains a plurality of vesicles containing a synthetic fusion protein at a concentration of about 0.3 ng / mL to 3 μg / mL of synthetic fusion protein. In 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, etc., each vesicle type can be provided at a fusion protein concentration of about 0.1 ng / mL to 1.5 μg / mL or about 0.3 ng / mL to 3 μg / mL.

[0016] In some embodiments, the immunogenic composition further comprises one or more pharmaceutically acceptable excipients. In one embodiment, the immunogenic composition does not comprise an adjuvant.

[0017] In some embodiments, the vesicles of the plurality of vesicles of the immunogenic composition have an average diameter of about 50-500 nm. In one embodiment, the vesicles are synthetic vesicles. In one embodiment, the vesicles are produced by cells. In one embodiment, the vesicles are extracellular vesicles. In one embodiment, the vesicles are microvesicles. In one embodiment, the vesicles are exosomes. In one embodiment, the vesicles are apoptotic bodies. In one embodiment, the vesicles express CD81 protein on their surface.

[0018] In one aspect, a method is provided for inducing an immune response in a subject by administering to the subject a dose of an immunogenic composition described in the previous aspects and embodiments. In some embodiments, the subject is administered multiple doses, e.g., a second dose administered a period of time after the first dose and / or subsequent booster doses. In some embodiments, the period (e.g., between doses) is 14 days to 1 year.

[0019] In one embodiment, a dose contains about 100 μL to 1 mL of the immunogenic composition. In some embodiments, the immunogenic composition contains about 0.1 ng / mL to 3 μg / mL of the synthetic fusion protein expressed on the surface of the vesicles. In some embodiments, the immunogenic composition contains about 2.81E9 to 2.81E13 vesicles / mL expressing the synthetic fusion protein on their surface.

[0020] In some embodiments, the immune response elicited in the subject is the production of neutralizing antibodies against a virus, e.g., SARS-CoV-2, influenza, RSV, etc. In some embodiments, the immune response elicited in the subject is the production of neutralizing antibodies against two or more SARS-CoV-2 variants, e.g., a delta variant, an omicron variant, or other variants of note or concern currently known or yet to be discovered.

[0021] In some embodiments, the immune response induced in the subject is the production of anti-spike antibodies. In some embodiments, the immune response induced in the subject is the production of anti-nucleocapsid antibodies. In some embodiments, the immune response induced in the subject is a spike-specific T cell response, for example, a CD4+ and / or CD8+ response. In some embodiments, the immune response induced in the subject is a nucleocapsid-specific T cell response, for example, a CD4+ and / or CD8+ response.

[0022] In some embodiments, the immune response induced in the subject is the production of anti-hemagglutinin antibody.In some embodiments, the immune response induced in the subject is spike-specific T cell response, for example, CD4+ and / or CD8+ response.In some embodiments, the immune response induced in the subject is hemagglutinin-specific T cell response, for example, CD4+ and / or CD8+ response.

[0023] In some embodiments, the immune response induced in the subject is the production of anti-RSV F antibodies. In some embodiments, the immune response induced in the subject is a spike-specific T cell response, for example, a CD4+ and / or CD8+ response. In some embodiments, the immune response induced in the subject is a RSV F-specific T cell response, for example, a CD4+ and / or CD8+ response.

[0024] In another aspect, a method of vaccinating a subject against influenza and SARS-CoV-2 is provided. In one embodiment, the subject is administered a composition containing (i) a plurality of vesicles expressing a SARS-CoV-2 protein polypeptide or antigen on their surface and (ii) a plurality of vesicles expressing an influenza protein polypeptide or antigen on their surface. In one embodiment, the SARS-CoV-2 protein is a spike glycoprotein, e.g., a SARS-CoV-2 delta variant spike protein or a genetically engineered variant thereof. In another aspect, the SARS-CoV-2 protein is a nucleocapsid protein or a genetically engineered variant thereof. In one embodiment, the influenza protein is a hemagglutinin protein, e.g., an H3 glycoprotein or a genetically engineered variant thereof. In another embodiment, the influenza protein is a neuraminidase protein or a genetically engineered variant thereof. In one embodiment, the vesicles are exosomes. In one embodiment, the viral protein polypeptide or antigen is fused to an exosomal protein, such as a tetraspanin, more specifically, the CD9 protein, or an engineered chimera containing elements of CD9 and other tetraspanins or transmembrane domains.

[0025] 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 expressing a SARS-CoV-2 protein polypeptide or antigen on their surface and (ii) a plurality of vesicles expressing a RSV protein polypeptide or antigen on their surface. In one embodiment, the SARS-CoV-2 protein is a spike glycoprotein, such as a SARS-CoV-2 delta variant spike protein or a genetically engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is a nucleocapsid protein or a genetically engineered variant thereof. In one embodiment, the RSV protein is an F protein or a pre-fusion F protein or a genetically engineered variant thereof. In one embodiment, the vesicle is an exosome. In one embodiment, the viral protein polypeptide or antigen is fused to an exosomal protein, such as a tetraspanin, more specifically, a CD9 protein, or a genetically engineered chimera containing elements of CD9 and other tetraspanin or transmembrane domains.

[0026] 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 expressing an RSV protein polypeptide or antigen on its surface, and (ii) a plurality of vesicles expressing an influenza protein polypeptide or antigen on its surface. In one embodiment, the RSV protein is an F protein or pre-fusion F protein or a genetically engineered variant thereof. In one embodiment, the influenza protein is a hemagglutinin protein, such as an H3 glycoprotein or a genetically engineered variant thereof. In another embodiment, the influenza protein is a neuraminidase protein or a genetically engineered variant thereof. In one aspect, the vesicles are exosomes. In one aspect, the viral protein polypeptide or antigen is fused with an exosome protein, such as a tetraspanin, more specifically, a CD9 protein, or a genetically engineered chimera containing a CD9 element and another tetraspanin or transmembrane domain.

[0027] In another aspect, a method of vaccinating a subject against influenza and RSV is provided. In one aspect, a subject is administered a composition containing (i) a plurality of vesicles expressing a SARS-CoV-2 protein polypeptide or antigen on their surface, (ii) a plurality of vesicles expressing an influenza protein polypeptide or antigen on their surface, and (iii) a plurality of vesicles expressing a RSV protein polypeptide or antigen on their surface. In one embodiment, the SARS-CoV-2 protein is a spike glycoprotein, such as a SARS-CoV-2 delta variant spike protein or a genetically engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is a nucleocapsid protein or a genetically engineered variant thereof. In one embodiment, the influenza protein is a hemagglutinin protein, such as an H3 glycoprotein or a genetically engineered variant thereof. In another embodiment, the influenza protein is a neuraminidase protein or a genetically engineered variant thereof. In one embodiment, the RSV protein is an F protein or a pre-fusion F protein or a genetically engineered variant thereof. In one embodiment, the vesicle is an exosome. In one embodiment, the viral protein polypeptide or antigen is fused with an exosome protein, such as a tetraspanin, more specifically, a CD9 protein, or a genetically engineered chimera containing CD9 elements and other tetraspanin or transmembrane domains.

[0028] In one aspect, a synthetic fusion protein is provided that contains a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosomal tetraspanin protein, the fusion protein being designed and engineered such that the SARS-CoV-2 protein antigen can be expressed on the surface of exosomes, enabling an immune response to be elicited when administered to a subject.

[0029] In one embodiment, the SARS-CoV-2 protein is a spike protein or a genetically engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is a nucleocapsid protein or a genetically engineered variant thereof.

[0030] In some embodiments where 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 shown in Figure 1A.

[0031] In some embodiments where the SARS-CoV-2 protein is a nucleocapsid protein and the exosomal tetraspanin protein is a CD9 protein, the nucleocapsid protein polypeptide is located at the N-terminus of the CD9 protein polypeptide. In some cases, a signal peptide is located at the N-terminus of the nucleocapsid protein polypeptide. In some cases, as shown in Figure 2A, a hinge peptide, a transmembrane domain peptide, and a linker peptide are located between the spike protein polypeptide and the CD9 protein polypeptide.

[0032] In one aspect, a synthetic fusion protein is provided that contains a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosomal tetraspanin protein, the fusion protein being designed and engineered such that the SARS-CoV-2 protein antigen can be expressed on the surface of exosomes, enabling an immune response to be elicited when administered to a subject.

[0033] In one embodiment, the SARS-CoV-2 protein is a spike protein or a genetically engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is a nucleocapsid protein or a genetically engineered variant thereof.

[0034] In some embodiments, where 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, as shown in Figure 1A (e.g., SEQ ID NO:2).

[0035] In some embodiments where the SARS-CoV-2 protein is a spike protein, the SARS-CoV-2 spike protein polypeptide contains one or more mutations, e.g., a furin cleavage site mutation (CSM[682RRAR685→682GSAG685]) and / or a diproline substitution (2P[986KV987→986PP987]). In a specific embodiment, the spike-containing fusion protein has the amino acid sequence set forth in SEQ ID NO:3.

[0036] In one aspect, a nucleic acid encoding a spike-CD9 fusion protein is provided. In one embodiment, the nucleic acid encoding the spike-CD9 fusion protein has the nucleic acid sequence of SEQ ID NO:4.

[0037] In some embodiments where 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-terminally of the CD9 protein polypeptide. In some cases, a signal peptide is positioned N-terminally of the nucleocapsid protein polypeptide. In some cases, as shown in Figure 2A, a hinge peptide, a transmembrane domain peptide (e.g., SEQ ID NO:9), and a linker peptide are positioned between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO:6). In a specific embodiment, the nucleocapsid-containing fusion protein has the amino acid sequence set forth in SEQ ID NO:7.

[0038] In one aspect, a nucleic acid encoding a nucleocapsid-CD9 fusion protein is provided. In one embodiment, the nucleic acid encoding the nucleocapsid-CD9 fusion protein has the nucleic acid sequence of SEQ ID NO:8.

[0039] In another aspect, a synthetic fusion protein is provided that contains a polypeptide sequence of an influenza protein fused to a polypeptide sequence of an exosomal tetraspanin protein, the fusion protein being designed and engineered such that the influenza protein antigen can be expressed on the surface of exosomes, enabling an immune response to be elicited when administered to a subject.

[0040] In one embodiment, the influenza protein is a hemagglutinin protein or a genetically engineered variant thereof. In a specific embodiment, the influenza protein is a hemagglutinin 3 (H3) protein or a genetically engineered variant thereof (e.g., SEQ ID NO:14).

[0041] In some embodiments where 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, as shown in Figure 3A, a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO: 15). In a specific embodiment, the H3-containing fusion protein has the amino acid sequence of SEQ ID NO: 16.

[0042] In one aspect, a nucleic acid encoding an H3-CD9 fusion protein is provided. In one embodiment, the nucleic acid encoding the H3-CD9 fusion protein has the nucleic acid sequence of SEQ ID NO:17.

[0043] In one aspect, a synthetic fusion protein is provided that contains a polypeptide sequence of a respiratory syncytial virus (RSV) protein fused with a polypeptide sequence of an exosomal tetraspanin protein. The fusion protein is designed and engineered so that the RSV protein antigen can be expressed on the surface of exosomes, thereby eliciting an immune response when administered to a subject.

[0044] In one embodiment, the RSV protein is a pre-fusion (F) protein or an engineered variant thereof (e.g., SEQ ID NO: 18).

[0045] In some embodiments where the RSV protein is a prefusion protein (RSV F) and the exosome tetraspanin protein is a CD9 protein (e.g., SEQ ID NO: 10), the RSV F protein polypeptide is located N-terminal to the CD9 protein polypeptide. In some cases, as shown in Figure 21A, a linker peptide is located between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO: 19). In a specific embodiment, the RSV F-containing fusion protein has the amino acid sequence of SEQ ID NO: 20.

[0046] In one aspect, a nucleic acid encoding a RSV F-CD9 fusion protein is provided. In one embodiment, the nucleic acid encoding the RSV F-CD9 fusion protein has the nucleic acid sequence of SEQ ID NO: 21.

[0047] In one aspect, a polynucleotide is provided that encodes a synthetic fusion protein containing a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosomal tetraspanin protein, the encoded fusion protein being designed and engineered such that the SARS-CoV-2 protein antigen can be expressed in cells and sorted onto the surface of exosomes, enabling an immune response to be elicited when the exosomes are administered to a subject.

[0048] 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 a furin cleavage site mutation (CSM[682RRAR685→682GSAG685]) and / or a diproline substitution (2P[986KV987→986PP987]). In a specific embodiment, the spike protein has an amino acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:1. In a specific embodiment, the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:11.

[0049] In some embodiments, where 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 such that it is positioned between the spike protein polypeptide and the CD9 protein polypeptide in the encoded synthetic fusion protein, as shown in FIG. 1A. In specific embodiments, the CD9 protein polypeptide has an amino acid sequence at least 80% identical to SEQ ID NO:10. In specific embodiments, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:13. In specific embodiments, the spike-CD9 fusion protein has an amino acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:3. In specific embodiments, 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 essentially identical to SEQ ID NO:4.

[0050] 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 at least 80% identical to SEQ ID NO:5 or essentially identical to SEQ ID NO:4. In a specific embodiment, the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence at least 80% identical to SEQ ID NO:12 or essentially identical to SEQ ID NO:12.

[0051] In some embodiments, where 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 N-terminal to the nucleocapsid protein polypeptide. In some cases, as shown in FIG. 2A, a hinge peptide, a transmembrane domain peptide, and a linker peptide are encoded by the polynucleotide such that they are positioned between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein. In specific embodiments, the CD9 protein polypeptide has an amino acid sequence at least 80% identical to SEQ ID NO:10. In specific embodiments, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence at least 80% identical to or essentially identical to SEQ ID NO:13. In specific embodiments, the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to, or essentially identical to, SEQ ID NO: 7. In specific embodiments, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to, or essentially identical to, SEQ ID NO: 8.

[0052] In another aspect, a polynucleotide is provided that encodes a synthetic fusion protein containing a polypeptide sequence of an influenza protein fused to a polypeptide sequence of an exosomal tetraspanin protein, the encoded fusion protein being designed and engineered such that the influenza protein antigen can be expressed in cells and sorted onto the surface of exosomes, enabling an immune response to be elicited when the exosomes are administered to a subject.

[0053] 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 at least 80% identical to, or essentially identical to, SEQ ID NO:14. In a specific embodiment, the polynucleotide encoding the H3 protein polypeptide has a nucleic acid sequence at least 80% identical to, or 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 at least 80% identical to, or essentially identical to, SEQ ID NO:10. In a specific embodiment, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:13.

[0054] In some embodiments, where 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 such that it is positioned between the hemagglutinin protein polypeptide and the CD9 protein polypeptide in the encoded synthetic fusion protein, as shown in FIG. 3A. In specific embodiments, the H3-CD9 fusion protein has an amino acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:16. In specific embodiments, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:17.

[0055] In another aspect, a polynucleotide encoding a synthetic fusion protein containing a polypeptide sequence of a respiratory syncytial virus (RSV) protein fused with a polypeptide sequence of an exosomal tetraspanin protein is provided. The encoded fusion protein is designed and engineered so that the RSV protein antigen can be expressed in cells and sorted onto the surface of exosomes, enabling an immune response to be elicited when the exosomes are administered to a subject.

[0056] 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 at least 80% identical to, or essentially identical to, SEQ ID NO: 18. In a specific embodiment, the polynucleotide encoding the RSV F protein polypeptide has a nucleic acid sequence at least 80% identical to, or 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 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 at least 80% identical to, or essentially identical to, SEQ ID NO: 13.

[0057] In some embodiments, in the encoded synthetic fusion protein, the encoded RSV F protein polypeptide is located N-terminal to the encoded CD9 protein polypeptide. In some embodiments, as shown in Figure 21A, a linker peptide is encoded by the polynucleotide such that it is located between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein. In a specific embodiment, the RSV F-CD9 fusion protein has an amino acid sequence that is at least 80% identical to, or 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, or essentially identical to, SEQ ID NO: 21.

[0058] In one aspect, a cell is provided that contains a polynucleotide encoding a synthetic fusion protein containing a polypeptide sequence of SARS-CoV-2 fused to a polypeptide sequence of an exosomal tetraspanin protein, the encoded fusion protein being designed and engineered such that the SARS-CoV-2 protein antigen can be expressed in cells and sorted onto the surface of exosomes, enabling an immune response to be elicited when the exosomes are administered to a subject.

[0059] In some embodiments of the cell aspect, 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 from 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.

[0060] 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 a furin cleavage site mutation (CSM[682RRAR685→682GSAG685]) and / or a diproline substitution (2P[986KV987→986PP987]). In a specific embodiment, the spike protein has an amino acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:1. In a specific embodiment, the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:11.

[0061] In some embodiments, where 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 such that it is positioned between the spike protein polypeptide and the CD9 protein polypeptide in the encoded synthetic fusion protein, as shown in FIG. 1A. In specific embodiments, the CD9 protein polypeptide has an amino acid sequence at least 80% identical to SEQ ID NO:10. In specific embodiments, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:13. In specific embodiments, the spike-CD9 fusion protein has an amino acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:3. In specific embodiments, 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 essentially identical to SEQ ID NO:4.

[0062] 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 at least 80% identical to SEQ ID NO:5 or essentially identical to SEQ ID NO:4. In a specific embodiment, the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence at least 80% identical to SEQ ID NO:12 or essentially identical to SEQ ID NO:12.

[0063] In some embodiments, where 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 N-terminal to the nucleocapsid protein polypeptide. In some cases, as shown in FIG. 2A, a hinge peptide, a transmembrane domain peptide, and a linker peptide are encoded by the polynucleotide such that they are positioned between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein. In specific embodiments, the CD9 protein polypeptide has an amino acid sequence at least 80% identical to SEQ ID NO:10. In specific embodiments, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence at least 80% identical to or essentially identical to SEQ ID NO:13. In specific embodiments, the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to, or essentially identical to, SEQ ID NO: 7. In specific embodiments, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to, or essentially identical to, SEQ ID NO: 8.

[0064] In another aspect, a cell is provided that contains a polynucleotide encoding a synthetic fusion protein containing an influenza virus polypeptide sequence fused to an exosomal tetraspanin protein polypeptide sequence, the encoded fusion protein being designed and engineered such that the influenza SARS-CoV-2 protein antigen can be expressed in the cell and sorted onto the surface of the exosome, enabling an immune response to be elicited when the exosome is administered to a subject.

[0065] In some embodiments of the cell aspect, 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 from 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.

[0066] 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 at least 80% identical to, or essentially identical to, SEQ ID NO:14. In a specific embodiment, the polynucleotide encoding the H3 protein polypeptide has a nucleic acid sequence at least 80% identical to, or 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 at least 80% identical to, or essentially identical to, SEQ ID NO:10. In a specific embodiment, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:13.

[0067] In some embodiments, where 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 such that it is positioned between the hemagglutinin protein polypeptide and the CD9 protein polypeptide in the encoded synthetic fusion protein, as shown in FIG. 3A. In specific embodiments, the H3-CD9 fusion protein has an amino acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:16. In specific embodiments, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence at least 80% identical to, or essentially identical to, SEQ ID NO:17.

[0068] In another aspect, a cell is provided that contains a polynucleotide encoding a synthetic fusion protein containing a polypeptide sequence of respiratory syncytial virus (RSV) fused with a polypeptide sequence of an exosomal tetraspanin protein. The encoded fusion protein is designed and engineered so that the RSV protein antigen can be expressed in cells and sorted onto the surface of exosomes, enabling an immune response to be elicited when the exosomes are administered to a subject.

[0069] In some embodiments of the cell aspect, 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 from 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.

[0070] 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 at least 80% identical to, or essentially identical to, SEQ ID NO: 18. In a specific embodiment, the polynucleotide encoding the RSV F protein polypeptide has a nucleic acid sequence at least 80% identical to, or 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 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 at least 80% identical to, or essentially identical to, SEQ ID NO: 13.

[0071] In some embodiments, the encoded RSV F protein polypeptide is located 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 such that it is located between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as shown in Figure 21A. In a specific embodiment, the RSV F-CD9 fusion protein has an amino acid sequence that is at least 80% identical to, or 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, or essentially identical to, SEQ ID NO: 21. [Brief explanation of the drawings]

[0072] [Figure 1] Figure 1A shows a linear representation of a SARS-CoV-2 spike-CD9 fusion protein with the amino terminus on the left and the carboxy terminus on the right, with a linker sequence positioned between the spike and CD9 protein polypeptides. Figure 1B shows the relationship of the SARS-CoV-2 spike-CD9 fusion protein of Figure 1A to a vesicle membrane with the amino terminus on the left and the carboxy terminus on the right. The spike protein polypeptide spans the membrane once, with its amino terminus facing the exterior of the vesicle. The linker is positioned on the luminal side of the membrane. The CD9 protein polypeptide spans the membrane four times, with its carboxy terminus facing the lumen of the vesicle. [Figure 2]Figure 2A shows a linear diagram of a SARS-CoV-2 nucleocapsid CD9 fusion protein, with the amino terminus on the left and the carboxy terminus on the right, in which an amino-terminal signal peptide is fused to a nucleocapsid protein polypeptide, which is fused to a hinge region peptide, which is fused to a transmembrane domain peptide, which is fused to a linker peptide, which is fused to a CD9 protein polypeptide. Figure 2B shows a diagram of the SARS-CoV-2 nucleocapsid CD9 fusion protein of Figure 2A, with the amino terminus on the left and the carboxy terminus on the right, in relation to a vesicle membrane. The nucleocapsid protein polypeptide with the amino-terminal signal sequence is positioned externally (cytoplasmically or externally), and a linker sequence connects the nucleocapsid protein polypeptide to the transmembrane domain peptide, which spans the membrane and is then connected to a lumenally positioned linker, which connects the carboxy terminus to the CD9 protein polypeptide, which spans the membrane four times toward the lumen of the vesicle. [Figure 3] Figure 3A shows a linear representation of an influenza hemagglutinin CD9 fusion protein with the amino terminus on the left and the carboxy terminus on the right, with a linker sequence positioned between the spike protein polypeptide and the CD9 protein polypeptide. Figure 3B shows a diagram of the influenza hemagglutinin CD9 fusion protein of Figure 3A in relation to a vesicle membrane with the amino terminus on the left and the carboxy terminus on the right. The hemagglutinin protein polypeptide spans the membrane once, with its amino terminus facing the outside of the vesicle. The linker is positioned on the luminal side of the membrane. The CD9 protein polypeptide spans the membrane four times, with its carboxy terminus facing into the lumen of the vesicle. [Figure 4]Figure 4A is a flow chart showing the elements and steps for lentiviral vector (304)-mediated production of cells expressing spike, nucleocapsid, influenza hemagglutinin, or other antigenic protein polypeptide fusion proteins using packaging cells (301) and host cells (311). Figure 4B is a histogram showing relative fluorescence intensity flow analysis of host cells expressing spike proteins on their surface. [Figure 5] Figure 5A is a graph showing 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. From left to right, the first column represents lanes loaded with size markers, the second column represents lanes loaded with untransduced 293F (host cell) proteins, the third column represents lanes loaded with proteins derived from exosomes from untransduced 293F cells, the fourth column represents lanes loaded with proteins derived from 293F cells constitutively expressing the spike fusion protein, the fifth column represents lanes loaded with proteins derived from exosomes from 293F cells constitutively expressing the spike fusion protein, and the sixth column represents lanes loaded with the spike fusion protein. Figure 5C is a histogram showing the relative fluorescence intensity flow analysis of exosomes expressing the spike on their surface. The left curve represents exosomes derived from 293F cells that do not express the spike-CD9 fusion protein, and the right curve represents exosomes derived from 293F cells that do express the spike-CD9 fusion protein. [Figure 6]Figure 6A shows a transmission electron micrograph of exosomes expressing the SARS-CoV-2 spike protein. The inset is a magnified image showing exosomes decorated with spike protein. The arrows point to the SARS-CoV-2 spikes around the exosomes. Figure 6B shows a higher magnification transmission electron micrograph of exosomes expressing the SARS-CoV-2 spike protein. The arrows point to the SARS-CoV-2 spikes around the exosomes. [Figure 7] Figure 7A is a graph showing 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. From left to right, the first column represents lanes loaded with size markers, the second column represents lanes loaded with nucleocapsid protein, the third column represents lanes loaded with protein from exosomes derived from non-transduced 293F cells, the fourth column represents lanes loaded with protein from non-transduced 293F cells, and the fifth column represents lanes loaded with protein from exosomes derived from 293F cells expressing the nucleocapsid fusion protein. Figure 7C is a histogram showing the relative fluorescence intensity flow analysis of exosomes with and without nucleocapsid protein expressed on their surface. 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 polynucleotide encoding a nucleocapsid-CD9 fusion protein, the third curve represents exosomes derived from 293F cells expressing a nucleocapsid-CD9 fusion protein, the third smaller curve represents exosomes derived from 293F cells expressing a nucleocapsid-CD9 fusion protein under tet induction, and the third larger curve represents exosomes derived from 293F cells harboring a CD9 knockout and expressing a nucleocapsid-CD9 fusion protein. [Figure 8-1]Figures 8A and 8B are histograms showing relative fluorescence intensity flow analysis of 293F cells with or without surface expression of influenza H3 hemagglutinin protein. From left to right in each panel, the first curve represents unmodified 293F cells, and the second curve represents 293F cells carrying and expressing a polynucleotide encoding an influenza H3-CD9 fusion protein. Panel A represents adherent 293F cells. Panel B represents non-adherent 293F cells. [Figure 8-2] Figure 8C shows Western blots stained for influenza H3 protein (panel 1) or CD9 (panel 2). For each panel, from left to right, the first column indicates the lane loaded with size marker, the second column indicates the lane loaded with unmodified 293F (host cell) protein, the third column indicates the lane loaded with protein derived from exosomes derived from unmodified 293F cells, the fourth column indicates the lane loaded with protein derived from 293F cells expressing an H3-CD9 fusion protein, and the fifth column indicates the lane loaded with protein derived from exosomes derived from 293F cells expressing an H3-CD9 fusion protein. Each sample was loaded at 0.6 mg / ml total protein. Figure 8D shows a flow cytometry dot plot of H3-expressing exosomes showing CD81 surface expression as a function of H3 surface expression. In this example, approximately 54.2% of the shed exosomes expressed both CD81 and H3, thus demonstrating that hemagglutinin polypeptides or antigens are expressed on the surface of vesicles when fused to exosomal proteins, such as tetraspanins, e.g., CD9. [Figure 9] Figure 9 is a timeline showing the process of generating an immune response by administering an immunogenic composition containing exosomes expressing a SARS-CoV-2 fusion protein on their surface. [Figure 10-1]Figures 10A-10D. STX-S exosomes elicited robust immune responses using significantly lower antigen concentrations than recombinant protein vaccines. Data are shown as mean ± SEM. ****p<0.0005, ***p<0.001, **p<0.01, *p<0.05, ns=non-significant, *one-way ANOVA; ##p<0.01, #p<0.05, #-two-tailed t-test. Figure 10A is a histogram showing the fold change in antibody titers against the spike protein in 1:100 diluted serum from mice on day 14 as a function of the dose in nanograms of spike-expressing exosomes. The leftmost bar represents the 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; and the rightmost bar represents serum from mice injected with a 32 ng dose of spike protein combined with adjuvant. Figure 10B is a histogram showing the fold change in antibody titer against spike protein in 1:100 diluted serum from mice on day 35 as a function of the dose in nanograms of spike-expressing exosomes. The leftmost bar represents the 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; and the rightmost bar represents serum from mice injected with a 32 ng dose of spike protein combined with adjuvant. FIG. 10C is a bar graph showing an ELISpot assay showing the number of spots producing IL4 in response to spike protein as a function of antigen dose.From left to right on the x-axis, the first bar represents non-spike-containing wells derived from PBS control splenocytes; the second bar represents reactivity to spike protein derived from PBS control splenocytes; the third bar represents non-spike-containing wells derived from splenocytes of a subject administered 10 ng STX-S; the fourth bar represents reactivity to spike protein derived from splenocytes of a subject administered 10 ng STX-S; the fifth bar represents non-spike-containing wells derived from splenocytes of a subject administered 32 ng STX-S; the sixth bar represents reactivity to spike protein derived from splenocytes of a subject administered 32 ng STX-S; the seventh bar represents non-spike-containing wells derived from splenocytes of a subject administered 32 ng spike protein with adjuvant; and the eighth bar represents reactivity to spike protein derived from splenocytes of a subject administered 32 ng spike protein with adjuvant. FIG. 10D is a bar graph showing an ELISpot assay showing the number of spots producing IFNγ in response to spike protein as a function of antigen dose. From left to right on the x-axis, the first bar represents non-spike-containing wells derived from PBS control splenocytes; the second bar represents reactivity to spike protein derived from PBS control splenocytes; the third bar represents non-spike-containing wells derived from splenocytes of a subject administered 10 ng STX-S; the fourth bar represents reactivity to spike protein derived from splenocytes of a subject administered 10 ng STX-S; the fifth bar represents non-spike-containing wells derived from splenocytes of a subject administered 32 ng STX-S; the sixth bar represents reactivity to spike protein derived from splenocytes of a subject administered 32 ng STX-S; the seventh bar represents non-spike-containing wells derived from splenocytes of a subject administered 32 ng spike protein with adjuvant; and the eighth bar represents reactivity to spike protein derived from splenocytes of a subject administered 32 ng spike protein with adjuvant. [Figure 10-2] See description of Figure 10-1. [Figure 11A]Figure 11, Panels A-C: Line graphs showing % SARS-CoV-2 neutralization as a function of immune serum at increasing serum dilutions. The graph shows the production of neutralizing antibodies after STX-S injection. Data are shown as mean ± SEM. COV-02-Delta = Plasma from a patient immunized with Moderna's mRNA vaccine who developed a breakthrough SARS-CoV-2 delta spike infection. Dose 4 = 9.8 ng S per injection; Dose 2 = 3.2 ng S per injection. Panel 11A. The STX-S vaccine produced potent neutralization against the SARS-CoV-2 delta spike (B.1.617.2). Panel 11B. The STX-S vaccine resulted in neutralization of the SARS-CoV-2 spike omicron BA.1. Panel 11C. The STX-S vaccine resulted in neutralization of the SARS-CoV-2 spike omicron BA.5.2.1. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 12-1]Figure 12, panels A-F, are histograms showing the amount (OD [panels A-C], fold change [panels B and E], or log10 titer [panels C and F]) of anti-H3 antibodies (panels A-C) and anti-spike antibodies (panels D-F) in serum from subjects at day 14 post-injection as a function of immunogenic composition (STX-H3 or STX-H3 + STX-S). Data are shown as mean ± SEM. *p<0.05, ***p<0.005, ****p<0.001, analysis of variance, corrected for multiple comparisons; ns=non-significant. Figure 12A is a histogram showing the OD of serum anti-H3 IgG on day 14 as a function of injected material dose, which are, 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 the SARS-CoV-2 spike (STX-S). Figure 12B is a histogram showing the fold change in serum anti-H3 IgG levels on day 14 as a function of injected material dose, which are, 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 the SARS-CoV-2 spike (STX-S). Figure 12C is a histogram showing the log10 titer of anti-H3 IgG in serum on day 14 as a function of injected material dose, which are, 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 the SARS-CoV-2 spike (STX-S). Figure 12D is a histogram showing the OD of anti-spike IgG in serum on day 14 as a function of injected material dose, which are, from left to right, PBS control, and a combination of STX-H3 and exosomes expressing the SARS-CoV-2 spike (STX-S).Figure 12E is a histogram showing the fold change in serum anti-H3 IgG levels on day 14 as a function of injected material dose, which are, 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 the SARS-CoV-2 spike (STX-S). Figure 12FE is a histogram showing the log10 titer of serum anti-H3 IgG on day 14 as a function of injected material dose, which are, 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 the SARS-CoV-2 spike (STX-S). [Figure 12-2] See description of Figure 12-1. [Figure 13-1] Figure 13, Panels A-D. The STX-N vaccine induces polyvalent immune responses. Data are shown as mean ± SEM. *p<0.05, ***p<0.005, ****p<0.001, analysis of variance, corrected for multiple comparisons; ns=non-significant. Dose 1 = 0.32 ng N per injection; Dose 2 = 3.2 ng N per injection; Dose 3 = 10 ng N per injection. Panels 13A and 13B: Histograms showing anti-nucleocapsid antibody titers in serum at day 35 post-immunization as a function of dose. The STX-N vaccine induced moderate expression of SARS-CoV-2 nucleocapsid antibodies in two sample bins (N1 and N2). PBS was used as a vehicle control. Panels 13C and 13D: Histograms showing anti-nucleocapsid IFNγ ELISpot-positive wells of splenocytes at day 40 post-immunization as a function of dose. [Figure 13-2] See description of Figure 13-1. [Figure 14]Figure 14 shows the expression of SARS-CoV-2 spike protein in 293 cells. Panel 14A is a histogram showing relative fluorescence intensity flow analysis of 293F cells expressing the SARS-CoV2 spike on their surface. The left curve represents 293F cells that do not express the spike-CD9 fusion protein. The right curve represents 293F cells that express the spike-CD9 fusion protein. Panel 14B is a Western blot stained for the SARS-CoV-2 spike protein. From left to right, the first column indicates lanes loaded with size markers, the second column represents lanes loaded with non-transduced 293F (host cell) proteins, the third column represents lanes loaded with proteins derived from exosomes derived from non-transduced 293F cells, the fourth column represents lanes loaded with proteins derived from 293F cells expressing spike fusion proteins, the fifth column represents lanes loaded with proteins derived from exosomes derived from 293F cells expressing spike fusion proteins, and the sixth column represents lanes loaded with spike fusion proteins. [Figure 15] Panel 15A is a histogram showing relative fluorescence intensity flow analysis of 293F cells expressing influenza hemagglutinin 3 (H3) on their surface. The left curve represents 293F cells that do not express the H3-CD9 fusion protein. The right curve represents 293F cells that express the H3-CD9 fusion protein. Panel 15B is a Western blot stained for influenza hemagglutinin 3 (H3) protein. From left to right, the first column represents lanes loaded with size markers, the second column represents lanes loaded with non-transduced 293F (host cell) proteins, the third column represents lanes loaded with proteins derived from exosomes from non-transduced 293F cells, the fourth column represents lanes loaded with proteins derived from 293F cells expressing the H3 fusion protein, and the fifth column represents lanes loaded with proteins derived from exosomes from 293F cells expressing the H3 fusion protein. [Figure 16]Figure 16 shows histograms showing the expression of surface markers in 293F-derived exosomes. Panel 16A shows a histogram showing relative fluorescence intensity flow analysis of exosomes derived from 293F cells expressing naturally occurring CD81 on their surface. The left curve represents 293F-derived exosomes decorated with an isotype control antibody. The right curve represents 293F-derived exosomes decorated with an anti-CD81 antibody. Panel 16B shows a histogram showing relative fluorescence intensity flow analysis of exosomes derived from 293F cells expressing spike on their surface. The left curve represents exosomes derived from 293F cells that do not express spike-CD9 fusion protein. The right curve represents exosomes derived from 293F cells that express spike-CD9 fusion protein. Panel 16C shows a histogram showing relative fluorescence intensity flow analysis of exosomes derived from transfected 293F cells expressing hemagglutinin 3 (H3) on their surface. The left curve represents exosomes derived from 293F cells that do not express the H3-CD9 fusion protein, while the right curve represents exosomes derived from 293F cells that do express the H3-CD9 fusion protein. [Figure 17] Figure 17 shows histograms depicting fold changes in antigen-specific IgG production. Panel 17A is a histogram depicting the fold change in serum anti-H3 IgG levels on day 14 as a function of injected material dose, which are, from left to right, PBS control and a combination of hemagglutinin 3-expressing exosomes (STX-H3) and SARS-CoV-2 spike-expressing exosomes (STX-S). Panel 17B is a histogram depicting the fold change in serum anti-spike IgG levels on day 14 as a function of injected material dose, which are, from left to right, PBS control and a combination of hemagglutinin 3-expressing exosomes (STX-H3) and SARS-CoV-2 spike-expressing exosomes (STX-S). [Figure 18]Figure 18 shows histograms depicting fold changes in antigen-specific IgG production. Panel 18A shows a histogram depicting the fold change in serum anti-H3 IgG levels on day 35 as a function of injected material dose, which are, from left to right, PBS control and a combination of hemagglutinin 3-expressing exosomes (STX-H3) and SARS-CoV-2 spike-expressing exosomes (STX-S). Panel 18B shows a histogram depicting the fold change in serum anti-spike IgG levels on day 35 as a function of injected material dose, which are, from left to right, PBS control and a combination of hemagglutinin 3-expressing exosomes (STX-H3) and SARS-CoV-2 spike-expressing exosomes (STX-S). [Figure 19]Figure 19 shows ELISpot histograms of IFNg production. Panel 19A is a histogram showing IFNg production by splenocytes as a function of immunogen and antigen. From left to right, the first histogram represents splenocytes from an animal immunized with PBS and not exposed to antigen; the second histogram represents splenocytes from an animal immunized with PBS and not exposed to H3 antigen; the third histogram represents splenocytes from an animal immunized with a combination of spike-expressing and H3-expressing exosomes and not exposed to antigen; and the fourth histogram represents splenocytes from an animal immunized with a combination of spike-expressing and H3-expressing exosomes and not exposed to H3 antigen. Panel 19A is a histogram showing IFNg production by splenocytes as a function of immunogen and antigen. From left to right, the first histogram represents splenocytes from an animal immunized with PBS and not exposed to antigen, the second histogram represents splenocytes from an animal immunized with PBS and exposed to H3 antigen, the third histogram represents splenocytes from an animal immunized with a combination of spike-expressing and H3-expressing exosomes and not exposed to antigen, and the fourth histogram represents splenocytes from an animal immunized with a combination of spike-expressing and H3-expressing exosomes and exposed to H3 antigen. Panel 19B is a histogram showing IFNg production by splenocytes as a function of immunogen and antigen. From left to right, the first histogram represents splenocytes from an animal immunized with PBS and not exposed to antigen, the second histogram represents splenocytes from an animal immunized with PBS and exposed to spike antigen, the third histogram represents splenocytes from an animal immunized with a combination of spike-expressing and H3-expressing exosomes and not exposed to antigen, and the fourth histogram represents splenocytes from an animal immunized with a combination of spike-expressing and H3-expressing exosomes and exposed to spike antigen. [Figure 20] Figure 20 shows a schematic diagram of the sequence encoding the respiratory syncytial virus fusion protein (RSFV F). Bar A represents unengineered RSV F. Bar B represents engineered RSV F version 1 (V1), which is DS-Cav1. Bar C represents engineered RSV F version 2 (V2), which is DS-Cav1 with a deleted polyA signal. Bar D represents engineered RSV F version 3 (V3), which is DS-Cav1 with a deleted polyA signal and two furin cleavage sites. Bar D represents engineered RSV F version 3 (V4), which is DS-Cav1 with a deleted polyA signal and the most N-terminal furin cleavage site. SP = signal peptide; F2 = RSV fusion protein subunit 2; p27 = RSV fusion protein p27 subunit; FP = hydrophobic fusion peptide (FP); F1 = RSV fusion protein subunit 1; TM = transmembrane domain. [Figure 22] Figure 22 is a histogram showing the fold change in anti-RSV antibody titer as a function of antigen (RSV version 3). The Y axis represents the fold change in antibody titer, the X axis represents the antigen, the left bar is the PBS control, and the right bar is the RSV F antigen. Panel 22A represents the fold change in antibody titer 14 days after injection. Panel 22B represents the fold change in antibody titer 35 days after injection, after a booster on day 21. [Figure 23] Figure 23 is a histogram showing the fold change in anti-RSV antibody titer as a function of antigen (RSV version 4). The Y-axis represents the fold change in antibody titer, and the X-axis represents the antigen. Panel 23A represents the fold change in antibody titer 14 days after injection. Panel 23B represents the fold change in antibody titer 35 days after injection, after a booster on day 21. For both panels, the X-axis labels 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, and 5 = 1E11 exosomes expressing RSV F V4-CD9. [Figure 24]Figure 24 shows a diagram depicting the combination of exosomes expressing SARS-CoV2 spike-tetraspanin (STX-S) and exosomes expressing influenza H3-tetraspanin (STX-H3). [Figure 25] Figure 25 shows the combination of exosomes expressing SARS-CoV2 spike-tetraspanin (STX-S) and exosomes expressing respiratory syncytial virus fusion protein-tetraspanin (STX-F). [Figure 26] Figure 26 is a diagram showing the combination of exosomes expressing influenza H3-tetraspanin (STX-H3) and exosomes expressing respiratory syncytial virus fusion protein-tetraspanin (STX-F). [Figure 27] Figure 27 shows the 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). [Figure 28]Figure 28 is a histogram showing the fold change in antibody titer as a function of antigen. The Y-axis represents the fold change in antibody titer, and the X-axis represents the antigen. Panel 28A represents the fold change in anti-spike antibodies 14 days after injection. Panel 28B represents the fold change in anti-H3 antibodies 14 days after injection. Panel 28C represents the fold change in anti-RSV antibodies 14 days after injection. For each panel, the x-axis labels are: 1 = PBS, 2 = spike-expressing exosomes (STX-S), 3 = H3-expressing exosomes (STX-H3), 4 = RSV F-expressing exosomes (STX-RSV), 5 = H3-expressing exosomes combined with spike-expressing exosomes (STX-H3 + STX-S), 6 = RSV F-expressing exosomes combined with spike-expressing exosomes (STX-RSV + STX-S), 7 = RSV F-expressing exosomes combined with H3-expressing exosomes (STX-RSV + STX-H3), 8 = RSV F-expressing exosomes combined with H3-expressing exosomes combined with spike-expressing exosomes (STX-RSV + STX-H3 + STX-S). [Figure 29]Figure 29 is a histogram showing the fold change in antibody titer as a function of antigen. The Y-axis represents the fold change in antibody titer, and the X-axis represents the antigen. Panel 29A represents the fold change in anti-spike antibodies after a booster at 21 days, 35 days after injection. Panel 28B represents the fold change in anti-H3 antibodies after a booster at 21 days, 35 days after injection. Panel 28C represents the fold change in anti-RSV antibodies after a booster at 21 days, 35 days after injection. For each panel, the x-axis labels are: 1 = PBS, 2 = spike-expressing exosomes (STX-S), 3 = H3-expressing exosomes (STX-H3), 4 = RSV F-expressing exosomes (STX-RSV), 5 = H3-expressing exosomes combined with spike-expressing exosomes (STX-H3 + STX-S), 6 = RSV F-expressing exosomes combined with spike-expressing exosomes (STX-RSV + STX-S), 7 = RSV F-expressing exosomes combined with H3-expressing exosomes (STX-RSV + STX-H3), 8 = RSV F-expressing exosomes combined with H3-expressing exosomes combined with spike-expressing exosomes (STX-RSV + STX-H3 + STX-S). DETAILED DESCRIPTION OF THE INVENTION

[0073] Detailed Description of the Embodiments definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, and / or steps, of the type described herein that will become apparent to those skilled in the art upon review of this disclosure, and so forth.

[0074] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, and also refers to and includes the absence of a combination when interpreted as alternatives (or).

[0075] The terms "about" and "approximately," as used herein, when referring to a measurable value, e.g., amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, location, length, etc., are meant to encompass a variation 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, location, length, etc. When the terms "about" and "approximately" are used in reference to the location or position of a region within a reference polypeptide, these terms encompass a variation 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.

[0076] The term "derived from" means that "A is derived from B" means that A is obtained from B in such a way that A is not identical to B.

[0077] The terms "treat," "therapeutic," "prophylactic," and "prevent" are not intended to be absolute terms. Treatment, prevention, and prophylaxis can refer to delaying onset, ameliorating symptoms, improving patient survival, increasing survival time or survival rate, etc. Treatment, prevention, and prophylaxis can be complete or partial. The term "prophylactic" refers not only to "preventing" but also to minimizing disease and illness. For example, a "prophylactic" agent can be administered to a subject, e.g., a human subject, to prevent infection or minimize the extent of disease and illness caused by such infection. The effect of treatment can be compared to an untreated individual or pool of individuals, or the same patient at different time points before or during treatment. In some aspects, the severity of the disease is reduced by at least 10%, for example, compared to the individual before administration or to an untreated control individual. In some aspects, the severity of the disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, becomes undetectable using standard diagnostic techniques.

[0078] As used herein, a treatment can be considered "effective" if, following treatment with the methods described herein, one or more of the signs or symptoms of a condition described herein are beneficially altered, other clinically accepted symptoms are improved or even ameliorated, or the desired response is induced, for example, by at least 2%, 3%, 4%, 5%, 10%, or more. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or prevalence of a condition treated according to the methods described herein, or any other suitable measurable parameter. Efficacy can also be measured by the lack of progression (e.g., cessation of disease progression) of an individual, as assessed by the need for hospitalization or medical intervention. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or animals), including (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) alleviating the severity of the disease, e.g., causing regression of symptoms. The effective amount for the treatment of a disease means that when administered to a subject in need thereof, it is sufficient to bring about an effective treatment (as this term is defined herein) for that disease.The effectiveness of an active substance can be determined by assessing the physical indicators of the condition or the desired response.Those skilled in the art can monitor the effectiveness of administration and / or treatment by measuring any one of these parameters or any combination of parameters.

[0079] The term "effective amount," as used herein, refers to the amount of a composition or agent necessary to alleviate at least one or more symptoms of a disease or disorder, and relates to the amount of a therapeutic composition sufficient to provide a desired effect. The term "therapeutically effective amount" refers to the amount of a composition or therapeutic agent sufficient to provide a particular effect when administered to a typical subject. As used herein, an effective amount can include, in various circumstances, an amount sufficient to delay the onset of disease symptoms, alter the course of a disease condition (e.g., but not limited to, slowing the progression of disease symptoms), or reverse disease symptoms. For example, for a given parameter, a therapeutically effective amount indicates an increase or decrease in therapeutic effect of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy may also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount may have an effect of at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more compared to a control. A therapeutically effective amount may be administered in one or more doses of a therapeutic agent. A therapeutically effective amount may be administered in a single dose or in multiple doses administered over a period of time.

[0080] "Administration," as used herein, can include any suitable route of administration of a therapeutic agent or composition disclosed herein. Suitable routes of administration include, but are not limited to, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, dermal, injection, or topical administration. Administration can be local or systemic.

[0081] 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. This term is used interchangeably with "physiologically acceptable" and "pharmacologically acceptable." Pharmaceutical compositions generally include agents for buffering and preservation during storage, and may include buffers and carriers for appropriate delivery, depending on the route of administration. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0082] The terms "dose" and "administration" are used interchangeably herein. Dose refers to the amount of active ingredient given to an individual at each administration. For the present invention, dose can refer to the concentration of extracellular vesicles or related components, such as the amount of therapeutic agent or the amount of radiolabel administered. Dose varies depending on many factors, including the frequency of administration; the size and tolerance of the individual; the severity of the condition; the risk of side effects; the administration route; and the imaging modality of the detectable moiety (if any). Those skilled in the art will recognize that dose can be modified depending on the above factors or based on the progress of treatment. The term "dosage form" refers to the specific form of the medicine and depends on the administration route. For example, the dosage form can be a liquid for injection, such as a saline solution.

[0083] The terms "subject," "patient," "individual," and the like are used interchangeably and, unless otherwise indicated, refer to mammals, e.g., 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 may be an individual seeking treatment, monitoring, adjustment or modification of an existing treatment regimen, and the like.

[0084] As used herein, the following meanings apply unless otherwise specified: The word "may" is used in its permissive sense (i.e., having the potential to), rather than its obligatory sense (i.e., meaning must). Words such as "include," "including," and "includes" 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, despite the use of other terms and phrases, e.g., "one or more," that mean one or more elements. The term "or" is non-exclusive, i.e., includes both "and" and "or," unless otherwise indicated. The term "any of" between a modifier and a series of words means that the modifier modifies every member of the series. Thus, for example, the phrase "any of at least 1, 2, or 3" means "at least 1, at least 2, or at least 3." The phrase "at least 1" includes "a plurality."

[0085] Definitions of common terms in cell 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.

[0086] The term "native form" refers to a polypeptide as understood to be encoded by the genome of an infectious pathogen. The term "exosomal form" refers to any derivative of a protein that is fused in whole or in part to an exosome-associated protein. The term "cytoplasmic form" refers to any derivative of a protein that is configured or designed in whole or in part to be expressed in the cytoplasm of a cell rather than entering the standard secretory pathway.

[0087] The phrase "configured, configured, or designed to be expressed" in a particular manner means that the nucleotide sequence encodes a particular amino acid sequence such that when the protein is expressed in a cell, the protein is expressed in a native, exosomal, or cytosolic form. For example, when the 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 being a transmembrane protein that includes an extracellular domain.

[0088] The term "extracellular vesicles" (EVs) refers to lipid bilayer-bound particles naturally released from cells. EV diameters range from approximately 20-30 nanometers to approximately 10 microns or larger. EVs can contain proteins, nucleic acids, lipids, and metabolites derived from the cells that produced them. EVs include exosomes (approximately 50-200 nm), microvesicles (approximately 100-300 nm), ectosomes (approximately 50-1000 nm), apoptotic bodies (approximately 50-5000 nm), and lipid-protein aggregates of similar dimensions.

[0089] 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'methoxyethyl, phosphoramidate, methylphosphonate, and phosphorothioate. Nucleic acids can 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, antisense DNA strands, shRNA, ribozymes, conjugated nucleic acids, and oligonucleotides. According to the present invention, nucleic acids can exist as single-stranded or double-stranded, and can exist as linear or covalently circularly closed molecules. Nucleic acids can be utilized for introduction into cells, e.g., transfection of cells, in the form of RNA, which can be prepared, for example, by in vitro transcription from a DNA template. RNA can be further modified by stabilizing sequences, capping, and polyadenylation before application. Generally, nucleic acids can be extracted, isolated, amplified, or analyzed by a variety of techniques, such as those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY, 2,028 pages (2012).

[0090] 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 bond. As used herein, a peptide can refer to a complete amino acid sequence encoding an entire protein or a portion thereof. A "protein coding sequence," or a sequence "encoding" a particular polypeptide or peptide, is a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, either 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 derived from prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence is typically located 3' to the coding sequence.

[0091] As used herein, the phrase "protein polypeptide" refers to a polypeptide sequence of a protein or a polypeptide sequence derived from a protein. For example, a CD9 protein polypeptide can be any polypeptide of a CD9 protein, such as 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, an intracellular (intraluminal) loop region of a CD9 protein, a C-terminal stretch of a CD9 protein, a combination thereof, etc. Here, a protein polypeptide can be at least 10 amino acids in length.

[0092] As used herein, the term "spike protein" includes any SARS-CoV-2 spike glycoprotein, fragments of SARS-CoV-2 spike glycoprotein, SARS-CoV-2 spike glycoprotein monomers, trimers of SARS-CoV-2 spike glycoprotein monomers, variants of SARS-CoV-2 spike glycoprotein, fusion or chimeric proteins containing a SARS-CoV-2 spike glycoprotein sequence and another non-SARS-CoV-2 spike glycoprotein sequence, SARS-CoV-2 spike glycoproteins with one or more deletions, additions, or substitutions of one or more amino acids, and conservatively substituted variations of SARS-CoV-2 spike glycoproteins with at least 80% amino acid sequence identity, for example, to at least the stem region, membrane-proximal stem helix region, or receptor-binding domain of the S2 subunit, or other similar domain.

[0093] Fragments of the SARS-CoV-2 spike glycoprotein include, for example, peptides or polypeptides that encompass, comprise, consist of, or overlap with antigenic epitopes, specific domains, such as the receptor binding domain (RBD) in the up or down conformational state, receptor binding fragment S1, 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), cytoplasmic tail (CT), etc.

[0094] Variants of the SARS-CoV-2 spike glycoprotein include any known or yet to be discovered, such as alpha, beta, gamma, delta, epsilon, eta, iota, kappa, 1.617.3, mu, zeta, omicron, or their subspecies, strains, and conservatively substituted spike protein sequences.

[0095] The SARS-CoV-2 spike glycoprotein can have additions, deletions, substitutions, or point mutations. For example, the spike protein can have a deletion of several (2-20) amino acids from the C-terminus (see Johnson et al., 2020 and Xiong et al., 2020) or a furin cleavage site alteration (see Johnson et al., 2020).

[0096] As used herein, the term "nucleocapsid protein" refers to a soluble coronavirus structural protein that binds to and forms a complex with RNA and the viral membrane protein (M), and is important for packaging the viral genome. The nucleocapsid protein contains (from the amino terminus to the carboxy terminus) an N-terminal domain containing an intrinsically disordered region (IDR) and an RNA-binding domain (NTD), a serine-arginine-rich linker region (LKR), a C-terminal domain containing an RNA-binding dimerization domain and a nuclear localization signal, followed by the C-terminal IDR. The nucleocapsid protein is generally described in, for example, 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.

[0097] Fusion or chimeric proteins of the SARS-CoV-2 spike glycoprotein or SARS-CoV-2 nucleocapsid protein include fusions of the spike protein or nucleocapsid protein sequence with the sequence of another protein that provides a particular outcome, such as improved sorting or targeting to endosomes and the resulting extracellular vesicles, e.g., exosomes. For example, the spike protein can be a fusion of the spike protein or nucleocapsid protein sequence with another glycoprotein known to sort into exosomes or useful in the production of pseudovirions, such as VSV glycoprotein or lentiviral glycoprotein. The spike protein or nucleocapsid protein can be a fusion of the spike protein sequence with another protein known to sort into exosomes, such as various tetraspanins (CD9, CD63, and CD81). The fusion protein may contain primarily SARS-CoV-2 sequences along with short sequences of other proteins (i.e., from dipeptides to peptides of 100 amino acids).

[0098] As used herein, the term "tetraspanin" or "tetraspanin protein" refers to any member of a family of proteins (or chimeras thereof) that have four transmembrane domains and, in some cases, are present in exosomal membranes. Tetraspanin proteins are known to regulate trafficking and cellular and membrane compartmentalization. Tetraspanins include, among others, CD9, CD37, CD63, CD81, CD82, CD151, TSPAN7, TSPAN8, TSPAN12, TSPAN33, peripherin, UP1a / 1b, TSP-15, TSP-12, TSP3A, TSP86D, TSP26D, TSP-2, and analogs, orthologs, and homologs thereof. It is contemplated that useful tetraspanins may include chimeras of any one or more standard tetraspanins, such as, for example, CD9 / CD81 chimeras. 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.

[0099] 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 two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence 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, the molecules are identical at that position. The percent identity between 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 × 100%). In one embodiment, the two sequences are the same length. Determining the percent identity between two sequences may be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 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, for example, using the NBLAST nucleotide program parameters set at score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application. BLAST protein searches can be performed with, for example, the XBLAST program parameters set to score-50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules described herein.To obtain gapped alignments for comparison purposes, Gapped BLAST, as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402, can be utilized. Alternatively, PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules (supra). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the NCBI website).

[0100] Another preferred, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into 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.

[0101] 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.

[0102] The percentage sequence identity for an antibody can be determined when the antibody sequences are maximally aligned by IMGT. After alignment, when a region of a subject antibody (e.g., the entire mature variable region of a heavy or light chain) is 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 regions, divided by the total number of aligned positions in the two regions, multiplied by 100, and converted to a percentage.

[0103] The percent amino acid sequence identity may 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 is available from the National Institutes of Health (Bethesda, Md). NCBI-BLAST2 uses several search parameters, all of which are set to default values, e.g., unmask=Yes, strand=All, expected occurrences=10, minimum low complexity length=15 / 5, multipass e-value=0.01, multipass constant=25, dropoff for final gap alignment=25, and scoring matrix=BLOSUM62.

[0104] In situations where NCBI-BLAST2 is utilized for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B is calculated as follows (alternatively, it may also be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B): 100 x fraction X / Y where X is the number of amino acid residues scored as an identical match by the sequence alignment program NCBI-BLAST2 in an alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if 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 be equal to 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 composed of naturally occurring bases, sugars, and intersugar (backbone) linkages, including cDNA. The term also includes modified or substituted sequences containing 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 contain 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-forms of adenine, guanine, cytosine, thymidine, and uracil; and xanthine and hypoxanthine. It is understood that polynucleotides containing non-transcribeable nucleotide bases can be useful as probes, for example, in hybridization assays. Nucleic acids can be either double-stranded or single-stranded, representing sense or antisense strands. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences, including codon-optimized equivalents or synonymous codon equivalents.

[0105] As used herein, the term "antibody" refers to immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that specifically binds an antigen. Antibodies are typically heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Light chains from vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Antibodies can have one or more effector functions, which refer to the biological activities attributable to the Fc region of an antibody (a native sequence Fc region, an amino acid sequence variant Fc region, or any other modified Fc region). Non-limiting examples of antibody effector functions include Clq binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors, such as B cell receptors (BCR); and cross-presentation of antigens by antigen-presenting cells or dendritic cells.

[0106] The term "neutralizing antibodies" (Nab) refers to antibodies that protect cells from pathogens or infectious particles by neutralizing their biological effects. Neutralization renders particles non-infectious or non-pathogenic. Neutralizing antibodies are part of the adaptive immune system's humoral response to viruses, intracellular bacteria, and microbial toxins. By specifically binding to surface antigens on infectious particles, neutralizing antibodies prevent the particles from interacting with host cells that they could infect and destroy.

[0107] Neutralizing antibody immunity is also known as sterilizing immunity because the immune system eliminates infectious particles before infection can occur.

[0108] The term "antigen" refers to any substance that induces an immune response. For example, an antigen relates to any substance, preferably a peptide or protein, that specifically reacts with antibodies or T lymphocytes (T cells). As used herein, the term "antigen" includes any molecule that contains at least one epitope. For example, an antigen is a molecule that, optionally after processing, induces an immune response. For example, any suitable antigen that is a candidate for an immune response can be used, where the immune response can be a cellular immune response. For example, the antigen can be presented by cells, which results in an immune response against the antigen. For example, the antigen corresponds to a naturally occurring antigen or is a product 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.

[0109] The term "pharmaceutical composition" refers to a formulation containing an active ingredient and, optionally, a pharmaceutically acceptable carrier, diluent, or excipient. The term "active ingredient" can refer interchangeably to "active ingredient" and refers to any agent capable of inducing a desired effect upon administration. "Pharmaceutically acceptable" means that the carrier, diluent, or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof and to the activity of the active ingredient of the formulation. Pharmaceutically acceptable carriers, excipients, or stabilizers are well known in the art (e.g., 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 include, but are not limited to, buffers, e.g., phosphate, citric acid, and other organic acids; antioxidants, e.g., ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, e.g., methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins. , such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).Examples of carriers include, but are not limited to, liposomes, nanoparticles, ointments, micelles, microspheres, microparticles, creams, emulsions, and gels.

[0110] Examples of excipients include, but are not limited to, anti-adherents such as magnesium stearate, binders such as sugars and their derivatives (sucrose, lactose, starch, cellulose, sugar alcohols, etc.), proteins such as 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 diluents include, but are not limited to, water, alcohol, saline solution, glycol, mineral oil, and dimethyl sulfoxide (DMSO).

[0111] 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, that recognizes and attacks pathogens or disease cells. The term "immune response" refers to the body's orchestrated response to an antigen, and refers to a cellular immune response and / or a humoral immune response. The immune response can be protective / preventative / prophylactic and / or therapeutic.

[0112] The term "cellular immune response" or "cell-mediated immune response" refers to any adaptive immune response in which antigen-specific T cells play a major role. This is practically defined as any adaptive immunity that cannot be transferred to a native recipient by serum antibodies. In contrast, the term "humoral immune response" refers to antibody-mediated immunity.

[0113] The cellular response involves cells called T cells or T lymphocytes that function as either "helpers" or "killers." Helper T cells (also called CD4+ T cells) play a central role by controlling the immune response, while killer cells (also called cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill diseased cells, such as cancer cells, and prevent the production of more diseased cells.

[0114] The terms "immunoreactive cell," "immune cell," or "immune effector cell" refer to cells that perform effector functions in immune responses. "Immune reactive cells" are preferably cells characterized by antigen presentation, or antigenic peptides derived from antigens, and can mediate immune responses. For example, such cells secrete cytokines and / or chemokines, secrete antibodies, recognize cancer cells, and optionally eliminate such cells. For example, immune reactive cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells.

[0115] The term "adjuvant" refers to a pharmacological or immunological agent that modifies the effect of another agent. Adjuvants can be added to the vaccine compositions of the present invention to boost the immune response so that more antibodies and longer-lasting immunity are produced, thereby minimizing the dose of antigen required. Depending on the purpose of the vaccine, adjuvants can also be used to enhance the effectiveness of vaccines by helping to modify the immune response to specific types of immune system cells, for example, by activating T cells rather than antibody-secreting B cells. Immunological adjuvants are added to vaccines to stimulate the immune system's response to the target antigen, but do not themselves provide immunity. Examples of adjuvants include, but are not limited to, analgesic adjuvants; inorganic compounds such as alum, aluminum hydroxide, aluminum phosphate, and calcium hydroxide phosphate; mineral oils such as paraffin oil; bacterial products such as killed bacteria (Bordetella pertussis, Mycobacterium bovis, and toxoids); non-bacterial organics such as squalene; delivery systems such as surfactants (Quil A); plant saponins derived from Quillaja, soybean, or Polygala senega; cytokines such as IL-1, IL-2, and IL-12; combinations such as Freund's complete and incomplete adjuvants; and food-based oils such as peanut oil-based adjuvant 65.

[0116] Aspects Aspects 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 the first fusion protein comprises a first viral polypeptide and an exosomal polypeptide, and the second fusion protein comprises a second viral 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 the first fusion protein comprises a first viral polypeptide and an exosomal polypeptide, the second fusion protein comprises a second viral polypeptide and an exosomal polypeptide, and the third fusion protein comprises a third viral polypeptide and an exosomal polypeptide. Embodiment 3 The immunogenic composition of embodiment 1 or embodiment 2, further comprising an excipient. Embodiment 4 The immunogenic composition of embodiment 3, wherein the excipient comprises a buffer. Embodiment 5 The immunogenic composition of embodiment 3 or 4, wherein the excipient comprises a cryoprotectant. Embodiment 6. The immunogenic composition of any one of Embodiments 1 to 5, which does not include an adjuvant. Embodiment 7 The immunogenic composition of any one of Embodiments 1 to 4, wherein the first fusion protein and the second fusion protein are present in the membrane of their respective vesicles. Embodiment 8. The immunogenic composition of any one of Embodiments 1 to 7, wherein some or all of each viral polypeptide is present at or on the outer surface of said vesicles. Embodiment 9. The immunogenic composition of any one of Embodiments 1 to 8, wherein each fusion protein is present in the composition at a concentration of about 1 ng / 100 μL to about 50 ng / 100 μL. Embodiment 10 The immunogenic composition of any one of embodiments 1 to 9, wherein the first viral polypeptide is a SARS-CoV-2 polypeptide and the second viral polypeptide is an influenza polypeptide. Embodiment 11 The immunogenic composition of any one of embodiments 1 to 10, wherein the first viral polypeptide is a SARS-CoV-2 spike protein polypeptide. Embodiment 12 The immunogenic composition of any one of embodiments 1 to 11, wherein the first viral polypeptide is a SARS-CoV-2 delta variant spike protein polypeptide. Embodiment 13 The immunogenic composition of any one of Embodiments 1 to 12, wherein the first viral polypeptide comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:1. Embodiment 14 The immunogenic composition of any one of embodiments 1 to 10, wherein the first viral polypeptide is a SARS-CoV-2 nucleocapsid protein polypeptide. Embodiment 15 The immunogenic composition of embodiment 14, wherein the first viral polypeptide comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:5. Embodiment 16 The immunogenic composition of any one of Embodiments 1 to 15, wherein the second viral polypeptide is an influenza hemagglutinin protein polypeptide. Embodiment 17 The immunogenic composition of any one of Embodiments 1 to 16, wherein the second viral polypeptide is an influenza hemagglutinin 3 (H3) protein polypeptide. Embodiment 18 The immunogenic composition of any one of embodiments 1 to 17, wherein the second viral polypeptide comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:14. Embodiment 19 The immunogenic composition of any one of Embodiments 2 to 18, wherein the third viral polypeptide is a respiratory syncytial virus (RSV) protein polypeptide. Embodiment 20 The immunogenic composition of any one of embodiments 2 to 19, wherein the third viral polypeptide is a RSV fusion (RSV F) protein polypeptide. Embodiment 21 The immunogenic composition of any one of Embodiments 2 to 20, wherein the third viral polypeptide comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:18. Embodiment 22 The immunogenic composition of any one of Embodiments 1 to 21, wherein the exosomal polypeptide is a tetraspanin protein polypeptide. Aspect 23 The immunogenic composition of any one of Aspects 1 to 22, wherein the exosomal polypeptide is a CD9 protein polypeptide. Embodiment 24 The immunogenic composition of any one of Embodiments 1 to 23, wherein the exosomal polypeptide comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:10. Embodiment 25 The immunogenic composition of any one of Embodiments 1 to 13 and 17 to 24, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:2. Embodiment 26 The immunogenic composition of any one of Embodiments 1 to 13 and 17 to 24, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:3. Embodiment 27 The immunogenic composition of any one of embodiments 1 to 10 and 14 to 23, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:6. Embodiment 28 The immunogenic composition of any one of embodiments 1 to 10, 14 to 23, and 27, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:7. Embodiment 29 The immunogenic composition of any one of embodiments 1 to 28, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:15. Embodiment 30 The immunogenic composition of any one of embodiments 1 to 29, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:16. Embodiment 31 The immunogenic composition of any one of Embodiments 2 to 30, wherein the third fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:19. Embodiment 32 The immunogenic composition of any one of Embodiments 2 to 31, wherein the third fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:20. Aspect 33. The immunogenic composition of any one of Aspects 1 to 32, wherein an immunogenic dose of the composition comprises from about 1 ng to about 50 ng of the first fusion protein or first viral polypeptide and from about 1 ng to about 50 ng of the second fusion protein or second viral polypeptide. Embodiment 34 The immunogenic composition of any one of Embodiments 2 to 33, wherein an immunogenic dose of the composition comprises from about 1 ng to about 50 ng of the third fusion protein or the third viral polypeptide. Embodiment 35. A method of immunizing a subject against a viral infection, comprising administering to the subject an immunogenically effective dose of the immunogenic composition of any one of Embodiments 1 to 34. Embodiment 36 The method of embodiment 35, wherein the immunogenic effective dose induces protective immunity in the subject against the first virus and the second virus. Embodiment 37 The method of embodiment 35 or 36, wherein the immunogenic effective dose induces protective immunity in the subject against a first virus, a second virus, and a third virus. Embodiment 38 The method of embodiment 35 or 36, wherein the immunogenic composition comprises a SARS-CoV-2 spike protein polypeptide and an influenza hemagglutinin polypeptide, and wherein the first virus is SARS-CoV2 and the second virus is an influenza virus. Embodiment 39. The method of any one of embodiments 35 to 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, wherein the first virus is SARS-CoV2, the second virus is an influenza virus, and the third virus is RSV. Embodiment 40 The method of any one of embodiments 35 to 39, wherein the immunogenic effective dose induces protective immunity against SARS-CoV-2 delta variant and SARS-CoV-2 omicron variant in the subject. Aspect 41. The method of any one of Aspects 35 to 40, wherein the immunogenic effective dose comprises between about 1 ng and about 50 ng of the first fusion protein, between about 1 ng and about 50 ng of the second fusion protein, and between about 1 ng and about 50 ng of the third fusion protein. Embodiment 42 The method of any one of Embodiments 35 to 41, further comprising administering to the subject a second effective dose of the immunogenic composition of any one of Embodiments 1 to 34. Embodiment 43 The method of any one of embodiments 35-42, wherein the protective immunity induced comprises (a) high antibody titers against the first, second, and third viruses, (b) a CD4+ T cell response against the first, second, and third viruses, and (c) a CD8+ cytotoxic T cell response against the first, second, and third viruses. Embodiment 44. A synthetic fusion protein comprising a viral polypeptide and an exosomal polypeptide. Embodiment 45. The synthetic fusion protein of embodiment 44, further comprising a linker polypeptide positioned between the viral polypeptide and the exosomal polypeptide. Embodiment 46 The synthetic fusion protein of embodiment 44 or 45, further comprising a hinge polypeptide positioned between the viral polypeptide and the exosomal polypeptide. Embodiment 47 The synthetic fusion protein of any one of Embodiments 44 to 46, further comprising a transmembrane domain polypeptide positioned between said viral polypeptide and said exosomal polypeptide. Embodiment 48 The synthetic fusion protein of any one of embodiments 44 to 47, wherein the exosomal polypeptide is a tetraspanin polypeptide. Embodiment 49 The synthetic fusion protein of any one of embodiments 44 to 48, wherein the exosomal polypeptide is a CD9 polypeptide. Embodiment 50. The synthetic fusion protein of any one of embodiments 44 to 49, wherein the viral polypeptide is a SARS-CoV-2 structural protein polynucleotide. Embodiment 51 The synthetic fusion protein of any one of embodiments 44 to 50, wherein the viral polypeptide is a SARS-CoV-2 spike protein polypeptide. Embodiment 52. The synthetic fusion protein of embodiment 51, wherein the SARS-CoV-2 spike protein polypeptide comprises one or more of a furin cleavage site mutation (CSM[682RRAR685→682GSAG685]) and a diproline substitution (2P[986KV987→986PP987]). Embodiment 53. The synthetic fusion protein of any one of Embodiments 44 to 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 The synthetic fusion protein of any one of embodiments 44 to 53, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:3. Embodiment 55. The synthetic fusion protein of any one of embodiments 44 to 54, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:3. Embodiment 56 The synthetic fusion protein of any one of embodiments 44 to 50, wherein the viral polypeptide is a SARS-CoV-2 nucleocapsid protein polypeptide. Embodiment 57. The synthetic fusion protein of any one of embodiments 44 to 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 The synthetic fusion protein of any one of embodiments 44 to 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. The synthetic fusion protein of any one of embodiments 44 to 50 and 56 to 58, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:7. Embodiment 60 The synthetic fusion protein of any one of embodiments 44 to 49, wherein the viral polypeptide is an influenza protein polypeptide. Embodiment 61 The synthetic fusion protein of embodiment 60, wherein the influenza protein polypeptide comprises hemagglutinin. Embodiment 62 The synthetic fusion protein of embodiment 60 or 61, wherein the influenza protein polypeptide comprises hemagglutinin 3 (H3). Embodiment 63 The synthetic fusion protein of any one of Embodiments 60 to 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 The synthetic fusion protein of any one of embodiments 60 to 63, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:16. Embodiment 65 The synthetic fusion protein of any one of embodiments 60 to 64, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:16. Embodiment 66 The synthetic fusion protein of any one of embodiments 44 to 49, wherein the viral polypeptide is a respiratory syncytial virus (RSV) protein polypeptide. 67. The synthetic fusion protein of embodiment 66, wherein the RSV protein polypeptide comprises a RSV fusion (RSV F) protein. Embodiment 68. The synthetic fusion protein of embodiment 66 or 67, wherein the fusion protein comprises, in order from the amino terminus to the carboxy terminus, a RSV F protein polypeptide, a linker polypeptide, and a CD9 polypeptide. Embodiment 69 The synthetic fusion protein of any one of embodiments 66 to 68, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:20. Embodiment 70 The synthetic fusion protein of any one of embodiments 60 to 64, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:20. Embodiment 71. A synthetic polynucleotide encoding the synthetic fusion protein of any one of Embodiments 44 to 70. Embodiment 72. The synthetic polynucleotide of embodiment 71, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 13. Embodiment 73. The synthetic polynucleotide of embodiment 71 or 72, comprising the nucleic acid sequence set forth in SEQ ID NO: 13. Embodiment 74. The synthetic polynucleotide of any one of Embodiments 71 to 73, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:11. Embodiment 75. The synthetic polynucleotide of any one of embodiments 71 to 74, comprising the nucleic acid sequence set forth in SEQ ID NO:11. Embodiment 76. The synthetic polynucleotide of any one of Embodiments 71 to 75, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:4. Embodiment 77. The synthetic polynucleotide of any one of embodiments 71 to 76, comprising the nucleic acid sequence set forth in SEQ ID NO:4. Embodiment 78. The synthetic polynucleotide of any one of embodiments 71 to 73, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 12. Embodiment 79. The synthetic polynucleotide of any one of embodiments 71 to 73 and 78, comprising the nucleic acid sequence set forth in SEQ ID NO: 12. Embodiment 80. The synthetic polynucleotide of any one of embodiments 71 to 73, 78, and 79, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:8. Embodiment 81. The synthetic polynucleotide of any one of embodiments 71 to 73 and 78 to 80, comprising the nucleic acid sequence set forth in SEQ ID NO:8. Embodiment 82 The synthetic polynucleotide of any one of Embodiments 71 to 73, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:22. Embodiment 83. The synthetic polynucleotide of any one of embodiments 71 to 73 and 82, comprising the nucleic acid sequence set forth in SEQ ID NO: 22. Embodiment 84. The synthetic polynucleotide of embodiments 71 to 73, 82, and 83, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 17. Embodiment 85. The synthetic polynucleotide of any one of embodiments 71 to 73 and 82 to 84, comprising the nucleic acid sequence set forth in SEQ ID NO: 17. Embodiment 86 The synthetic polynucleotide of any one of Embodiments 71 to 73, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:23. Embodiment 87. The synthetic polynucleotide of any one of embodiments 71 to 73 and 86, comprising the nucleic acid sequence set forth in SEQ ID NO: 23. Embodiment 88. The synthetic polynucleotide of any one of embodiments 71 to 73, 86, and 87, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:21. Embodiment 89. The synthetic polynucleotide of any one of embodiments 71 to 73 and 86 to 88, comprising the nucleic acid sequence set forth in SEQ ID NO: 21. Embodiment 90. A cell comprising the synthetic polynucleotide of any one of embodiments 71 to 89. Embodiment 91. The cell of embodiment 90, which is a metazoan cell. Embodiment 92. The cell of embodiment 90 or 91, which is a vertebrate cell. Embodiment 93. The cell of any one of embodiments 90 to 92, which is a mammalian cell. Embodiment 94. The cell of any one of embodiments 90 to 93, which is a primate cell. Embodiment 95. The cell of any one of embodiments 90 to 94, which is a human cell. Embodiment 96. The cell of any one of embodiments 90 to 95, which is a primary cell. Embodiment 97. The cell of any one of embodiments 90 to 95, which is a human embryonic kidney cell. Embodiment 98. The cell of embodiment 97, which is a 293 cell. Embodiment 99. The cell of any one of embodiments 90 to 98, produced by transducing said cell with a lentivirus comprising said synthetic polynucleotide. Embodiment 100. A cell according to any one of embodiments 90 to 99, comprising the synthetic fusion protein according to any one of embodiments 44 to 70. Embodiment 101. A vesicle comprising the synthetic fusion protein of any one of embodiments 44 to 70. Embodiment 102. The vesicle of embodiment 101, which is an exosome. Embodiment 103. The vesicle of embodiment 101 or 102, having a diameter of about 50 to 500 nm. Embodiment 104. The vesicle of any one of embodiments 101 to 103, wherein a SARS-CoV-2 spike protein polypeptide is expressed on the outer surface of the vesicle. Embodiment 105. The vesicle of any one of embodiments 101 to 103, which expresses a SARS-CoV-2 nucleocapsid protein polypeptide on its surface. Embodiment 106. The vesicle of any one of embodiments 101 to 103, which expresses an influenza hemagglutinin protein polypeptide on its surface. Embodiment 107. The vesicle of any one of embodiments 101 to 103, which expresses a respiratory syncytial virus fusion protein polypeptide on its surface. Embodiment 108. A method for producing a vesicle according to any one of embodiments 101 to 107, comprising culturing a cell according to any one of embodiments 90 to 100 in a cell culture medium, collecting the cell culture medium, and purifying a plurality of vesicles comprising the vesicles from the cell culture medium. Embodiment 109. The method of embodiment 108, further comprising the step of inducing expression of the synthetic polynucleotide of any one of embodiments 71 to 89 to produce the synthetic fusion protein of any one of embodiments 44 to 70. Embodiment 110 The method of embodiment 105, wherein the inducing step comprises contacting the cells with tetracycline, doxycycline, or an analog thereof. Embodiment 111 The method of embodiment 105, wherein the inducing step comprises removing tetracycline, doxycycline, or an analog thereof from the cells. Embodiment 112. A method of eliciting an immune response in a subject, comprising: A plurality of vesicles, including a vesicle according to any one of embodiments 101 to 107 or a vesicle made by the method according to any one of embodiments 108 to 111. administering to the subject a first dose of an immunogenic composition comprising: wherein the synthetic fusion protein according to any one of embodiments 44 to 70 is expressed on the outer surface of the vesicle. The method. Embodiment 113 The method of embodiment 112, further comprising administering a second dose of the immunogenic composition to the subject a period of time after administering the first dose. Embodiment 114. The method of embodiment 113, wherein the period is 14 days to 1 year. Embodiment 115. The method of any one of embodiments 112 to 114, wherein the first dose or the second dose comprises between about 100 μL and 1 mL of the immunogenic composition, and the immunogenic composition comprises between about 0.3 ng / mL and 3 μg / mL of the synthetic fusion protein. Embodiment 116. The method of any one of embodiments 112 to 115, wherein the first dose or the second dose comprises between about 100 μL and 1 mL of an immunogenic composition containing between about 2E9 vesicles / mL and 3E13 vesicles / mL. Embodiment 117 The method of any one of embodiments 112 to 115, wherein the synthetic fusion protein comprises a SARS-CoV-2 spike protein polypeptide. Embodiment 118 The method of any one of embodiments 112 to 117, wherein the synthetic fusion protein comprises a SARS-CoV-2 nucleocapsid protein polypeptide. Embodiment 119 The method of any one of embodiments 112 to 117, wherein the synthetic fusion protein comprises an influenza hemagglutinin protein polypeptide. Embodiment 120 The method of any one of embodiments 112 to 117, wherein the synthetic fusion protein comprises a respiratory syncytial virus fusion (RSV F) protein polypeptide. Embodiment 121. The method of any one of embodiments 112 to 120, wherein the immunogenic composition comprises a vesicle expressing a SARS-CoV-2 spike protein polypeptide on its outer surface, a vesicle expressing an influenza hemagglutinin protein polypeptide on its outer surface, and a vesicle expressing a RSV F protein polypeptide on its outer surface. Embodiment 122 The method of any one of embodiments 112 to 121, wherein the immune response elicited comprises producing neutralizing antibodies against an antigen present in the synthetic fusion protein. Embodiment 123 The method of any one of embodiments 112 to 123, wherein the elicited immune response comprises producing anti-spike antibodies. Embodiment 124 The method of any one of embodiments 112 to 123, wherein the elicited immune response comprises a spike-specific T cell response. Embodiment 125 The method of any one of embodiments 112 to 124, wherein the elicited immune response comprises producing anti-nucleocapsid antibodies. Embodiment 126 The method of any one of embodiments 112 to 125, wherein the elicited immune response comprises a nucleocapsid-specific T cell response. Embodiment 127 The method of any one of embodiments 112 to 126, wherein the elicited immune response comprises producing anti-hemagglutinin antibodies. Embodiment 128 The method of any one of embodiments 112 to 127, wherein the elicited immune response comprises a hemagglutinin-specific T cell response. Embodiment 129 The method of any one of embodiments 112 to 128, wherein the elicited immune response comprises producing anti-RSV F antibodies. Embodiment 130. The method of any one of embodiments 112 to 129, wherein the elicited immune response comprises a RSV F-specific T cell response. Embodiment 131 The method of any one of embodiments 112 to 130, wherein the immune response persists in the subject for up to 9 months. Embodiment 132 The method of any one of embodiments 112 to 130, wherein the immune response persists in the subject for at least 9 months.

[0117] Extracellular vesicles and exosomes A variety of host cells are known in the art and are suitable for protein expression and extracellular vesicle production. Non-limiting examples of typical cells used for transfection include, but are not limited to, bacterial cells, eukaryotic cells, yeast cells, insect cells, or plant cells. For example, human embryonic kidney 293 (HEK293), Escherichia 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. For a description of recombinant proteins in 293 cells in suspension culture, 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.

[0118] Extracellular vesicles (EVs) are lipid-bound vesicles secreted by cells into the extracellular space. Three major subtypes of EVs are microvesicles (MVs), exosomes, and apoptotic bodies, which are distinguished based on their biogenesis, release pathway, size, contents, and function. For a review of extracellular vesicles, see, for example, 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.

[0119] Exosomes, in particular, include small secretory vesicles approximately 20-200 nm in diameter that are released by mammalian cells and are generated either by budding into endosomes or from the cell's plasma membrane. In some cases, exosomes have a characteristic buoyant density of approximately 1.1-1.2 g / mL and a characteristic lipid composition. The lipid membrane is typically cholesterol-rich and contains sphingomyelin, ceramide, lipid rafts, and exposed phosphatidylserine. Exosomes express certain marker proteins, such as integrins and cell adhesion molecules, but generally lack lysosomal, mitochondrial, or caveolar markers. In some embodiments, exosomes contain cell-derived components, including, but not limited to, proteins, DNA, and RNA (e.g., microRNAs [miRs] and non-coding RNAs). In some embodiments, exosomes can be obtained from cells obtained from sources that are allogeneic, autologous, xenogeneic, or syngeneic to the exosome recipient.

[0120] Certain types of RNA, such as microRNAs (miRNAs), are known to be transported by exosomes. miRNAs often function as post-transcriptional regulators by binding to complementary sequences on target messenger RNA transcripts (mRNAs), thereby resulting in translational repression, target mRNA degradation, and / or gene silencing.

[0121] Useful exosomes can be obtained from any cellular source, including prokaryotic, plant, fungal, metazoan, vertebrate, mammalian, primate, human, autologous, 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 can be derived from mesenchymal stem cells, embryonic stem cells, iPS cells, immune cells, PBMCs, neural stem cells, HEK293 cells, as described in, for example, Dumont et al., "Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives," Crit Rev Biotechnol 2016;36:1110-22, HEK293T cells, as described in, for example, Li et al., "Identification and characterization of 293T cell-derived exosomes by profiling the protein, mRNA, and MicroRNA components," PLoS One 2016;11:e0163043, and 293F cells (Stenkamp et al., "Exosomes represent a novel mechanism of regulatory T cell suppression (P1079)," J Immunol May 1, 2013, 190(1 Supplementary Note 121.11), amniocytes, CAR-T cells, e.g., cardiospheres and cardiosphere-derived cells (CDCs), etc., as described in WO2014028493, WO2022006178A1, US20210032598A1, US9828603B2, EP2914273A1, US20200316226A1, US20120315252A1, US20170360842A1, and references therein.

[0122] Briefly, methods for preparing exosomes can include culturing cells in a medium, isolating the cells from the medium, e.g., by serial centrifugation, and purifying the exosomes, optionally using a density gradient, e.g., a sucrose density gradient. In some cases, the isolated and purified exosomes are essentially free of non-exosome components, e.g., cellular components or whole cells. The exosomes can be resuspended in a buffer, e.g., sterile PBS buffer containing 0.01-1% human serum albumin. The exosomes can be frozen and stored for future use.

[0123] Exosomes can be collected, concentrated, and / or purified using methods known in the art. For example, differential centrifugation has become the primary technique for isolating secreted exosomes from the supernatant of cultured cells. This approach leverages size to separate exosomes from larger extracellular vesicles and most non-particulate contaminants. Exosomes can be prepared as described in numerous publications, 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 al., "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 al., "Higher-order oligomerization targets plasma membrane proteins and HIV gag to exosomes," PLoS Biol., 5:el58 (2007). Exosomes can be isolated using commercially available kits, including, but not limited to, ExoSpin™ Exosome Purification Kit, Invitrogen® Total Exosome Purification Kit, PureExo® Exosome Isolation Kit, and ExoCap™ Exosome Isolation Kit. Methods for isolating exosomes from stem cells can be found, for example, in Tan et al., Journal of Extracellular Vesicles, 2:22614 (2013); Ono et al., Sci Signal, 7(332):ra63 (2014), and U.S. Application Publication Nos. 2012 / 0093885 and 2014 / 0004601.Methods for isolating exosomes from cardiosphere-derived cells can be found, for example, in Ibrahim et al., "Exosomes as critical agents of cardiac regeneration triggered by cell therapy," Stem Cell Reports, 2014. Specific methodologies include ultracentrifugation, density gradients, HPLC, affinity-based attachment to substrates, or size-exclusion-based filtration.

[0124] Size exclusion allows for the separation of biochemically similar but biophysically distinct microvesicles with relatively large diameters up to 1,000 nm. Differences in flotation velocity further allow for the separation of exosomes of different sizes. Typically, exosomes range in diameter from 30 to 200 nm, e.g., 40 to 100 nm. Further purification can rely on the specific properties of the particular exosome of interest. This includes, for example, the use of immunoadsorption with a protein of interest to select for specific vesicles with exoplasmic or outward orientation.

[0125] Among current methods, such as differential centrifugation, discontinuous density gradients, immunoaffinity, ultrafiltration, and high-performance liquid chromatography (HPLC), differential centrifugation is the most commonly used for exosome isolation. This technique utilizes increasing centrifugal forces from 2,000 x g to 10,000 x g to separate medium-sized particles and cellular debris from the exosome pellet at 100,000 x g. Centrifugation alone allows for substantial separation / collection of exosomes from conditioned medium, but may be insufficient to remove common contaminants such as various protein aggregates, genetic material, medium-derived particulates, and cellular debris. Enhanced specificity of exosome purification can be achieved by utilizing continuous centrifugation combined with ultrafiltration, or equilibrium density gradient centrifugation on a sucrose density gradient to provide higher purity of exosome preparations (buoyant density 1.1–1.2 g / mL), or by applying a discontinuous sugar cushion to the preparation.

[0126] Ultrafiltration can be used to purify exosomes without compromising biological activity. Membranes with different pore sizes, e.g., a 100 kDa molecular weight cutoff (MWCO), and gel filtration to exclude smaller particles have been used to avoid the use of non-neutral pH or non-physiological salt concentrations. Currently available tangential flow filtration (TFF) systems are scalable (up to over 10,000 L) and allow for the purification and concentration of exosome fractions, with such approaches requiring less time than differential centrifugation. HPLC can also be used to purify exosomes into more uniformly sized particle preparations and preserve biological activity, as the preparation is maintained at physiological pH and salt concentration. Other chemical methods utilize differences in exosome solubility for precipitation techniques, the addition of volume-exclusion polymers (e.g., polyethylene glycol (PEG)), and additional rounds of centrifugation or filtration, which may be combined. For example, the precipitation reagent ExoQuick® can be added to conditioned cell culture media to rapidly precipitate exosome populations, but resuspension of pellets prepared via this technique can be difficult.Flow field-flow fractionation (FlFFF) is an elution-based technique that has been used to separate and characterize macromolecules (e.g., proteins) and nano- to micro-sized particles (e.g., organelles and cells) and has been successfully applied to fractionate exosomes from culture media.

[0127] In addition to these techniques that rely on common biochemical and biophysical features, focused techniques can be applied to isolate specific exosomes of interest. These include relying on antibody immunoaffinity to recognize certain exosome-associated antigens. As noted, exosomes also express the extracellular domains of membrane-bound receptors on their surface. This provides an opportunity to isolate and separate exosomes based on shared antigen profiles and in relation to their parental cellular origin. Conjugation to magnetic beads (e.g., anti-CD81 magnetic beads), chromatography matrices, plates, or microfluidic devices allows for the isolation of specific exosome populations of interest, which may be related to their production from the parental cell of interest or to associated cellular regulatory states. Other affinity capture methods use lectins that bind to specific carbohydrate residues on the exosome surface.

[0128] For example, exosomes (and other extracellular vesicles) can be produced via 293F cells transfected with (or transduced with a lentivirus carrying) a polynucleotide encoding the spike protein, or nucleocapsid protein, or influenza hemagglutinin protein described herein, or a chimeric fusion thereof (see FIG. 4A), and capable of expressing the spike protein or nucleocapsid protein such that the spike protein or nucleocapsid is sorted and presented in exosomes isolated therefrom. An exemplary procedure for producing exosomes derived from 293F cells may include the following steps: 293F cells (Gibco™, catalog number 51-0029, ThermoFisher Scientific, Waltham, MA) may be pathogen-tested and found to be free of viral (cytomegalovirus, human immunodeficiency virus I and II, Epstein-Barr virus, hepatitis B virus, and parvovirus B19) and bacterial (mycoplasma) contamination. Cells may be maintained in FreeStyle™ 293 Expression Medium (Gibco, catalog number 12338-018, ThermoFisher Scientific, Waltham, MA) and incubated at 37°C in 8% CO2. For exosome production, 293F cells may be seeded into shaker flasks at a density of 1.5E6 cells / ml in approximately one-quarter of the flask volume and grown at a shaking speed of approximately 110 rpm. HEK293 cells can be grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum.

[0129] To purify exosomes, 293F cells can be cultured in shaker flasks for 3 days. Cells and large cell debris can be removed by centrifugation at 300 x g for 5 minutes, followed by 3,000 x g for 15 minutes. The resulting supernatant can be passed through a 0.22 μm sterile filter unit (Thermo Fisher, catalog number 566-0020) to produce clarified tissue culture supernatant (CTCS). CTCS can be concentrated by centrifugal filtration (Centricon Plus-70, Ultracel-PL Membrane, 100 kDa size exclusion, Millipore Sigma, catalog number UFC710008, St. Louis, MO) to reduce the volume of approximately 120 mL of CTCS to approximately 0.5 mL. The enriched CTCS can then be purified by size-exclusion chromatography (SEC) (qEV Original Column / 35 nm: Izon Science, Catalog No. SP5) in 1x PBS. The exosomes present in each 0.5 mL starting sample can be eluted into three 0.5 mL fractions. The purified exosomes can be re-enriched using an Amicon® Ultra-4 100 kDa cutoff spin column (Cat. No. UFC810024). This process yields a population of exosomes / small EVs with the expected ultrastructural and size distribution profile of human exosomes and containing the exosome marker proteins CD9 and CD63, resulting in a 500-fold enrichment effect at a final concentration of 1E10–2E12 exosomes / mL. The concentration and size of the isolated extracellular vesicles can be measured using a NANOSIGHT nanoparticle tracking analysis system (Malvern Panalytical, Malvern, UK).

[0130] SARS-CoV-2 Proteins Membrane-bound vesicles containing one or more populations of SARS-CoV-2 structural proteins are disclosed. By "containing," we mean that the contained proteins are within the lumen of the vesicle, displayed on the surface of the vesicle, or both within the lumen and on the surface. Here, fusion proteins containing tetraspanin protein polypeptide sequences are primarily displayed on the surface of the vesicle. These proteins displayed on the surface of the vesicle can have a portion of the protein within the lumen, a portion of the protein spanning the membrane of the vesicle (i.e., a transmembrane region or domain), and a portion of the protein extending outside the vesicle. In one embodiment, the SARS-CoV-2 structural protein is the spike glycoprotein (S), nucleocapsid (N), membrane (M), or 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, for example, Figures 1A, 1B, 2A, and 2C, which show spike / CD9 and nucleocapsid / CD9 fusion proteins for expression of spike or nucleocapsid antigens on the surface of exosomes.

[0131] In one embodiment, the antigenic protein is the SARS-CoV-2 spike glycoprotein (also known as spike protein or simply "spike"). The spike protein can be of any variant of SARS-CoV-2, such as the Wuhan-1 strain, an omicron variant (e.g., a BA.2 variant), a delta variant (e.g., a B.1.617.2, AY.3, AY.103, AY.44, AY.43 variant, etc.), and an epsilon variant (e.g., a B.1.427 or B.1.429 variant), or any currently known or undiscovered variant. As used herein, the term spike refers to any SARS-CoV-2 spike glycoprotein, chimera, or fragment thereof, unless otherwise specified.

[0132] In some embodiments, the SARS-CoV-2 spike protein is a 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; a Wuhan-1 strain SARS-CoV-2 spike protein comprising an amino acid change of D614G; a Wuhan-1 strain SARS-CoV-2 spike protein comprising a diproline substitution of 986KV987→986PP987 (S-2P); and / or a Wuhan-1 strain SARS-CoV-2 spike protein comprising a cleavage site mutation of 682RRAR685→682GSAG685, or equivalents (S-CSM).

[0133] Extracellular vesicles displaying viral proteins In one embodiment, the present invention provides extracellular vesicles that express (also known as "displaying") a spike protein or nucleocapsid on their surface, useful as vaccines against multiple variants of SARS-CoV-2. In one embodiment, the present invention provides extracellular vesicles that express (also known as "displaying") a hemagglutinin protein or neuraminidase protein on their surface, useful as vaccines against multiple variants of influenza. In one embodiment, the present invention provides a combination of vesicles, some of which display SARS-CoV-2 proteins and some of which display influenza proteins. The spike protein can be a delta variant having any one or more of a trimer-stabilizing mutation, a prefusion conformation-stabilizing mutation (e.g., a diproline-stabilizing mutation), and a furin cleavage site mutation.For example, 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.

[0134] Here, we generated synthetic fusion proteins containing a C-terminal tetraspanin protein and either the SARS-CoV-2 spike protein (Figures 1A and 1B), the SARS-CoV-2 nucleocapsid protein (Figures 2A and 2B), or the influenza hemagglutinin protein. It is important to note that the nucleocapsid protein is a soluble protein that is not expressed on the viral surface; therefore, engineered fusion proteins containing tetraspanins and other transmembrane domains allow for the localization of the nucleocapsid protein to the vesicle surface, providing a readily accessible antigen for immunization (see Figure 2B).

[0135] In one embodiment, exosomes expressing spike or nucleocapsid proteins on their surface were generated from 293F cells expressing spike / nucleocapsid proteins. Referring to Figure 4A, in a specific exemplary embodiment, packaging cells (300) were transfected with a plasmid (301a) encoding a spike-, nucleocapsid-, or hemagglutinin-tetraspanin fusion protein and plasmids (302a and 303a) encoding lentiviral structural proteins. Lentiviral proteins were produced (302b and 303b), which incorporated the fusion protein RNA, forming a lentiviral vector (304) containing the fusion protein RNA. Host cells (311) were transduced with lentivirus (304) carrying the fusion protein RNA, which allowed the SARS-CoV-2 spike or nucleocapsid-tetraspanin fusion protein (307a) to be produced and sorted to the plasma membrane (307b) (Figure 4B) and the surface of exosomes produced by the transduced host cells (Figures 4C, 5A, 5B, and 6C).

[0136] Referring to Figures 5A-C and 6A and 6B, exosomes were isolated from 293F cells harboring the spike-CD9 construct (Figures 1A and 1B). Figure 5A shows the size distribution of these exosomes, ranging from approximately 50 nm to approximately 270 nm, with a median of approximately 100-150 nm. Figure 5B shows the expression of the spike-containing fusion protein in transduced 293F cells (lane 4) and the enriched expression / presentation of the spike-containing fusion protein in exosomes derived from transduced 293F cells (lane 5). Figure 5C shows significant expression of the spike-containing fusion protein in these exosomes, as determined by flow cytometry.

[0137] Transmission electron microscopy confirmed that spike protein was expressed on the surface of exosomes derived from spike-CD9-transduced 293F cells (arrows in Figures 5A and 5B point to spikes on the surface of the vesicles).

[0138] Referring to Figures 7A-C, exosomes were isolated from 293F cells harboring the nucleocapsid-CD9 construct (Figures 2A and 2B). Figure 7A shows the size distribution of these exosomes, ranging from approximately 50 nm to approximately 270 nm, with a median of approximately 100-150 nm. Figure 7B shows the expression of the nucleocapsid-containing fusion protein in exosomes derived from transduced 293F cells (lane 5). Figure 7C shows significant expression of the spike-containing fusion protein in these exosomes, as determined by spike flow cytometry. In one embodiment, expression of the nucleocapsid-CD9 fusion protein was placed under the control of a tet-inducible promoter. Here, exosomes derived from transduced but uninduced host cells did not express nucleocapsid (see the second curve from the left in Figure 7C).

[0139] Referring to Figures 8A-8D, 293F cells were transfected or transduced with constructs or virions encoding hemagglutinin (e.g., H3)-CD9 constructs (Figures 3A and 3B). Figures 8A and 8B show significant expression of influenza H3-containing fusion proteins (approximately 90%-96% in this example) in adherent and suspension 293F cells, respectively, as determined by anti-H3 flow cytometry. Exosomes produced by 293F cells expressing the H3 fusion protein were purified and assessed for H3 and H3-CD9 by Western blot (Figure 8C, panels 1 and 2, respectively). Dual detection of both H3 and CD90 just below 180 kDa in lanes 6 of panels 1 and 2 indicates significant loading of the H3-CD9 fusion protein in 293F-derived exosomes. Furthermore, Figure 8D shows significant expression of spike-containing fusion proteins in these exosomes, as determined by spike flow cytometry. In one embodiment, exosomes produced from 293F cells expressing influenza H3-CD9 fusion proteins were combined with micron-sized magnetic beads coupled with anti-human CD81 (an exosome marker) and subjected to flow cytometry. Approximately 54% of CD81-expressing exosomes express influenza H3 antigen (Figure 8D).

[0140] Referring to Figure 9, on day 1, subject mice were administered vesicles containing a 1x dose (i.e., 3E10 vesicles) or a 10x dose (i.e., 3E11 vesicles), or about 0.3 ng, about 3 ng, or about 10 ng of fusion protein, comprising a spike-CD9 fusion protein, a nucleocapsid-CD9 fusion protein, or an influenza H3-CD9 fusion protein, via intramuscular injection (710). On day 14, blood was collected from the administered mice and assessed for initial humoral immune responses (720) (see Figures 10A and 12A-F). On day 21, subject mice received a second dose of spike- or nucleocapsid-expressing exosomes of the invention (730). On day 35, blood was collected from the administered mice and assessed for humoral immune responses, including antibody production (see Figures 10B and 11A-C) and neutralizing antibody production (740). On day 40, splenocytes were collected from the administered mice and assessed for cellular immune responses via ELISpot assay (750) (see Figures 10C, 10D, 13C, and 13D).

[0141] Referring to Figures 10A and 10B, humoral immune responses to COVID-19 spike antigen-expressing exosomes (STX-S) were induced in subjects as early as two weeks after the first injection (Figure 10A) and persisted until at least day 35 post-injection (Figure 10B). Furthermore, robust and sustained anti-spike antibody responses were elicited using nanogram amounts of spike protein expressed on exosomes without the inclusion of an adjuvant. Here, for example, as little as 10 ng of exosome-expressed spike protein induced significant and useful antibody responses measured on days 14 and 35 without an adjuvant. Furthermore, 32 ng of exosome-expressed spike protein induced a more than 30-fold greater antibody response without an adjuvant than 32 ng of purified spike protein with or without an adjuvant (exosome-less). It is generally known in the art that prior art spike protein vaccines are administered with adjuvants in microgram amounts to generate antibody responses similar to those observed with nanogram amounts of the exosome-expressed spike protein antigen of the present invention.

[0142] Referring to Figures 10C and 10D, immune responses to STX-S at the cellular level were induced in subjects, as shown in ELISpot assays performed on splenocytes obtained on days 35-40. Here, a dose of as little as 10 ng of adjuvant-less exosome-expressed spike antigen induced a significant CD4+ T cell response to the spike protein in subjects, as evidenced by an IL4 ELISpot assay of splenocytes from the subjects (Figure 10C). Here, doses of 10 ng and 32 ng of adjuvant-less exosome-expressed spike antigen also induced a significant CD8+ cytotoxic T cell response to the spike protein in subjects, as evidenced by an IFNγ ELISpot assay of splenocytes from the subjects (Figure 10D).

[0143] Referring to Figures 11A-C, administration of nanogram amounts of STX-S to subjects induced potent neutralization of both the delta and omicron variants of SARS-CoV-2 induced by STX-S exosome injection. In one embodiment, day 40 serum from a subject administered approximately 3.2 ng of STX-S per injection (dose 2, Figure 11A) and day 14 and day 40 serum from a subject administered approximately 9.8 ng of STX-S per injection (dose 4, Figure 11A) were tested for neutralizing antibodies against the SARS-CoV-2 delta variant. Here, STX-S induced potent neutralizing activity in each sample tested, comparable to SARS-CoV-2 delta-positive vaccine sera (Figure 11A). STX-S showed dose-dependent neutralization of the virus, as estimated by its ability to protect infected cells from virus-induced cytopathic effects (compare dose 4 with dose 2 in serum on day 40, Figure 11A).

[0144] Thus, according to one embodiment, the STX-S engineered exosome vaccine induced neutralizing antibodies against the delta spike in subjects by day 14 (after a single im injection). By day 40, approximately four weeks after the STX-S boost, robust neutralization was observed in all subjects, regardless of dose (Figure 11A). Here, a single injection of approximately 9 ng of STX-S spike (dose 4, Figure 11A) resulted in approximately 65-75% neutralization against the delta variant. Furthermore, full immunization (i.e., a primary injection and at least one booster, or two im injections) resulted in approximately 80-85% neutralization against the delta variant with a dose of approximately 3-9 ng of spike delivered by STX-S exosomes.

[0145] Delta variant neutralization induced in subjects by administration of STX-S induced responses comparable to human control plasma (CoV02-delta, plasma from a patient immunized with Moderna's mRNA vaccine who developed a breakthrough Delta infection), with full neutralization responses induced at higher dilutions (e.g., 1:320, see day 40 of STX-S dose 4) (Figure 11A).

[0146] Further, referring to Figures 11B and 11C, at a dose of approximately 9-10 ng of STX-S (where the spike protein of STX-S is of the delta variant, STX-S δ Day 40 serum samples from subjects receiving STX-S were also tested for neutralizing antibodies against SARS-CoV-2 Omicron variants (Omicron BA.1 and BA.5.2.1). As shown in Figures 11B and 11C, STX-S δ Strong cross-neutralization was observed for sera obtained from subjects treated with STX-S exosomes, in which approximately 84% neutralization of the Omicron BA1 variant (Figure 11B) and a range of 16% to 97% neutralization of the Omicron BA5 variant (Figure 11C) were achieved after full immunization (i.e., at least two im injections) with an approximately 9-10 ng dose of delta spike delivered by STX-S exosomes. Thus, in some embodiments, administration of immunogenic compositions containing single-variant spike STX-S exosomes provides some level of protective immunity against other SARS-CoV-2 variants.

[0147] Because such small amounts of antigen-expressing exosomes (nanogram amounts 1,000-fold less than standard protein or subunit vaccines) are required to induce immunity as measured by neutralization, antibody titers, and IL4 / IFNγ, it is possible to combine exosomes expressing specific, distinct antigens to provide a composite vaccine or immunogenic composition. For example, SARS-CoV-2 antigen-expressing exosomes (e.g., STX-S) can be combined with influenza antigen-expressing exosomes (e.g., hemagglutinin, e.g., H3).

[0148] Referring to Figures 12A-F, a vaccine combination containing both STX-S and exosomes (STX-H3) expressing influenza hemagglutinin 3 antigen on their surface was administered to mouse subjects. Serum obtained from mice 14 days after injection of a low dose of STX-H3 alone (Figures 12A-C, second and third bars from the left) shows significant levels of anti-H3 IgG. As with the STX-S and STX-N dose sizes (Figures 13A-D below), the lower doses (3E10-3E11 exosomes expressing low nanogram amounts of H3 antigen) elicited significant IgG responses in mice.

[0149] Low nanogram amounts of STX-S and STX-H3 were combined and administered to mouse subjects. Day 14 serum obtained from these mice showed significant induction of IgG against both the H3 antigen (Figures 12A-C, fourth bar from the left) and the spike antigen (Figures 12D-F, right bar). Therefore, it is possible to combine low nanogram doses of viral antigen-expressing exosomes to create a combination vaccine. Here, for example, nanogram amounts of STX-S and STX-H3 were combined to create an immunogenic composition capable of eliciting potent antibody responses against both SARS-CoV-2 and influenza.

[0150] Similar nanogram doses of adjuvant-less exosomes 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 (e.g., influenza hemagglutinin, influenza neuraminidase, respiratory syncytial virus, and / or other viral glycoproteins or other proteins or fragments thereof) expressed on the surface of exosomes according to the present disclosure will elicit humoral and / or cell-mediated immune responses in recipient subjects.

[0151] Thus, in some embodiments, 1 ng to 1 μg (inclusive) of viral antigen (e.g., SARS-CoV-2 spike, SARS-CoV-2 nucleocapsid, or influenza hemagglutinin, or a combination thereof), 1 to 900 ng, 1 to 800 ng, 1 to 700 ng, 1 to 600 ng, 1 to 500 ng, 1 to 400 ng, 1 to 300 ng, 1 to 200 ng, 1 to 300 ng, 1 to 400 ng, 1 to 500 ng, 1 to 600 ng, 1 to 700 ng, 1 to 800 ng, 1 to 900 ng, 1 to 1000 ng, 1 to 1200 ng, 1 to 1400 ng, 1 to 16 ng, 1~100ng, 1~90ng, 1~80ng, 1~70ng, 1~60ng, 1~50ng, 1~40ng, 5~100ng, 5~90ng, 5~80ng, 5~70ng, 5~6 0ng, 5~50ng, 5~40ng, 100μg or less, 50μg or less, 25μg or less, 20μg or less, 10μg or less, 1μg or less, 900ng or less, 800ng or less, 700ng or less, 60 0ng or less, 500ng or less, 400ng or less, 300ng or less, 200ng or less, 100ng or less, 90ng or less, 80ng or less, 70ng or less, 60ng or less, 50ng or less, 40ng Below, about 1ng, about 1.5ng, about 2ng, about 2.5ng, about 3ng, about 3.5ng, about 4ng, about 4.5ng, about 5ng, about 6ng, about 7ng, about 8ng, about 9ng, about 10ng, about 11n Low dose immunogenic compositions or vaccines containing an immunogenic dose of about 100 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) are provided.

[0152] Referring to Figures 13A and 13B, humoral immune responses to COVID-19 nucleocapsid antigen-expressing exosomes (STX-N) were induced in subjects and detected 35 days after injection (Figures 13A and 13B). Furthermore, robust and sustained anti-spike antibody responses were elicited using nanogram amounts of spike protein expressed on exosomes without the inclusion of an adjuvant. Here, for example, as little as 3.2 ng of exosome-expressed nucleocapsid protein elicited a significant beneficial antibody response measured at 35 days without an adjuvant (Figure 13A, STX-N dose 2). Thus, in one embodiment, a complete immunization cycle (two im injections) induced a significant increase in IgG against the SARS-CoV-2 nucleocapsid protein (N) compared to PBS, 3-fold for a dose of approximately 3 ng / injection (Dose 2, Figure 9A) and as much as 10-fold for 10 ng / injection (Dose 3, Figure 13B).

[0153] Referring to Figures 13C and 13D, to characterize T cell responses to STX-N, antigen-specific T cell responses to nucleocapsid protein were measured by ELISpot assay performed on splenocytes obtained on day 40. Here, vaccination of subjects with STX-N induced polyfunctional antigen-specific T cell responses to SARS-CoV-2 nucleocapsid protein on day 40 (after the boost (second) injection). STX-N administration resulted in a strong IFNγ response (Figures 13C and 13D). Here, evaluation of cells secreting IFNγ in response to ex vivo nucleocapsid protein stimulation showed a 6-fold increase in spleens immunized with STX-N (Figures 13C and 13D), thereby suggesting a Th1-biased CD8+ T cell response. Because the nucleocapsid protein is not a surface protein and is most likely presented to immune cells only after the infecting virus has been processed within the infected cell, this response is crucial for the development of immunogenic compositions expressing exosomes that have the potential to protect against a wide range of SARS-CoV-2 variants.

[0154] Similar to the humoral antibody responses against the nucleocapsid protein (Figures 13A and 13B), in this specific embodiment, administration of doses of STX-N in the range of 3 to 10 ng, approximately three orders of magnitude lower than prior art protein subunit vaccines, resulted in significant CD8+ / IFNγ responses against the nucleocapsid protein in subjects without an adjuvant.

[0155] In addition to eliciting superior immune responses, spike-exosome vaccines, nucleocapsid-exosome vaccines, hemagglutinin-exosome vaccines, and RSV-exosome vaccines each offer several other advantages over currently available vaccines. First, the exosome-based vaccines of the present invention deliver antigens through a fully internalized, self-assembled lipid bilayer, which can easily integrate into host cell membranes and facilitate presentation of engineered antigens to immune cells. Membrane-bound antigens are easily presented to circulating immune cells and can rapidly activate responses, while free antigens contained within exosomes can be further processed by the lysosomal system and activate cytotoxic T lymphocyte responses. Therefore, the use of natural delivery systems promotes efficient delivery and responses compared to synthetic lipid nanoparticle technology.

[0156] Second, spike exosome vaccines, nucleocapsid exosome vaccines, RSV exosome vaccines, and hemagglutinin exosome vaccines are protein-based vaccines, and therefore antigens are readily available to the subject's immune system without the translation in host cells required for RNA-based vaccines. Incomplete translation and / or incorrect folding of the mRNA encoding the spike protein, nucleocapsid protein, or hemagglutinin protein limits the amount of antigen available after vaccination, thus resulting in highly variable immune responses and reduced efficacy.

[0157] Third, the exosome-based spike fusion protein vaccine, nucleocapsid fusion protein vaccine, hemagglutinin fusion protein vaccine, and RSV fusion protein vaccine of the present invention do not require adjuvants or synthetic lipid nanoparticles (LNPs) for delivery and immune response. Traditional protein-based vaccines require adjuvants (e.g., aluminum salts and squalene oil-in-water emulsion systems MF59 (Novartis) and AS03 (GlaxoSmithKline)) to enhance and coordinate immune responses and influence the affinity, specificity, magnitude, and functional profile of B cell and T cell responses (see, e.g., Wong, S.S., and R.J. Webby, "Traditional and new influenza vaccines," Clin Microbiol Rev, 2013.26(3):476-92). Current mRNA vaccines do not use adjuvants, which, combined with the need for mRNA translation before antigens become available, may reduce long-term efficacy. Currently approved COVID vaccines use LNPs to deliver mRNA or adjuvants for proteins, and unwanted side effects have been reported.

[0158] Fourth, the immunogenic compositions of spike fusion proteins, nucleocapsid fusion proteins, hemagglutinin fusion proteins, and RSV fusion proteins of the present invention exhibit greater immunogenic efficacy at doses significantly lower, i.e., three orders of magnitude lower, than currently available LNP mRNA and protein vaccines. For example, clinically approved protein candidate vaccines use approximately 5µg to 25µg of antigen along with an adjuvant to induce immunization (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.e1003769; 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 antibody and CD4 T cell responses after a single immunization in (See mice. EBioMedicine, 2021. 63:103197; and references therein.) Here, immunological data from subjects immunized with either spike-fused exosomes or nucleocapsid-fused exosomes demonstrate that approximately 1 / 1000th the amount of protein antigen currently administered in clinically approved vaccines (i.e., nanogram amounts versus microgram amounts) elicits complete immunization correlated with high antibody levels, potent virus neutralization, broad multivariate activity, and both B cell and T cell memory.

[0159] Fifth, a further limitation of available vaccines is the need for refrigeration and the short shelf life of the product at elevated temperatures. Due to their high stability at physiological pH and temperature, exosomes containing display exosomes can be stored at 4°C for extended periods of time. Exosome compositions expressing spike fusions or nucleocapsid fusions may be lyophilized for long-term storage at convenient temperatures (see, e.g., U.S. Patent Application No. 2017 / 0360842 A1).

[0160] Thus, here, when spike protein, nucleocapsid protein, or hemagglutinin protein was delivered via exosomes, it induced not only a strong CD8+ T cell response but also a strong B cell response, as evidenced by IgG production and potential neutralizing antibodies. Without wishing to be bound by theory, this result may be explained by the role of extracellular vesicles in cell-to-cell communication and antigen presentation. Specifically, multiple copies of spike protein (Figures 6A and 6B), nucleocapsid protein, or hemagglutinin protein may be present on the vesicle surface, facilitating cross-linking to B cell receptors. Furthermore, the spike protein of extracellular vesicle-based vaccines may indirectly activate B cells and CD8(+) T cells through antigen cross-presentation.

[0161] It is further contemplated that the fusion protein-expressing exosomes of the present invention can be engineered to express an antigen of interest to target new COVID variants and / or problematic influenza strains. The antigen of interest can be easily exchanged and adapted to meet needs. To enable presentation of the antigen in exosomes for delivery to the host subject's immune system, the antigen can be expressed as a fusion protein (e.g., chimera) comprising, for example, an exosomal expression or presentation domain fused to an antigen domain.

[0162] It is further envisioned that exosomes can be genetically engineered to selectively target organs or tissues of interest and to allow for safe and targeted delivery of antigens to specific immune subsystems to elicit specific types of responses in a subject.

[0163] Cellular and exosomal characterization of STX-S and STX-H3 Referring to Figure 14A, high expression of spike on the cell surface was detected by flow cytometry (right curve). Unengineered parental 293F cells, as expected, do not express the SARS-CoV-2 spike protein.

[0164] Referring to Figure 14B, Jess automated Western blot confirmed enrichment of spike protein in exosomes (lane 5: STX-S exo), where 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.

[0165] Referring to Figure 15A, high expression of influenza hemagglutinin 3 (H3) on the cell surface was detected by flow cytometry (right curve). The parental unengineered 293F cells (left curve) do not express influenza H3 protein, as expected.

[0166] Referring to Figure 15B, Jess automated Western blot confirmed enrichment of H3 protein in exosomes (lane 5: STX-H3 exo), where lane 1: marker, lane 2: non-engineered 293F cells, lane 3: non-engineered 293F exosomes, lane 4: STX-H3 cells, and lane 5: STX-H3 exo.

[0167] Referring to FIG. 16A, CD81 was detected in STX exosomes by flow cytometry using a bead assay (right curve), whereas there was no signal from an isotype control antibody (left curve).

[0168] Referring to FIG. 16B, spike protein was detected in STX-S exosomes (right curve) but not in 293F parental exosomes (left curve).

[0169] Referring to FIG. 16C, H3 protein was detected in STX-H3 exosomes (right curve) but not in 293F parental exosomes (left curve).

[0170] Figures 17-19 show the immune response to intramuscular injection of a combination vaccine of spike-expressing exosomes (STX-S) and H3-expressing exosomes (STX-H3) (combined as STX-S+H3) in a mouse model. Here, mice received an injection 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 obtained on day 40. The STX-S+H3 vaccine induced robust expression of antibodies against influenza H3 and SARS-CoV-2 spike in mice after the first (day 14) and second (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 T cell responses against both H3 and spike after in vitro stimulation, as demonstrated by IFNg ELISPOT in 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=non-significant, one-way ANOVA, where N=10 / experimental group.

[0171] Referring to Figure 17A, IgG against H3 was observed on day 14 after a single IM injection.

[0172] Referring to Figure 17B, IgG against the spike was observed on day 14 after a single IM injection.

[0173] Referring to Figure 18A, IgG against H3 was observed on day 35 after full immunization (two IM injections).

[0174] Referring to Figure 18B, IgG against the spike was observed on day 35 after full immunization (two IM injections).

[0175] Referring to Figure 19A, an IFNg response to H3 in vitro stimulation was observed.

[0176] Referring to Figure 19B, the IFNg response to spike in vitro stimulation was observed.

[0177] Multivalent vaccine against covid19, influenza, and / or respiratory syncytial virus Respiratory syncytial virus-F-expressing vesicles In particular, various constructs of the RSV F protein were constructed to find a stable native conformation of the F protein that confers robust and stable immunity to subjects when displayed on the surface of vesicles such as exosomes. Figure 20 shows five construct versions of RSV F. RSV F version 1 ("V1") is a DS-Cav1 engineered variant 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") is a V1 DS-Cav1 engineered variant in which the polyadenylation signal in the F gene has been removed. RSV F version 3 ("V3") is a genetically engineered V2 variant with both furin cleavage sites mutated, as described by 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") is an engineered version of V2 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, in which the N-terminal most furin cleavage site has been deleted. A specific version of the V4 construct is exemplified in SEQ ID NO:20.

[0178] Various engineered RSV constructs were expressed in cells to determine protein stability, expression levels, and exosome loading. The V1 construct was observed to exhibit low RSV F protein expression in multiple transduced 293F cell lines. Cells that exhibited high RSV F expression (approximately 10%) were sorted by FACS, but 50% of these cells lost expression within one week. The V2 construct, in which the polyadenylation signal spanning the F gene was removed, was also observed to be poorly expressed in transduced 293F cell lines.

[0179] The V3 construct, in which both furin cleavage sites were deleted, showed a high percentage (approximately 96-97%) of full-length RSV F protein and high expression levels in 293F-transduced cells. However, V3 expression was observed to be unstable. Cells transfected with RSV F V3-CD9 chimeras with the top 10% of RSV F expression were FACS-sorted. CD9 and RSV F protein expression was measured 1 and 2 weeks after FACS sorting. Both the unsorted and sorted pools showed a loss of RSV F protein expression (using anti-AM22), while CD9 expression remained high, indicating a loss of V3 expression. 293F-generated exosomes produced from the sorted pools showed very little or no RSV F on the exosomes.

[0180] Furthermore, RSV F V3-CD9-expressing exosomes were tested for in vivo humoral immune responses, and mice showed low antibody responses to the RSV F protein on day 35 after two injections (Figure 22, panels A and B). Two lots of exosomes expressing the V3 RSV F-CD9 chimeric fusion protein were used: Lot #1, which produced 2.0E12 exosomes per mL at a protein concentration of approximately 43 ng / mL, and Lot #2, which produced 1.8E12 exosomes per mL at a protein concentration of approximately 26 ng / mL. Lot #1 (2 mL) and Lot #2 (1.3 mL) were combined to obtain a combined lot #1.2 (3.3 mL) with a final concentration of 25.27 ng / mL. Ten mice received two injections of approximately 3 ng / injection (using a 100 μl stock). Although antibodies were detected 14 days (FIG. 22A) and 35 days (FIG. 22B) after injection, the immune response was not sufficient to induce good humoral immunity.

[0181] The V4 construct is TIFF2025532934000002.tif3128 was observed to have improved expression levels and stability (compared to V3) in transduced 293F cells as determined by anti-RSV F flow cytometry using antigenic site antibodies (AM22, D25) and two RSV neutralization site II monoclonal antibodies (palivizumab, motavizumab), as well as JESS Western blotting.

[0182] RSV F V4-CD9-expressing exosomes were tested for in vivo humoral immune responses and were observed to induce strong immune responses on days 14 and 35 (Figure 23, panels A and B), with a clear boosting effect observed on day 35 (Figure 23B). Here, the RSV concentration on the exosomes was approximately 250 μg / mL, and the total protein was 990 μg / mL per 1.9E12 exosomes / mL.

[0183] [Table 1]

[0184] In some embodiments, the RSV F protein is fused with a tetraspanin protein. In a specific embodiment, the RSV F protein is fused with CD9, i.e., is part of a genetically engineered chimeric protein with CD9. Here, the genetically engineered chimeric protein contains an N-terminal RSV F polypeptide, preferably the RSV F V4 construct (SEQ ID NO: 18), followed by a linker, and then a C-terminal CD9 protein (FIG. 21A) that allows the RSV F protein to be expressed on the outer surface of the vesicle (FIG. 21B). In a specific embodiment, the RSV F-CD9 chimeric construct has the amino acid sequence of SEQ ID NO: 20.

[0185] Combination vaccines In one embodiment, a vaccine or immunogenic composition of the invention contains a combination of SARS-CoV2 spike-expressing vesicles and influenza hemagglutinin-expressing vesicles (Figure 24). In another embodiment, a vaccine or immunogenic composition of the invention contains a combination of SARS-CoV2 spike-expressing vesicles and respiratory syncytial virus fusion protein (RSV F)-expressing vesicles (Figure 25). In another embodiment, a vaccine or immunogenic composition of the invention contains a combination of influenza hemagglutinin-expressing vesicles and respiratory syncytial virus fusion protein (RSV F)-expressing vesicles (Figure 26). In another embodiment, a vaccine or immunogenic composition of the invention contains a combination of SARS-CoV2 spike-expressing vesicles, RSV F-expressing vesicles, and influenza hemagglutinin-expressing vesicles (Figure 27).

[0186] 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) was injected into mice either as a single formulation or in combination with each other to verify the induction of antibody responses. For spike and H3, 10-15 ng of protein was used for immunization. For RSV, 130 ng of RSV protein was used for immunization. Antibody responses were analyzed for all groups on day 14 (after one injection) (Figure 28) and day 35 (after a booster injection on day 21, full immunization) (Figure 29).

[0187] Here, we observed that injection of STX-S, STX-H3, and STX-RSV induced the production of virus-type-specific antibodies, both when injected as exosomes expressing a single virus antigen and when injected in combination with one or more other exosomes, each expressing a different virus antigen. A clear booster effect was also observed. Furthermore, no immune interference was observed when more than one exosome-virus species was injected.

[0188] All patents and other publications cited throughout this application, including, for example, references, issued patents, published patent applications, and co-pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing the methodologies described in such publications, which may 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 are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0189] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes; however, as those skilled in the relevant art will recognize, various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or may perform the functions substantially simultaneously. The teachings of the present disclosure provided herein may be applied to other approaches or methods, as appropriate. The various embodiments described herein may be combined to provide additional embodiments. Aspects of the present disclosure may be modified, as appropriate, to utilize the compositions, functions, and concepts of the above-mentioned references and applications to provide additional embodiments of the present disclosure. Furthermore, considerations of biological functional equivalence allow some changes to be made to protein structure without affecting the type or amount of biological or chemical action. These and other changes may be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0190] Specific elements of any of the above aspects may be combined with or substituted for elements in other aspects. Additionally, although advantages associated with certain aspects of the present disclosure have been described with respect to those aspects, other aspects may also exhibit such advantages, and not all aspects need exhibit such advantages to fall within the scope of the present disclosure.

Claims

1. An immunogenic composition comprising a first vesicle and a first fusion protein and a second vesicle and a second fusion protein, wherein the first fusion protein comprises a first viral polypeptide and an exosomal polypeptide, and the second fusion protein comprises a second viral 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 the first fusion protein comprises a first viral polypeptide and an exosomal polypeptide, the second fusion protein comprises a second viral polypeptide and an exosomal polypeptide, and the third fusion protein comprises a third viral polypeptide and an exosomal polypeptide.

3. 3. The immunogenic composition of claim 1 or claim 2, further comprising an excipient.

4. 4. The immunogenic composition of claim 3, wherein the excipient comprises a buffer.

5. 4. The immunogenic composition of claim 3, wherein the excipient comprises a cryoprotectant.

6. 3. The immunogenic composition of claim 1 or claim 2, which does not contain an adjuvant.

7. 3. The immunogenic composition of claim 1 or claim 2, wherein the first fusion protein and the second fusion protein are present in the membrane of their respective vesicles.

8. The immunogenic composition of claim 7, wherein some or all of each viral polypeptide is present at or on the outer surface of the vesicle.

9. 3. 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 μL to about 50 ng / 100 μL.

10. 3. The immunogenic composition of claim 1 or claim 2, wherein the first viral polypeptide is a SARS-CoV-2 polypeptide and the second viral polypeptide is an influenza polypeptide.

11. The immunogenic composition of claim 10, wherein the first viral polypeptide is a SARS-CoV-2 spike protein polypeptide.

12. The immunogenic composition of claim 10, wherein the first viral polypeptide is a SARS-CoV-2 delta variant spike protein polypeptide.

13. 12. The immunogenic composition of claim 11, wherein the first viral polypeptide comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:

1.

14. The immunogenic composition of claim 10, wherein the first viral polypeptide is a SARS-CoV-2 nucleocapsid protein polypeptide.

15. 15. The immunogenic composition of claim 14, wherein the first viral polypeptide comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:

5.

16. 11. The immunogenic composition of claim 10, wherein the second viral polypeptide is an influenza hemagglutinin protein polypeptide.

17. 11. The immunogenic composition of claim 10, wherein the second viral polypeptide is an influenza hemagglutinin 3 (H3) protein polypeptide.

18. 18. The immunogenic composition of claim 17, wherein the second viral polypeptide comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:

14.

19. The immunogenic composition of claim 2, wherein the third viral polypeptide is a respiratory syncytial virus (RSV) protein polypeptide.

20. The immunogenic composition of any one of claims 2 or 19, wherein the third viral polypeptide is a RSV fusion (RSV F) protein polypeptide.

21. 21. The immunogenic composition of claim 20, wherein the third viral polypeptide comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:

18.

22. The immunogenic composition of claim 1 or claim 2, wherein the exosome polypeptide is a tetraspanin protein polypeptide.

23. The immunogenic composition of claim 22, wherein the exosome polypeptide is a CD9 protein polypeptide.

24. 23. The immunogenic composition of claim 22, wherein the exosomal polypeptide comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:

10.

25. The immunogenic composition of any one of claims 1 to 13 and 17 to 24, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:

2.

26. 23. The immunogenic composition of claim 22, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:

3.

27. 11. The immunogenic composition of claim 10, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:

6.

28. 11. The immunogenic composition of claim 10, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:

7.

29. 11. The immunogenic composition of claim 10, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:

15.

30. 11. The immunogenic composition of claim 10, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:

16.

31. 21. The immunogenic composition of claim 20, wherein the third fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:

19.

32. 21. The immunogenic composition of claim 20, wherein the third fusion protein comprises an amino acid sequence that is 80% identical to or identical to SEQ ID NO:

20.

33. 3. The immunogenic composition of claim 1 or claim 2, wherein an immunogenic dose of the composition comprises about 1 ng to about 50 ng of the first fusion protein or the first viral polypeptide and about 1 ng to about 50 ng of the second fusion protein or the second viral polypeptide.

34. The immunogenic composition of claim 2, wherein an immunogenic dose of the composition comprises from about 1 ng to about 50 ng of the third fusion protein or the third viral polypeptide.

35. 10. A method of immunizing a subject against a viral infection, comprising administering to the subject an immunogenically effective dose of the immunogenic composition of claim 1 or claim 2.

36. 36. The method of claim 35, wherein the immunogenic effective dose induces protective immunity in the subject against the first virus and the second virus.

37. 36. The method of claim 35, wherein the immunogenic effective dose induces protective immunity in the subject against the first virus, the second virus, and the third virus.

38. 36. The method of claim 35, wherein the immunogenic composition comprises a SARS-CoV-2 spike protein polypeptide and an influenza hemagglutinin polypeptide, wherein the first virus is SARS-CoV2 and the second virus is an influenza virus.

39. 36. 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, wherein the first virus is SARS-CoV2, the second virus is an influenza virus, and the third virus is RSV.

40. 36. The method of claim 35, wherein the immunogenic effective dose induces protective immunity against SARS-CoV-2 delta variant and SARS-CoV-2 omicron variant in the subject.

41. 36. The method of claim 35, wherein the immunogenic effective dose comprises about 1 ng to about 50 ng of the first fusion protein, about 1 ng to about 50 ng of the second fusion protein, and about 1 ng to about 50 ng of the third fusion protein.

42. 36. 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. 36. The method of claim 35, wherein the protective immunity induced comprises: (a) high antibody titers against the first, second, and third viruses; (b) CD4+ T cell responses against the first, second, and third viruses; and (c) CD8+ cytotoxic T cell responses against the first, second, and third viruses.

44. A synthetic fusion protein comprising a viral polypeptide and an exosomal polypeptide.

45. 45. The synthetic fusion protein of claim 44, further comprising a linker polypeptide positioned between said viral polypeptide and said exosomal polypeptide.

46. 46. ​​The synthetic fusion protein of claim 45, further comprising a hinge polypeptide positioned between said viral polypeptide and said exosomal polypeptide.

47. 47. The synthetic fusion protein of claim 46, further comprising a transmembrane domain polypeptide positioned between said viral polypeptide and said exosomal polypeptide.

48. 45. The synthetic fusion protein of claim 44, wherein the exosomal polypeptide is a tetraspanin polypeptide.

49. 49. The synthetic fusion protein of any one of claims 44 or 48, wherein said exosomal polypeptide is a CD9 polypeptide.

50. 45. The synthetic fusion protein of claim 44, wherein the viral polypeptide is a SARS-CoV-2 structural protein polynucleotide.

51. 51. The synthetic fusion protein of claim 44 or 50, wherein the viral polypeptide is a SARS-CoV-2 spike protein polypeptide.

52. 52. The synthetic fusion protein of claim 51, wherein said SARS-CoV-2 spike protein polypeptide comprises one or more of a furin cleavage site mutation (CSM[682RRAR685→682GSAG685]) and a diproline substitution (2P[986KV987→986PP987]).

53. 52. 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. 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. 55. The synthetic fusion protein of claim 53 or 54, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:

3.

56. 51. The synthetic fusion protein of claim 44 or 50, wherein the viral polypeptide is a SARS-CoV-2 nucleocapsid protein polypeptide.

57. 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. 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. 59. The synthetic fusion protein of claim 57 or 58, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:

7.

60. 46. ​​The synthetic fusion protein of claim 44 or 45, wherein the viral polypeptide is an influenza protein polypeptide.

61. 61. The synthetic fusion protein of claim 60, wherein said influenza protein polypeptide comprises hemagglutinin.

62. 62. The synthetic fusion protein of claim 61, wherein said influenza protein polypeptide comprises hemagglutinin 3 (H3).

63. 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. 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. 64. The synthetic fusion protein of claim 63, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:

16.

66. 46. ​​The synthetic fusion protein of claim 44 or 45, wherein the viral polypeptide is a respiratory syncytial virus (RSV) protein polypeptide.

67. 67. The synthetic fusion protein of claim 66, wherein the RSV protein polypeptide comprises a RSV fusion (RSV F) protein.

68. The synthetic fusion protein of claim 66, wherein the fusion protein comprises, in order from the amino terminus to the carboxy terminus, a RSV F protein polypeptide, a linker polypeptide, and a CD9 polypeptide.

69. 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. 69. The synthetic fusion protein of claim 68, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:

20.

71. 45. A synthetic polynucleotide encoding the synthetic fusion protein of claim 44.

72. 72. The synthetic polynucleotide of claim 71, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:

13.

73. 72. The synthetic polynucleotide of claim 71, comprising the nucleic acid sequence set forth in SEQ ID NO:

13.

74. 72. The synthetic polynucleotide of claim 71, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:

11.

75. 72. The synthetic polynucleotide of claim 71, comprising the nucleic acid sequence set forth in SEQ ID NO:

11.

76. 72. The synthetic polynucleotide of claim 71, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:

4.

77. 72. The synthetic polynucleotide of claim 71, comprising the nucleic acid sequence set forth in SEQ ID NO:

4.

78. 72. The synthetic polynucleotide of claim 71, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:

12.

79. 72. The synthetic polynucleotide of claim 71, comprising the nucleic acid sequence set forth in SEQ ID NO:

12.

80. 72. The synthetic polynucleotide of claim 71, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:

8.

81. 72. The synthetic polynucleotide of claim 71, comprising the nucleic acid sequence set forth in SEQ ID NO:

8.

82. 72. The synthetic polynucleotide of claim 71, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:

22.

83. 72. The synthetic polynucleotide of claim 71, comprising the nucleic acid sequence set forth in SEQ ID NO:

22.

84. 72. The synthetic polynucleotide of claim 71, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:

17.

85. 72. The synthetic polynucleotide of claim 71, comprising the nucleic acid sequence set forth in SEQ ID NO:

17.

86. 72. The synthetic polynucleotide of claim 71, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:

23.

87. 72. The synthetic polynucleotide of claim 71, comprising the nucleic acid sequence set forth in SEQ ID NO:

23.

88. 72. The synthetic polynucleotide of claim 71, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:

21.

89. 72. The synthetic polynucleotide of claim 71, comprising the nucleic acid sequence set forth in SEQ ID NO:

21.

90. 72. A cell comprising the synthetic polynucleotide of claim 71.

91. 91. The cell of claim 90, which is a metazoan cell.

92. 92. The cell of claim 91, which is a vertebrate cell.

93. 93. The cell of claim 92, which is a mammalian cell.

94. 94. The cell of claim 93, which is a primate cell.

95. 95. The cell of claim 94, which is a human cell.

96. 96. The cell of claim 90 or 95, which is a primary cell.

97. 96. The cell of claim 90 or 95, which is a human embryonic kidney cell.

98. 98. The cell of claim 97, which is a 293 cell.

99. 96. The cell of claim 90 or 95, produced by transducing said cell with a lentivirus comprising said synthetic polynucleotide.

100. 91. The cell of claim 90, comprising the synthetic fusion protein of claim 44.

101. 45. A vesicle comprising the synthetic fusion protein of claim 44.

102. 102. The vesicle of claim 101, which is an exosome.

103. 103. The vesicle of claim 101 or 102, having a diameter of about 50 to 500 nm.

104. The vesicle of claim 101 or 102, wherein the SARS-CoV-2 spike protein polypeptide is expressed on the outer surface of the vesicle.

105. The vesicle of claim 101 or 102, which expresses a SARS-CoV-2 nucleocapsid protein polypeptide on its surface.

106. 103. The vesicle of claim 101 or 102, which expresses an influenza hemagglutinin protein polypeptide on its surface.

107. 103. The vesicle of claim 101 or 102, which expresses a respiratory syncytial virus fusion protein polypeptide on its surface.

108. 102. A method for producing a vesicle according to claim 101, comprising culturing a cell according to claim 90 in a cell culture medium, collecting the cell culture medium, and purifying a plurality of vesicles comprising the vesicle from the cell culture medium.

109. 109. The method of claim 108, further comprising inducing expression of the synthetic polynucleotide of claim 71 to produce the synthetic fusion protein of claim 44.

110. 106. The method of claim 105, wherein said inducing step comprises contacting said cells with tetracycline, doxycycline, or an analog thereof.

111. 106. The method of claim 105, wherein said inducing step comprises removing tetracycline, doxycycline, or an analog thereof from said cells.

112. 1. A method of eliciting an immune response in a subject, comprising: A plurality of vesicles comprising a vesicle of claim 101 or a vesicle made by the method of claim 108. administering to the subject a first dose of an immunogenic composition comprising: wherein the synthetic fusion protein of claim 44 is expressed on the outer surface of the vesicle. The method.

113. 113. The method of claim 112, further comprising administering a second dose of the immunogenic composition to the subject a period of time after administering the first dose.

114. 114. The method of claim 113, wherein the period is from 14 days to 1 year.

115. 115. The method of claim 113 or 114, wherein the first dose or the second dose comprises about 100 μL to 1 mL of the immunogenic composition, wherein the immunogenic composition comprises about 0.3 ng / mL to 3 μg / mL of the synthetic fusion protein.

116. 115. The method of any one of claims 113 or 114, wherein the first dose or the second dose comprises about 100 μL to 1 mL of an immunogenic composition comprising about 2E9 vesicles / mL to 3E13 vesicles / mL.

117. 113. The method of claim 112, wherein said synthetic fusion protein comprises a SARS-CoV-2 spike protein polypeptide.

118. 113. The method of claim 112, wherein the synthetic fusion protein comprises a SARS-CoV-2 nucleocapsid protein polypeptide.

119. 113. The method of claim 112, wherein the synthetic fusion protein comprises an influenza hemagglutinin protein polypeptide.

120. The method of claim 112, wherein the synthetic fusion protein comprises a respiratory syncytial virus fusion (RSV F) protein polypeptide.

121. The method of claim 112, wherein the immunogenic composition comprises vesicles expressing a SARS-CoV-2 spike protein polypeptide on the outer surface, vesicles expressing an influenza hemagglutinin protein polypeptide on the outer surface, and vesicles expressing a RSV F protein polypeptide on the outer surface.

122. 122. The method of claim 112 or 121, wherein the immune response elicited comprises producing neutralizing antibodies against an antigen present in said synthetic fusion protein.

123. 122. The method of claim 112 or 121, wherein the immune response elicited comprises producing anti-spike antibodies.

124. The method of claim 112 or 121, wherein the immune response elicited comprises a spike-specific T cell response.

125. 122. The method of claim 112 or 121, wherein the immune response elicited comprises producing anti-nucleocapsid antibodies.

126. 122. The method of claim 112 or 121, wherein the immune response elicited comprises a nucleocapsid-specific T cell response.

127. 122. The method of claim 112 or 121, wherein the immune response elicited comprises producing anti-hemagglutinin antibodies.

128. 122. The method of claim 112 or 121, wherein the immune response elicited 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 immune response induced comprises a RSV F-specific T cell response.

131. 122. The method of claim 112 or 121, wherein the immune response persists in the subject for up to 9 months.

132. 122. The method of claim 112 or 121, wherein the immune response persists in the subject for at least 9 months.

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