Combination exosome immunogenic compositions and methods

Abstract

The present disclosure relates to compositions and methods for vaccinating a subject against a variety of SARS-CoV-2 variants and other respiratory viruses, the compositions and methods involve preparing and delivering extracellular vesicles expressing on its surface an engineered spike protein, an engineered nucleocapsid protein, an engineered hemagglutinin protein, and / or an engineered respiratory syncytial virus pre-fusion or fusion (RSV F) protein. The invention also relates to compositions and methods for designing, preparing, making, formulating and / or using spike display, nucleocapsid display, hemagglutinin display and / or RSV F display vesicle vaccines, the spike display, nucleocapsid display, hemagglutinin display, and / or RSV F display vesicle vaccines are designed to elicit potent humoral and cellular immune responses against a variety of respiratory viruses and variants.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of the priority dates of U.S. Provisional Application No. 63 / 412,226, filed on September 30, 2022, and U.S. Provisional Application No. 63 / 455,902, filed on March 30, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0003] Sequence Listing

[0004] This application includes a sequence listing that has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on September 27, 2023, is named 516PCTseqlst and is 49,339 bytes in size.

[0005] Background

[0006] The emergence, re-emergence, and mutation of severe respiratory viral infections, such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), influenza, and Respiratory Syncytial Virus (RSV), create an urgent need for the production of safe, effective, readily available and accessible vaccines that can be produced quickly and efficiently to counter the emergence of evolving variants. In the case of SARS-CoV-2, many vaccines have been developed that attempt to achieve immunity against the virus, mainly by directing the immune response towards the surface spike protein, which binds to the host cell receptor angiotensin-converting enzyme 2 (ACE2) and mediates viral entry and infection of cells. The spike (S) protein is a class I fusion glycoprotein, the major surface protein on the SARS-CoV-2 virus and the main target for neutralizing antibodies. The spike is also the major site of mutations identified in the SARS-CoV-2 virus, reducing the efficacy of the vaccine-induced immune response. Since the start of the COVID-19 pandemic, five Variants of Concern (VOCs) and eight Variants of Interest (VOIs) have been reported, posing challenges to current vaccines and creating a need for more effective vaccines. Vaccines against the SARS-CoV-2 nucleocapsid protein (the internal soluble coronavirus protein) have been largely unsuccessful to date.

[0007] Current leading vaccines have employed mRNA-lipid nanoparticle or viral vector technologies, and recent vaccines have utilized recombinant proteins (see, e.g., Krammer, F., SARS-CoV-2 vaccines in development. Nature, 2020. 586(7830): pp. 516-527). Compared to leading mRNA vaccines, recombinant or inactivated protein vaccines are safe and reliable immunization methods but generally suffer from weaker immunogenicity and thus need to be formulated with appropriate adjuvants. While all current candidate mRNA vaccines have achieved strong initial immune responses against the virus, thereby reducing hospitalization rates, they all lack long-term protection. It has been reported that antibody levels decline sharply over time and thus the efficacy of preventing infection also declines sharply (see, e.g., Xiang, T., et al., Declining Levels of Neutralizing Antibodies Against SARS-CoV-2 in Convalescent COVID-19 Patients One Year Post Symptom Onset. Front Immunol, 2021. 12: p. 708523). In addition, mRNA vaccines have thus far lacked cross-reactivity against new 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, faster, and more effective manufacturing and globally deployable vaccines for influenza, RSV, and SARS-CoV-2 that provide improved, broader, more durable neutralization, more robust T cell responses, and an enhanced safety profile against these viruses.

[0008] Briefly, problems that the present invention can relate to include the emergence of SARS-CoV-2 variants of concern that are more infectious and less immunogenic compared to some less-concerning variants. This creates ongoing public health and medical problems where vaccines are no longer effective against new strains and new strains with lower immunogenicity (both long-term and short-term immunogenicity).

[0009] This problem is similar to that of influenza, where new variants emerge each year or different variants pose problems in different seasons. Thus, there are problems with the development and delivery of combination vaccines (e.g., seasonal vaccines) against both SARS-CoV-2 variants and influenza variants.

[0010] A solution to this problem is disclosed, which includes a safe (LNP-free, adjuvant-free) and effective combination vaccine that confers long-term humoral and cell-mediated immunity against multiple, emerging, and stubborn variants of SARS-CoV-2, influenza, and / or RSV. This solution comprises: extracellular vesicles displaying the spike protein or nucleocapsid protein of a single variant of SARS-CoV-2, which confer robust humoral and / or cellular immunity against several SARS-CoV-2 variants of concern or interest; a combination of extracellular vesicles displaying the hemagglutinin protein of a single variant of influenza, which also confers immunity against influenza variants of interest, and / or a combination of extracellular vesicles displaying the RSV protein of a single variant of RSV, which also confers immunity against RSV variants of interest.

[0011] Overview

[0012] In one aspect, there is provided an immunogenic composition comprising a combination of two or more vesicles expressing viral antigenic substances. In one embodiment, the immunogenic composition comprises (i) more than one vesicle expressing a SARS-CoV-2 protein polypeptide or antigen on its surface, and (ii) more than one vesicle expressing an influenza protein polypeptide or antigen on its surface.

[0013] In another aspect, there is provided an immunogenic composition comprising a combination of two or more vesicles expressing viral antigenic substances. In one embodiment, the immunogenic composition comprises (i) more than one vesicle expressing a SARS-CoV-2 protein polypeptide or antigen on its surface, and (ii) more than one vesicle expressing an RSV protein polypeptide or antigen on its surface.

[0014] In another aspect, there is provided an immunogenic composition comprising a combination of two or more vesicles expressing viral antigenic substances. In one embodiment, the immunogenic composition comprises (i) more than one vesicle expressing an influenza protein polypeptide or antigen on its surface, and (ii) more than one vesicle expressing an RSV protein polypeptide or antigen on its surface.

[0015] In another aspect, there is provided an immunogenic composition comprising a combination of two or more vesicles expressing viral antigenic substances. In one embodiment, the immunogenic composition comprises (i) more than one vesicle expressing a SARS-CoV-2 protein polypeptide or antigen on its surface, (ii) more than one vesicle expressing an influenza protein polypeptide or antigen on its surface, and (iii) more than one vesicle expressing an RSV protein polypeptide or antigen on its surface.

[0016] In one embodiment of various immunogenic compositions, the SARS-CoV-2 protein is the spike glycoprotein, such as, for example, the SARS-CoV-2 Delta spike protein or an engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is the nucleocapsid protein or an engineered variant thereof. In one embodiment, the influenza protein is the hemagglutinin protein or an engineered variant of the hemagglutinin protein, such as, for example, the H3 glycoprotein or an engineered variant thereof. In another embodiment, the influenza protein is the neuraminidase protein or an engineered variant thereof. In one embodiment, the RSV protein is the RSV fusion (F) protein, such as, for example, the RSV F protein or the prefusion F protein (see, for example, Simoes et al., N Engl J Med 2022;386:1615-1626). In one embodiment, the vesicle is an exosome. In one aspect, the viral protein polypeptide or antigen is fused to an exosome protein, such as, for example, a tetraspanin, or more specifically, the CD9 protein or an engineered chimera containing CD9 and elements of other tetraspanins or transmembrane domains.

[0017] In some embodiments of the foregoing immunogenic compositions, the immunogenic composition contains more than one vesicle, and the more than one vesicle contains a synthetic fusion protein at a concentration of about 2E9 vesicles / mL - 3E13 vesicles / mL. In those embodiments where the immunogenic composition contains two or more types of vesicles (i.e., one type contains a first synthetic fusion protein and another type contains a second synthetic fusion protein, etc.), each vesicle type can be provided at a concentration of about 1E9 vesicles / mL - 2E13 vesicles / mL or 2E9 vesicles / mL - 3E13 vesicles / mL.

[0018] In some embodiments, the immunogenic composition contains more than one vesicle, and the vesicle contains a synthetic fusion protein at a concentration of about 0.3 ng / mL - 3 μg / mL. In those embodiments where the immunogenic composition contains two or more types of vesicles (i.e., one type contains a first synthetic fusion protein and another type contains a second synthetic fusion protein, etc.), each vesicle type can be provided at a fusion protein concentration of about 0.1 ng / mL - 1.5 μg / mL or about 0.3 ng / mL - 3 μg / mL.

[0019] In some embodiments, the immunogenic composition further contains one or more pharmaceutically acceptable excipients. In one embodiment, the immunogenic composition does not contain an adjuvant.

[0020] In some embodiments, the vesicles in more than one vesicle 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 the CD81 protein on their surface.

[0021] In one aspect, a method of eliciting an immune response in a subject is provided, the method comprising administering to the subject a dose of the immunogenic composition described in the foregoing aspects and embodiments. In some embodiments, more than one dose is administered to the subject, such as a second dose and / or a subsequent booster dose administered at a certain time period after the first dose. In some embodiments, the time period (e.g., between doses) is 14 days - 1 year.

[0022] In one embodiment, the dose comprises about 100 μL - 1 mL of the immunogenic composition. In some embodiments, the immunogenic composition contains about 0.1 ng / mL - 3 μg / mL of the synthetic fusion protein in a form expressed on the surface of the vesicles. In some embodiments, the immunogenic composition contains about 2.81E9 vesicles / mL - 2.81E13 vesicles / mL of the vesicles expressing the synthetic fusion protein on their surface.

[0023] In some embodiments, the immune response elicited in the subject is the production of neutralizing antibodies against a virus such as 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 (such as, for example, the Delta variant, the Omicron variant, or other variants of interest or concern that are now known or not yet discovered).

[0024] In some embodiments, the immune response elicited in the subject is the production of anti-spike antibodies. In some embodiments, the immune response elicited in the subject is the production of anti-nucleocapsid antibodies. In some embodiments, the immune response elicited in the subject is a spike-specific T cell response, such as a CD4+ response and / or a CD8+ response. In some embodiments, the immune response elicited in the subject is a nucleocapsid-specific T cell response, such as a CD4+ response and / or a CD8+ response.

[0025] In some embodiments, the immune response elicited in a subject is the production of anti-hemagglutinin antibodies. In some embodiments, the immune response elicited in a subject is a spike-specific T cell response, such as a CD4+ response and / or a CD8+ response. In some embodiments, the immune response elicited in a subject is a hemagglutinin-specific T cell response, such as a CD4+ response and / or a CD8+ response.

[0026] In some embodiments, the immune response elicited in a subject is the production of anti-RSV F antibodies. In some embodiments, the immune response elicited in a subject is a spike-specific T cell response, such as a CD4+ response and / or a CD8+ response. In some embodiments, the immune response elicited in a subject is an RSV F-specific T cell response, such as a CD4+ response and / or a CD8+ response.

[0027] In another aspect, a method for vaccinating a subject against influenza and SARS-CoV-2 is provided. In one embodiment, a composition is administered to the subject, the composition comprising (i) more than one vesicle expressing a SARS-CoV-2 protein polypeptide or antigen on its surface, and (ii) more than one vesicle expressing an influenza protein polypeptide or antigen on its surface. In one embodiment, the SARS-CoV-2 protein is a spike glycoprotein, such as, for example, the SARS-CoV-2 Delta variant spike protein or an engineered variant thereof. In another aspect, the SARS-CoV-2 protein is a nucleocapsid protein or an engineered variant thereof. In one embodiment, the influenza protein is a hemagglutinin protein, such as, for example, an H3 glycoprotein or an engineered variant thereof. In another embodiment, the influenza protein is a neuraminidase protein or an engineered variant thereof. In one embodiment, the vesicle is an exosome. In one embodiment, the viral protein polypeptide or antigen is fused to an exosome protein, such as, for example, a tetraspanin protein, or more specifically, a CD9 protein or an engineered chimera containing a CD9 and other tetraspanin proteins or transmembrane domains.

[0028] On the other hand, a method of vaccinating a subject against RSV and SARS-CoV-2 is provided. In one embodiment, a composition is administered to the subject, the composition comprising (i) more than one vesicle expressing a SARS-CoV-2 protein polypeptide or antigen on its surface, and (ii) more than one vesicle expressing an RSV protein polypeptide or antigen on its surface. In one embodiment, the SARS-CoV-2 protein is the spike glycoprotein, such as, for example, the SARS-CoV-2 Delta variant spike protein or an engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is the nucleocapsid protein or an engineered variant thereof. In one embodiment, the RSV protein is the F protein or the prefusion F protein or an engineered variant thereof. In one embodiment, the vesicle is an exosome. In one embodiment, the viral protein polypeptide or antigen is fused to an exosome protein, such as, for example, a tetraspanin protein, or more specifically, the CD9 protein or an engineered chimera containing CD9 and elements of other tetraspanin proteins or transmembrane domains.

[0029] On the other hand, a method of vaccinating a subject against influenza and RSV is provided. In one embodiment, a composition is administered to the subject, the composition comprising (i) more than one vesicle expressing an RSV protein polypeptide or antigen on its surface, and (ii) more than one vesicle expressing an influenza protein polypeptide or antigen on its surface. In one embodiment, the RSV protein is the F protein or the prefusion F protein or an engineered variant thereof. In one embodiment, the influenza protein is the hemagglutinin protein, such as, for example, the H3 glycoprotein or an engineered variant thereof. In another embodiment, the influenza protein is the neuraminidase protein or an engineered variant thereof. In one aspect, the vesicle is an exosome. In one aspect, the viral protein polypeptide or antigen is fused to an exosome protein, such as, for example, a tetraspanin protein, or more specifically, the CD9 protein or an engineered chimera containing CD9 and elements of other tetraspanin proteins or transmembrane domains.

[0030] On the other hand, a method for vaccinating a subject against influenza and RSV is provided. In one aspect, a composition is administered to the subject, the composition comprising (i) more than one vesicle expressing a SARS-CoV-2 protein polypeptide or antigen on its surface, (ii) more than one vesicle expressing an influenza protein polypeptide or antigen on its surface, and (iii) more than one vesicle expressing an RSV protein polypeptide or antigen on its surface. In one embodiment, the SARS-CoV-2 protein is the spike glycoprotein, such as, for example, the SARS-CoV-2 Delta variant spike protein or an engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is the nucleocapsid protein or an engineered variant thereof. In one embodiment, the influenza protein is the hemagglutinin protein, such as, for example, the H3 glycoprotein or an engineered variant thereof. In another embodiment, the influenza protein is the neuraminidase protein or an engineered variant thereof. In one embodiment, the RSV protein is the F protein or the prefusion F protein or an engineered variant thereof. In one embodiment, the vesicle is an exosome. In one embodiment, the viral protein polypeptide or antigen is fused to an exosome protein, such as, for example, a tetraspanin protein, or more specifically, the CD9 protein or an engineered chimera containing the CD9 and other tetraspanin proteins or transmembrane domain elements.

[0031] In one aspect, a synthetic fusion protein is provided, the synthetic fusion protein containing the polypeptide sequence of a SARS-CoV-2 protein fused to the polypeptide sequence of an exosome tetraspanin protein. The fusion protein is designed and prepared such that the SARS-CoV-2 protein antigen can be expressed on the surface of the exosome so as to elicit an immune response when administered to a subject.

[0032] In one embodiment, the SARS-CoV-2 protein is the spike protein or an engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is the nucleocapsid protein or an engineered variant thereof.

[0033] In some embodiments where the SARS-CoV-2 protein is the spike protein and the exosome tetraspanin protein is the CD9 protein, the spike protein polypeptide is located at the N-terminus of the CD9 protein polypeptide. In some cases, a linker peptide is located between the spike protein polypeptide and the CD9 protein polypeptide, as Figure 1A depicted.

[0034] In some embodiments where the SARS-CoV-2 protein is the nucleocapsid protein and the exosome tetraspanin protein is the 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, a hinge peptide, a transmembrane domain peptide, and a linker peptide are located between the spike protein polypeptide and the CD9 protein polypeptide, as Figure 2Adepicted in

[0035] In one aspect, there is provided a synthetic fusion protein comprising a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosomal tetraspanin protein. Such a fusion protein is designed and prepared such that the SARS-CoV-2 protein antigen can be expressed on the surface of the exosome so as to be able to elicit an immune response when administered to a subject.

[0036] In one embodiment, the SARS-CoV-2 protein is the spike protein or an engineered variant thereof. In another embodiment, the SARS-CoV-2 protein is the nucleocapsid protein or an engineered variant thereof.

[0037] In some embodiments where the SARS-CoV-2 protein is the spike protein (e.g., SEQ ID NO: 1) and the exosomal tetraspanin protein is the CD9 protein (SEQ ID NO: 10), the spike protein polypeptide is located at the N-terminus of the CD9 protein polypeptide. In some cases, a linker peptide is located between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO: 2), as Figure 1A depicted in

[0038] In some embodiments where the SARS-CoV-2 protein is the spike protein, the SARS-CoV-2 spike protein polypeptide contains one or more mutations, such as, for example, the furin cleavage site mutation (CSM[682RRAR685-to-682GSAG685]) and / or the diproline substitution (2P[986KV987-to-986PP987]). In a particular embodiment, the spike-containing fusion protein has the amino acid sequence listed in SEQ ID NO: 3.

[0039] In one aspect, there is provided a nucleic acid encoding a spike-CD9 fusion protein. In one embodiment, the nucleic acid encoding the spike-CD9 fusion protein has the nucleic acid sequence of SEQ ID NO: 4.

[0040] In some embodiments where the SARS-CoV-2 protein is the nucleocapsid protein (e.g., SEQ ID NO: 5) and the exosomal tetraspanin protein is the CD9 protein (e.g., SEQ ID NO: 10), 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, a hinge peptide, a transmembrane domain peptide (e.g., SEQ ID NO: 9) and a linker peptide are located between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO: 6), as Figure 2ADepicted in. In certain embodiments, the nucleocapsid-containing fusion protein has the amino acid sequence set forth in SEQ ID NO:7.

[0041] In one aspect, provided is a nucleic acid encoding a nucleocapsid-CD9 fusion protein. In one embodiment, the nucleic acid encoding the nucleocapsid-CD9 fusion protein has the nucleic acid sequence of SEQ ID NO:8.

[0042] In another aspect, provided is a synthetic fusion protein comprising a polypeptide sequence of an influenza protein fused to a polypeptide sequence of an exosomal tetraspanin protein. Such a fusion protein is designed and prepared such that the influenza protein antigen can be expressed on the surface of the exosome to elicit an immune response when administered to a subject.

[0043] In one embodiment, the influenza protein is a hemagglutinin protein or an engineered variant thereof. In certain embodiments, the influenza protein is a hemagglutinin 3 (H3) protein or an engineered variant thereof (e.g., SEQ ID NO:14).

[0044] 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 located at the N-terminus of the CD9 protein polypeptide. In some cases, a linker peptide is located between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO:15), as Figure 3A Depicted in. In certain embodiments, the H3-containing fusion protein has the amino acid sequence of SEQ ID NO:16.

[0045] In one aspect, provided is a nucleic acid encoding an H3-CD9 fusion protein. In one embodiment, the nucleic acid encoding the H3-CD9 fusion protein has the nucleic acid sequence of SEQ ID NO:17.

[0046] In one aspect, provided is a synthetic fusion protein comprising a polypeptide sequence of a respiratory syncytial virus (RSV) protein fused to a polypeptide sequence of an exosomal tetraspanin protein. Such a fusion protein is designed and prepared such that the RSV protein antigen can be expressed on the surface of the exosome to elicit an immune response when administered to a subject.

[0047] In one embodiment, the RSV protein is a prefusion (F) protein or an engineered variant thereof (e.g., SEQ ID NO:18).

[0048] In some embodiments where the RSV protein is the prefusion protein (RSV F) and the exosome tetraspanin protein is the CD9 protein (e.g., SEQ ID NO:10), the RSV F protein polypeptide is located at the N-terminus of the CD9 protein polypeptide. In some cases, a linker peptide is located between the spike protein polypeptide and the CD9 protein polypeptide (e.g., SEQ ID NO:19), as depicted in Figure 21A In certain embodiments, the RSV F-containing fusion protein has the amino acid sequence of SEQ ID NO:20.

[0049] In one aspect, a nucleic acid encoding an 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.

[0050] In one aspect, a polynucleotide encoding a synthetic fusion protein is provided, the synthetic fusion protein comprising a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosome tetraspanin protein. The encoded fusion protein is designed and prepared such that the SARS-CoV-2 protein antigen can be expressed in cells and sorted to the surface of exosomes so as to elicit an immune response when the exosomes are administered to a subject.

[0051] 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, for example, a furin cleavage site mutation (CSM[682RRAR685-to-682GSAG685]) and / or a di-proline substitution (2P[986KV987-to-986PP987]). In certain embodiments, the spike protein has an amino acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:1. In certain embodiments, the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:11.

[0052] In some embodiments where the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide and the encoded exosome tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded spike protein polypeptide is located at the N-terminus of the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some cases, a linker peptide is encoded by the polynucleotide to be located between the spike protein polypeptide and the CD9 protein polypeptide in the encoded synthetic fusion protein, as depicted in Figure 1ADepicted in. In certain embodiments, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO:10. In certain embodiments, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:13 or is substantially identical to SEQ ID NO:13. In certain embodiments, the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:3 or is substantially identical to SEQ ID NO:3. In certain 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 is substantially identical to SEQ ID NO:4.

[0053] In another embodiment, the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide. In certain embodiments, the nucleocapsid protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:5 or is substantially identical to SEQ ID NO:5. In certain embodiments, the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:12 or is substantially identical to SEQ ID NO:12.

[0054] In some embodiments where the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide and the encoded exosome tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded nucleocapsid protein polypeptide is located at the N-terminus of the encoded CD9 protein polypeptide. In some cases, the signal peptide is located at the N-terminus of the nucleocapsid protein polypeptide. In some cases, the hinge peptide, transmembrane domain peptide, and linker peptide are encoded by the polynucleotide to be located between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as Figure 2A Depicted in. In certain embodiments, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO:10. In certain embodiments, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ IDNO:13 or is substantially identical to SEQ ID NO:13. In certain embodiments, the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:7 or is substantially identical to SEQ ID NO:7. In certain embodiments, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:8 or is substantially identical to SEQ ID NO:8.

[0055] In another aspect, there is provided a polynucleotide encoding a synthetic fusion protein comprising a polypeptide sequence of an influenza protein fused to a polypeptide sequence of an exosomal tetraspanin protein. The encoded fusion protein is designed and prepared such that the influenza protein antigen can be expressed in cells and sorted onto the surface of exosomes so as to be able to elicit an immune response when the exosomes are administered to a subject.

[0056] 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 certain embodiments, the encoded H3 protein polypeptide has an amino acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:14. In certain embodiments, the polynucleotide encoding the H3 protein polypeptide has a nucleic acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:22. In one embodiment, the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide. In certain embodiments, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:10. In certain embodiments, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:13.

[0057] 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 located at the N-terminus of the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some embodiments, a linker peptide is encoded by the polynucleotide to be located between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as Figure 3A depicted. In certain embodiments, the H3-CD9 fusion protein has an amino acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:16. In certain embodiments, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:17.

[0058] In another aspect, there is provided a polynucleotide encoding a synthetic fusion protein comprising a polypeptide sequence of a respiratory syncytial virus (RSV) protein fused to a polypeptide sequence of an exosomal tetraspanin protein. The encoded fusion protein is designed and prepared such that the RSV protein antigen can be expressed in cells and sorted onto the surface of exosomes so as to elicit an immune response when the exosomes are administered to a subject.

[0059] In one embodiment, the encoded RSV protein polypeptide is a prefusion protein polypeptide (RSV F). In certain embodiments, the encoded RSV F protein polypeptide has an amino acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:18. In certain embodiments, the polynucleotide encoding the RSV F protein polypeptide has a nucleic acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:23. In one embodiment, the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide. In certain embodiments, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO:10. In certain embodiments, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:13.

[0060] In some embodiments, the encoded RSV F protein polypeptide is located at the N-terminus of the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some embodiments, a linker peptide is encoded by the polynucleotide to be located between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as Figure 21A depicted. In certain embodiments, the RSV F-CD9 fusion protein has an amino acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:20. In certain embodiments, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:21.

[0061] In one aspect, there is provided a cell comprising a polynucleotide encoding a synthetic fusion protein comprising a polypeptide sequence of SARS-CoV-2 fused to a polypeptide sequence of an exosomal tetraspanin protein. The encoded fusion protein is designed and prepared such that the SARS-CoV-2 protein antigen can be expressed in cells and sorted onto the surface of exosomes so as to elicit an immune response when the exosomes are administered to a subject.

[0062] In some embodiments in terms of cells, the cells are metazoan cells. In some embodiments, the cells are vertebrate cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are primate cells. In some embodiments, the cells are human cells. In some embodiments, the cells are primary cells. In some embodiments, the cells are established cell lines. In some embodiments, the cells are human embryonic kidney cells. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are 293F cells.

[0063] 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, for example, furin cleavage site mutations (CSM[682RRAR685-to-682GSAG685]) and / or diproline substitutions (2P[986KV987-to-986PP987]). In certain embodiments, the spike protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:1 or is substantially identical to SEQ ID NO:1. In certain embodiments, the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:11 or is substantially identical to SEQ ID NO:11.

[0064] In some embodiments where the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide and the encoded exosome tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded spike protein polypeptide is located at the N-terminus of the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some cases, a linker peptide is encoded by a polynucleotide to be located between the spike protein polypeptide and the CD9 protein polypeptide in the encoded synthetic fusion protein, as Figure 1A depicted. In certain embodiments, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO:10. In certain embodiments, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:13 or is substantially identical to SEQ ID NO:13. In certain embodiments, the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:3 or is substantially identical to SEQ ID NO:3. In certain 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 is substantially identical to SEQ ID NO:4.

[0065] In another embodiment, the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide. In certain embodiments, the nucleocapsid protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:5 or is substantially identical to SEQ ID NO:5. In certain embodiments, the polynucleotide encoding the spike protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:12 or is substantially identical to SEQ ID NO:12.

[0066] In some embodiments where the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide and the encoded exosome tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded nucleocapsid protein polypeptide is located at the N-terminus of the encoded CD9 protein polypeptide. In some cases, a signal peptide is located at the N-terminus of the nucleocapsid protein polypeptide. In some cases, a hinge peptide, transmembrane domain peptide, and linker peptide are encoded by the polynucleotide to be located between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as Figure 2A depicted in. In certain embodiments, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO:10. In certain embodiments, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical to SEQ IDNO:13 or is substantially identical to SEQ ID NO:13. In certain embodiments, the spike-CD9 fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:7 or is substantially identical to SEQ ID NO:7. In certain embodiments, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical to SEQ ID NO:8 or is substantially identical to SEQ ID NO:8.

[0067] In another aspect, a cell containing a polynucleotide encoding a synthetic fusion protein is provided, the synthetic fusion protein containing a polypeptide sequence of an influenza virus fused to a polypeptide sequence of an exosome tetraspanin protein. Such an encoded fusion protein is designed and prepared such that the influenza SARS-CoV-2 protein antigen can be expressed in the cell and sorted onto the surface of the exosome so that an immune response can be elicited when the exosome is administered to a subject.

[0068] In some embodiments in terms of cells, the cells are metazoan cells. In some embodiments, the cells are vertebrate cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are primate cells. In some embodiments, the cells are human cells. In some embodiments, the cells are primary cells. In some embodiments, the cells are established cell lines. In some embodiments, the cells are human embryonic kidney cells. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are 293F cells.

[0069] 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 certain embodiments, the encoded H3 protein polypeptide has an amino acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:14. In certain embodiments, the polynucleotide encoding the H3 protein polypeptide has a nucleic acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:22. In one embodiment, the encoded exosome tetraspanin protein polypeptide is a CD9 protein polypeptide. In certain embodiments, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:10. In certain embodiments, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:13.

[0070] In some embodiments where the encoded influenza protein polypeptide is a hemagglutinin 3 (H3) protein polypeptide and the encoded exosome tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded hemagglutinin protein polypeptide is located at the N-terminus of the CD9 protein polypeptide encoded in the encoded synthetic fusion protein. In some embodiments, a linker peptide is encoded by a polynucleotide to be located between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as Figure 3A depicted. In certain embodiments, the H3-CD9 fusion protein has an amino acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:16. In certain embodiments, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:17.

[0071] In another aspect, there is provided a cell comprising a polynucleotide encoding a synthetic fusion protein, the synthetic fusion protein comprising a polypeptide sequence of respiratory syncytial virus (RSV) fused to a polypeptide sequence of an exosomal tetraspanin protein. The encoded fusion protein is designed and prepared such that the RSV protein antigen can be expressed in the cell and sorted to the surface of exosomes so as to be able to elicit an immune response when the exosomes are administered to a subject.

[0072] In some embodiments in terms of the cell, 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 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.

[0073] In one embodiment, the encoded RSV protein polypeptide is a prefusion protein polypeptide (RSV F). In certain embodiments, the encoded RSV F protein polypeptide has an amino acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:18. In certain embodiments, the polynucleotide encoding the RSV F protein polypeptide has a nucleic acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:23. In one embodiment, the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide. In certain embodiments, the CD9 protein polypeptide has an amino acid sequence that is at least 80% identical to SEQ ID NO:10. In certain embodiments, the polynucleotide encoding the CD9 protein polypeptide has a nucleic acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:13.

[0074] In some embodiments, the encoded RSV F protein polypeptide is located at the N-terminus of the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some embodiments, a linker peptide is encoded by the polynucleotide to be located between the hemagglutinin protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as Figure 21A depicted. In certain embodiments, the RSV F-CD9 fusion protein has an amino acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:20. In certain embodiments, the polynucleotide encoding the spike-CD9 fusion protein has a nucleic acid sequence that is at least 80% identical or substantially identical to SEQ ID NO:21.

[0075] Attached photos

[0076] Figure 1A A linear cartoon of the SARS-CoV-2 spike CD9 fusion protein is depicted in an amino-terminal to carboxyl-terminal orientation from left to right, with a linker sequence located between the spike protein polypeptide and the CD9 protein polypeptide.

[0077] Figure 1B Depicted Figure 1A Cartoon of the SARS-CoV-2 spike CD9 fusion protein relative to the vesicle membrane, oriented from left to right, amino-terminal to carboxyl-terminal. The spike protein polypeptide spans the membrane once and its amino terminus is oriented on the outside of the vesicle. The linker is on the luminal side of the membrane. The CD9 protein polypeptide spans the membrane 4 times and its carboxyl terminus is oriented in the lumen of the vesicle.

[0078] Figure 2A A linear cartoon of the SARS-CoV-2 nucleocapsid CD9 fusion protein is depicted in an amino-terminal to carboxyl-terminal orientation from left to right, wherein the amino-terminal signal peptide is fused to the nucleocapsid protein polypeptide, the nucleocapsid protein polypeptide is fused to the hinge region peptide, the hinge region peptide is fused to the transmembrane domain peptide, the transmembrane domain peptide is fused to the linker peptide, and the linker peptide is fused to the CD9 protein polypeptide.

[0079] Figure 2B Depicted Figure 2A From left to right, a cartoon of the SARS-CoV-2 nucleocapsid CD9 fusion protein oriented from the amino terminus to the carboxyl terminus relative to the vesicle membrane. The nucleocapsid protein polypeptide with the amino terminal signal sequence is located on the outside (cytoplasmic or external side), the hinge sequence connects the nucleocapsid protein polypeptide to the transmembrane domain peptide, which spans the membrane and is in turn connected to the linker located in the cavity, and the linker is connected to the CD9 protein polypeptide, which spans the membrane 4 times and its carboxyl terminus is oriented in the cavity of the vesicle.

[0080] Figure 3A Depicted are linear cartoons of influenza hemagglutinin CD9 fusion proteins oriented from left to right in amino-terminal to carboxy-terminal orientation, with a linker sequence positioned between the spike protein polypeptide and the CD9 protein polypeptide.

[0081] Figure 3B Depicted Figure 3A Cartoon of influenza hemagglutinin CD9 fusion protein relative to the vesicle membrane, oriented from left to right, amino-terminal to carboxyl-terminal. The hemagglutinin protein polypeptide spans the membrane once and its amino-terminal end is oriented on the outside of the vesicle. The linker is located on the luminal side of the membrane. The CD9 protein polypeptide spans the membrane 4 times and its carboxyl-terminal end is oriented in the lumen of the vesicle.

[0082] Figure 4AIt is a flowchart depicting the components and steps for generating cells expressing a polypeptide fusion protein of spike, nucleocapsid, influenza hemagglutinin, or other antigenic proteins mediated by a lentiviral vector (304) using packaging cells (301) and host cells (311).

[0083] Figure 4B It is a histogram depicting the flow analysis of the relative fluorescence intensity of host cells expressing the spike protein on their surface.

[0084] Figure 5A It is a graph depicting the change in the concentration of exosomes expressing spike (number of exosomes / ml) with the diameter of exosomes in nanometers.

[0085] Figure 5B It is a Western blot stained for the SARS-CoV-2 spike protein. The first column from left to right depicts the lane loaded with size markers, the second column represents the lane loaded with protein from untransduced 293F (host cells), the third column represents the lane loaded with protein from exosomes derived from untransduced 293F cells, the fourth column represents the lane loaded with protein from 293F cells constitutively expressing the spike fusion protein, the fifth column represents the lane loaded with protein from exosomes derived from 293F cells constitutively expressing the spike fusion protein, and the sixth column represents the lane loaded with the spike fusion protein.

[0086] Figure 5C It is a histogram depicting the flow analysis of the relative fluorescence intensity of exosomes with spike expressed on their surface. The left curve represents exosomes derived from 293F cells not expressing the spike-CD9 fusion protein. The right curve represents exosomes derived from 293F cells expressing the spike-CD9 fusion protein.

[0087] Figure 6A It depicts a transmission electron micrograph of exosomes expressing the SARS-CoV-2 spike protein. The inset is a magnified view showing exosomes decorated with the spike protein. Arrows point to SARS-CoV-2 spikes around the exosomes.

[0088] Figure 6B It depicts a transmission electron micrograph of exosomes expressing the SARS-CoV-2 spike protein at a higher magnification. Arrows point to SARS-CoV-2 spikes around the exosomes.

[0089] Figure 7A It is a graph depicting the change in the concentration of exosomes expressing nucleocapsid (number of exosomes / ml) with the diameter of exosomes in nanometers.

[0090] Figure 7BIs a Western blot stained for SARS-CoV-2 nucleocapsid protein. The first column from left to right depicts the lane loaded with size markers, the second column represents the lane loaded with nucleocapsid protein, the third column represents the lane loaded with proteins from exosomes derived from untransduced 293F cells, the fourth column represents the lane loaded with proteins from untransduced 293F cells, and the fifth column represents the lane loaded with proteins from exosomes derived from 293F cells expressing nucleocapsid fusion protein.

[0091] Figure 7C Is a histogram depicting flow analysis of the relative fluorescence intensity of exosomes with or without nucleocapsid protein expressed on the 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 carrying a polynucleotide encoding nucleocapsid-CD9 fusion protein, the third curve represents exosomes derived from 293F cells expressing nucleocapsid-CD9 fusion protein, the smaller third curve represents exosomes derived from 293F cells expressing nucleocapsid-CD9 fusion protein under tet induction, and the larger third curve represents exosomes derived from 293F cells carrying CD9 knockout and expressing nucleocapsid-CD9 fusion protein.

[0092] Figure 8A and Figure 8B Is a histogram depicting flow analysis of the relative fluorescence intensity of 293F cells expressing or not expressing influenza H3 hemagglutinin protein on the surface. On each graph from left to right, the first curve represents unmodified 293F cells, and the second curve represents 293F cells carrying and expressing a polynucleotide encoding influenza H3-CD9 fusion protein. Figure 8A Represents adherent 293F cells. Figure 8B Represents 293F cells in suspension.

[0093] Figure 8C Is a Western blot stained for influenza H3 protein (inset 1) or CD9 (inset 2). For each graph, the first column from left to right depicts the lane loaded with size markers, the second column represents the lane loaded with proteins from unmodified 293F (host cell), the third column represents the lane loaded with proteins from exosomes derived from unmodified 293F cells, the fourth column represents the lane loaded with proteins from 293F cells expressing H3-CD9 fusion protein, and the fifth column represents the lane loaded with proteins from exosomes derived from 293F cells expressing H3-CD9 fusion protein. Each sample was loaded at 0.6 mg / ml total protein.

[0094] Figure 8DIt is a flow cytometry dot plot of exosomes expressing H3, depicting the change in CD81 surface expression with H3 surface expression. In this example, approximately 54.2% of the flowing exosomes express both CD81 and H3, thus showing that when fused with exosomal proteins such as the tetraspanin CD9, the hemagglutinin polypeptide or antigen is expressed at the vesicle surface.

[0095] Figure 9 It is a timeline depicting the process of generating an immune response by administering an immunogenic composition containing exosomes expressing the SARS-CoV-2 fusion protein on their surface.

[0096] Figures 10A - 10D . STX-S exosomes elicited a robust immune response with significantly lower antigen than the recombinant protein vaccine. Data are shown as mean ± SEM. **** p < 0.0005, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant, * One-way ANOVA; ## p < 0.01, # p < 0.05, # -Two-tailed t-test.

[0097] Figure 10A It is a histogram depicting the fold change in the serum antibody titer against the spike protein at a 1:100 dilution on day 14 in mice as a function of the dose of spike-expressing exosomes (in nanograms). The leftmost bar represents the PBS control; the second bar represents the 10 ng dose of spike-expressing exosomes (STX-S); the third bar represents the 32 ng dose of STX-S; the rightmost bar represents the serum from mice injected with a 32 ng dose of spike protein combined with an adjuvant.

[0098] Figure 10B It is a histogram depicting the fold change in the serum antibody titer against the spike protein at a 1:100 dilution on day 35 in mice as a function of the dose of spike-expressing exosomes (in nanograms). The leftmost bar represents the PBS control; the second bar represents the 10 ng dose of spike-expressing exosomes (STX-S); the third bar represents the 32 ng dose of STX-S; the rightmost bar represents the serum from mice injected with a 32 ng dose of spike protein combined with an adjuvant.

[0099] Figure 10CIt is a bar graph depicting an ELISpot assay showing the number of IL4-producing spots in response to the spike protein varying with the antigen dose. From left to right on the x-axis, the first bar represents the spike-free wells from PBS control splenocytes; the second bar represents the spike protein reactivity from PBS control splenocytes; the third bar represents the spike-free wells from splenocytes of subjects administered 10 ng STX-S; the fourth bar represents the spike protein reactivity from splenocytes of subjects administered 10 ng STX-S; the fifth bar represents the spike-free wells from splenocytes of subjects administered 32 ng STX-S; the sixth bar represents the spike protein reactivity from splenocytes of subjects administered 32 ng STX-S; the seventh bar represents the spike-free wells from splenocytes of subjects administered 32 ng spike protein with adjuvant; the eighth bar represents the spike protein reactivity from splenocytes of subjects administered 32 ng spike protein with adjuvant.

[0100] Figure 10D It is a bar graph depicting an ELISpot assay showing the number of IFNγ-producing spots in response to the spike protein varying with the antigen dose. From left to right on the x-axis, the first bar represents the spike-free wells from PBS control splenocytes; the second bar represents the spike protein reactivity from PBS control splenocytes; the third bar represents the spike-free wells from splenocytes of subjects administered 10 ng STX-S; the fourth bar represents the spike protein reactivity from splenocytes of subjects administered 10 ng STX-S; the fifth bar represents the spike-free wells from splenocytes of subjects administered 32 ng STX-S; the sixth bar represents the spike protein reactivity from splenocytes of subjects administered 32 ng STX-S; the seventh bar represents the spike-free wells from splenocytes of subjects administered 32 ng spike protein with adjuvant; the eighth bar represents the spike protein reactivity from splenocytes of subjects administered 32 ng spike protein with adjuvant.

[0101] Figure 11, Panels A - C: Line graphs depicting the percentage of SARS-CoV-2 neutralization varying with immune sera as the serum dilution increases. These graphs depict the production of neutralizing antibodies after STX-S injection. Data are shown as mean ± SEM. COV-02-Delta = plasma from patients with breakthrough SARS-CoV-2 Delta spike infection immunized with Moderna's mRNA vaccine. Dose 4 = 9.8 ng S per injection; Dose 2 = 3.2 ng S per injection.

[0102] Figure 11A .The STX-S vaccine produces strong neutralization against the SARS-CoV-2 Delta spike (B.1.617.2).

[0103] Figure 11B.The STX-S vaccine induces neutralization of SARS-CoV-2 spike Omicron BA.1.

[0104] Figure 11C .The STX-S vaccine induces neutralization of SARS-CoV-2 spike Omicron BA.5.2.1.

[0105] Figure 12, Figures A - F are histograms depicting the amount of anti-H3 (Figures A - C) and anti-spike (Figures D - F) antibodies (OD [Figures A and D], fold change [Figures B and E], or log10 titer [Figures C and F]) in sera on day 14 post-injection from subjects as a function of the immunogenic composition (STX-H3 or STX-H3 plus STX-S). Data are shown as mean ± SEM. * p < 0.05, *** p < 0.005, **** p < 0.001, ANOVA, corrected for multiple comparisons; ns = not significant.

[0106] Figure 12A is a histogram depicting the OD of anti-H3 IgG in sera on day 14 as a function of the dose of injection material, where the doses of injection material from left to right are PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3, and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).

[0107] Figure 12B is a histogram depicting the fold change in the level of anti-H3 IgG in sera on day 14 as a function of the dose of injection material, where the doses of injection material from left to right are PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3, and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).

[0108] Figure 12C is a histogram depicting the log10 titer of anti-H3 IgG in sera on day 14 as a function of the dose of injection material, where the doses of injection material from left to right are PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3, and a combination of STX-H3 and exosomes expressing SARS-CoV-2 spike (STX-S).

[0109] Figure 12DIs a histogram depicting the change in OD of serum anti-spike IgG on day 14 with the dose of the injection material, where the injection material doses from left to right are PBS control, and the combination of STX-H3 and exosomes expressing the SARS-CoV-2 spike (STX-S).

[0110] Figure 12E Is a histogram depicting the fold change in the level of serum anti-H3 IgG on day 14 with the dose of the injection material, where the injection material doses from left to right are PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3, and the combination of STX-H3 and exosomes expressing the SARS-CoV-2 spike (STX-S).

[0111] Figure 12F Figure E is a histogram depicting the change in log10 titer of serum anti-H3 IgG on day 14 with the dose of the injection material, where the injection material doses from left to right are PBS control, 3E10 exosomes expressing influenza H3 (STX-H3), 3E11 STX-H3, and the combination of STX-H3 and exosomes expressing the SARS-CoV-2 spike (STX-S).

[0112] Figure 13, Figures A - D. STX-N vaccine elicits a multivalent immune response. Data are shown as mean ± SEM. * p < 0.05, *** p < 0.005, **** p < 0.001, ANOVA, corrected multiple comparisons; ns = not significant. Dose 1 = 0.32 ng N per injection; Dose 2 = 3.2 ng N per injection; Dose 3 = 10 ng N per injection.

[0113] Figure 13A and Figure 13B : A histogram depicting the change in the anti-nucleocapsid antibody titer of serum on day 35 post-immunization with the dose. The STX-N vaccine induces moderate expression of SARS-CoV-2 nucleocapsid antibodies in two sample bins (N1 and N2). PBS is used as a vehicle control.

[0114] Figure 13C and Figure 13D : A histogram depicting the change in the number of anti-nucleocapsid IFNγ ELISpot positive wells of splenocytes on day 40 post-immunization with the dose.

[0115] Figure 14 depicts the expression of the SARS-CoV-2 spike protein on 293 cells.

[0116] Figure 14AIt is a histogram depicting the flow analysis of the relative fluorescence intensity of 293F cells expressing SARS-CoV2 spike on the surface. The left curve represents 293F cells not expressing the spike-CD9 fusion protein. The right curve represents 293F cells expressing the spike-CD9 fusion protein.

[0117] Figure 14B It is a Western blot stained for the SARS-CoV-2 spike protein. The first column from left to right depicts the lane loaded with size markers, the second column represents the lane loaded with protein from untransduced 293F (host cells), the third column represents the lane loaded with protein from exosomes derived from untransduced 293F cells, the fourth column represents the lane loaded with protein from 293F cells expressing the spike fusion protein, the fifth column represents the lane loaded with protein from exosomes derived from 293F cells expressing the spike fusion protein, and the sixth column represents the lane loaded with the spike fusion protein.

[0118] Figure 15A It is a histogram depicting the flow analysis of the relative fluorescence intensity of 293F cells expressing influenza hemagglutinin 3 (H3) on the surface. The left curve represents 293F cells not expressing the H3-CD9 fusion protein. The right curve represents 293F cells expressing the H3-CD9 fusion protein.

[0119] Figure 15B It is a Western blot stained for the influenza hemagglutinin 3 (H3) protein. The first column from left to right depicts the lane loaded with size markers, the second column represents the lane loaded with protein from untransduced 293F (host cells), the third column represents the lane loaded with protein from exosomes derived from untransduced 293F cells, the fourth column represents the lane loaded with protein from 293F cells expressing the H3 fusion protein, and the fifth column represents the lane loaded with protein from exosomes derived from 293F cells expressing the H3 fusion protein.

[0120] Figure 16 is a histogram depicting the surface marker expression on 293F-derived exosomes.

[0121] Figure 16A It is a histogram depicting the flow analysis of the relative fluorescence intensity of exosomes derived from 293F cells expressing naturally occurring CD81 on the surface. The left curve represents 293F-derived exosomes modified with an isotype control antibody. The right curve represents 293F-derived exosomes modified with an anti-CD81 antibody.

[0122] Figure 16BIt is a histogram depicting the flow analysis of the relative fluorescence intensity of exosomes derived from 293F cells expressing spike on the surface. The left curve represents exosomes derived from 293F cells not expressing the spike-CD9 fusion protein. The right curve represents exosomes derived from 293F cells expressing the spike-CD9 fusion protein.

[0123] Figure 16C It is a histogram depicting the flow analysis of the relative fluorescence intensity of exosomes derived from 293F cells transfected with hemagglutinin 3 (H3) expressed on the surface. The left curve represents exosomes derived from 293F cells not expressing the H3-CD9 fusion protein. The right curve represents exosomes derived from 293F cells expressing the H3-CD9 fusion protein.

[0124] Figure 17 presents a histogram depicting the fold change in antigen-specific IgG production.

[0125] Figure 17A It is a histogram depicting the fold change in the serum anti-H3 IgG level on day 14 as it varies with the injection material dose, where the injection material doses from left to right are the PBS control, and a combination of exosomes expressing hemagglutinin 3 (STX-H3) and exosomes expressing the SARS-CoV-2 spike (STX-S).

[0126] Figure 17B It is a histogram depicting the fold change in the serum anti-spike IgG level on day 14 as it varies with the injection material dose, where the injection material doses from left to right are the PBS control, and a combination of exosomes expressing hemagglutinin 3 (STX-H3) and exosomes expressing the SARS-CoV-2 spike (STX-S).

[0127] Figure 18 presents a histogram depicting the fold change in antigen-specific IgG production.

[0128] Figure 18A It is a histogram depicting the fold change in the serum anti-H3 IgG level on day 35 as it varies with the injection material dose, where the injection material doses from left to right are the PBS control, and a combination of exosomes expressing hemagglutinin 3 (STX-H3) and exosomes expressing the SARS-CoV-2 spike (STX-S).

[0129] Figure 18B It is a histogram depicting the fold change in the serum anti-spike IgG level on day 35 as it varies with the injection material dose, where the injection material doses from left to right are the PBS control, and a combination of exosomes expressing hemagglutinin 3 (STX-H3) and exosomes expressing the SARS-CoV-2 spike (STX-S).

[0130] Figure 19 presents an ELISpot histogram of IFNγ production.

[0131] Figure 19A It is a histogram depicting the change in IFNγ production by splenocytes with respect to immunogens and antigens. From left to right, the first histogram represents splenocytes from animals immunized with PBS and not exposed to antigen, the second histogram represents splenocytes from animals immunized with PBS and exposed to H3 antigen, the third histogram represents splenocytes from animals immunized with a combination of spike-expressing exosomes and H3-expressing exosomes and not exposed to antigen, and the fourth histogram represents splenocytes from animals immunized with a combination of spike-expressing exosomes and H3-expressing exosomes and exposed to H3 antigen.

[0132] Figure 19A It is a histogram depicting the change in IFNγ production by splenocytes with respect to immunogens and antigens. From left to right, the first histogram represents splenocytes from animals immunized with PBS and not exposed to antigen, the second histogram represents splenocytes from animals immunized with PBS and exposed to H3 antigen, the third histogram represents splenocytes from animals immunized with a combination of spike-expressing exosomes and H3-expressing exosomes and not exposed to antigen, and the fourth histogram represents splenocytes from animals immunized with a combination of spike-expressing exosomes and H3-expressing exosomes and exposed to H3 antigen.

[0133] Figure 19B It is a histogram depicting the change in IFNγ production by splenocytes with respect to immunogens and antigens. From left to right, the first histogram represents splenocytes from animals immunized with PBS and not exposed to antigen, the second histogram represents splenocytes from animals immunized with PBS and exposed to spike antigen, the third histogram represents splenocytes from animals immunized with a combination of spike-expressing exosomes and H3-expressing exosomes and not exposed to antigen, and the fourth histogram represents splenocytes from animals immunized with a combination of spike-expressing exosomes and H3-expressing exosomes and exposed to spike antigen.

[0134] Figure 20Depicts a graphical representation of the coding sequence of respiratory syncytial virus fusion protein (RSFV F). Bar A represents non-engineered RSV F. Bar B represents engineered RSV F form 1 (V1), which is DS-Cav1. Bar C represents engineered RSV F form 2 (V2), which is DS-Cav1 with a deleted polyA signal. Bar D represents engineered RSV F form 3 (V3), which is DS-Cav1 with a deleted polyA signal and two furin cleavage sites. Bar D represents engineered RSV F form 3 (V4), which is DS-Cav1 with a deleted polyA signal and most of the N-terminal furin cleavage sites. 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.

[0135] Figure 22 is a histogram depicting the fold change in anti-RSV antibody titers as a function of antigen (RSV form 3). The Y-axis represents the fold change in antibody titers, and the X-axis represents the antigen. The left bar is the PBS control, and the right bar is the RSV F antigen. Figure 22A Represents the fold change in antibodies 14 days after injection. Figure 22B Represents the fold change in antibodies 35 days after injection and after boosting on day 21.

[0136] Figure 23 is a histogram depicting the fold change in anti-RSV antibody titers as a function of antigen (RSV form 4). The Y-axis represents the fold change in antibody titers, and the X-axis represents the antigen. Figure 23A Represents the fold change in antibodies 14 days after injection. Figure 23B Represents the fold change in antibodies 35 days after injection and after boosting on day 21. The X-axis labels for both figures are: 1 = PBS, 2 = 1E9 exosomes expressing RSV F V4-CD9, 3 = 1E10 exosomes expressing RSV F V4-CD9, 4 = 3E10 exosomes expressing RSV F V4-CD9, 5 = 1E11 exosomes expressing RSV F V4-CD9.

[0137] Figure 24 Is a figure showing a combination of exosomes expressing SARS-CoV2 spike-tetraspanin (STX-S) and exosomes expressing influenza H3-tetraspanin (STX-H3).

[0138] Figure 25 Is a figure showing a combination of exosomes expressing SARS-CoV2 spike-tetraspanin (STX-S) and exosomes expressing respiratory syncytial virus fusion protein-tetraspanin (STX-F).

[0139] Figure 26 This is a diagram showing a combination of exosomes expressing influenza H3 - tetraspanin (STX - H3) and exosomes expressing respiratory syncytial virus fusion protein - tetraspanin (STX - F).

[0140] Figure 27 This is a diagram showing a combination of exosomes expressing SARS - CoV2 spike - tetraspanin (STX - S), exosomes expressing influenza H3 - tetraspanin (STX - H3), and exosomes expressing respiratory syncytial virus fusion protein - tetraspanin (STX - F).

[0141] Figure 28 is a histogram depicting 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. Figure 28A Represents the fold change in anti - spike antibody 14 days after injection. Figure 28B Represents the fold change in anti - H3 antibody 14 days after injection. Figure 28C Represents the fold change in anti - RSV antibody 14 days after injection. The X - axis of each figure is labeled as: 1 = PBS, 2 = exosomes expressing spike (STX - S), 3 = exosomes expressing H3 (STX - H3), 4 = exosomes expressing RSV F (STX - RSV), 5 = combination of exosomes expressing H3 and exosomes expressing spike (STX - H3+STX - S), 6 = combination of exosomes expressing RSV F and exosomes expressing spike (STX - RSV+STX - S), 7 = combination of exosomes expressing RSV F and exosomes expressing H3 (STX - RSV+STX - H3), 8 = combination of exosomes expressing RSV F, exosomes expressing H3, and exosomes expressing spike (STX - RSV+STX - H3+STX - S).

[0142] Figure 29 is a histogram depicting 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. Figure 29A Represents the fold change in anti - spike antibody 35 days after injection and after a boost on day 21. Figure 28B Represents the fold change in anti - H3 antibody 35 days after injection and after a boost on day 21. Figure 28CShows the fold change in anti-RSV antibodies 35 days after injection and after a boost on day 21. The X-axis of each graph is labeled as follows: 1 = PBS, 2 = spike-expressing exosomes (STX-S), 3 = H3-expressing exosomes (STX-H3), 4 = RSV F-expressing exosomes (STX-RSV), 5 = combination of H3-expressing exosomes and spike-expressing exosomes (STX-H3+STX-S), 6 = combination of RSV F-expressing exosomes and spike-expressing exosomes (STX-RSV+STX-S), 7 = combination of RSV F-expressing exosomes and H3-expressing exosomes (STX-RSV+STX-H3), 8 = combination of RSV F-expressing exosomes, H3-expressing exosomes, and spike-expressing exosomes (STX-RSV+STX-H3+STX-S). Detailed implementation

[0143] Definition

[0144] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein, which will become apparent to those skilled in the art upon reading this disclosure and the like.

[0145] As used herein, "and / or" means and encompasses any and all possible combinations of one or more of the related listed items, as well as the absence of a combination when interpreted in the alternative (or).

[0146] As used herein, the terms "about" or "approximate" when referring to a measurable value, such as an amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, quantity, weight, position, length, etc., mean 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, quantity, weight, position, length, etc. In cases where the terms "about" and "approximate" are used in combination with the positioning or location 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.

[0147] In the case of "A is derived from B", the term "derived from" means that A is obtained from B in such a way that A is not identical to B.

[0148] The terms "treat", "therapeutic", "prophylactic", and "prevent" are not intended to be absolute terms. Treatment, prevention, and prophylaxis can refer to any delay in the onset of disease, improvement in symptoms, improvement in patient survival, increase in survival time or survival rate, etc. Treatment, prevention, and prophylaxis can be complete or partial. The term "prophylactic" not only means "prevent" but also minimizes the morbidity and disease. For example, a "prophylactic" agent can be administered to a subject (e.g., a human subject) to prevent an infection or to minimize the extent of morbidity and disease caused by such infection. The effectiveness of treatment can be compared to an individual or a group of individuals who have not received treatment, or to the same patient at different times before or during treatment. In some aspects, the severity of the disease is reduced by at least 10% compared to, for example, the individual before administration or a control individual who has not undergone treatment. In some aspects, the severity of the disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, is no longer detectable using standard diagnostic techniques.

[0149] As used herein, if, after treatment according to the methods described herein, one or more signs or symptoms of the conditions described herein change in a beneficial manner, other clinically acceptable symptoms are improved or even ameliorated, or a desired response such as at least 2%, 3%, 4%, 5%, 10% or more is induced, the treatment can be considered "effective". Efficacy, for example, can be evaluated by measuring markers, metrics, symptoms, and / or incidence of the conditions treated according to the methods described herein or any other suitable measurable parameter. Efficacy can also be measured by the individual not deteriorating, as evaluated by hospitalization or the need for medical intervention (e.g., the progression of the disease stops). Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include humans or animals) and includes: (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of the disease, e.g., causing the symptoms to resolve. An effective amount for treating a disease means an amount sufficient to result in an effective treatment of the disease (as defined herein) when administered to a subject in need thereof. The efficacy of an agent can be determined by evaluating physical indicators of the condition or the desired response. One of ordinary skill in the art can monitor the efficacy of administration and / or treatment by measuring any one or any combination of such parameters.

[0150] As used herein, the term "effective amount" refers to the amount of a composition or agent needed 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 the 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. In various instances, an effective amount as used herein can include an amount sufficient to delay the development of disease symptoms, alter the course of disease symptoms (e.g., but not limited to, slow the progression of disease symptoms) or reverse disease symptoms. For example, for a particular parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90% or at least 100% of a therapeutic effect. Therapeutic efficacy can also be expressed as a "-fold" increase or decrease. For example, a therapeutically effective amount can have an effect of at least 1.2-fold, 1.5-fold, 2-fold, 5-fold or more fold compared to a control. A therapeutically effective amount can be administered in one or more doses of a therapeutic agent. A therapeutically effective amount can be administered as a single dose or in multiple doses over a period of time.

[0151] "Administer" as used herein can include any suitable route for administering a therapeutic agent or composition as disclosed herein. Suitable routes of administration include, but are not limited to, oral administration, parenteral administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway (aerosol) administration, pulmonary administration, dermal administration, injection administration or topical administration. Administration can be local or systemic.

[0152] As used herein, the term "pharmaceutically acceptable" refers to a carrier that is compatible with the other ingredients of the formulation and not harmful to its recipient. This term is used synonymously with "physiologically acceptable" and "pharmacologically acceptable". Depending on the route of administration, a pharmaceutical composition will generally contain agents for buffering and preservation during storage, and may contain buffering agents and carriers for proper delivery. The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms that are suitable, within the scope of sound medical judgment, for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0153] The terms "dose" and "dosage" are used interchangeably herein. A dose refers to the amount of active ingredient administered to an individual per administration. For the present invention, the dose may refer to the concentration of extracellular vesicles or related components, such as the amount of therapeutic agent or the dose of a radiolabel. The dose will vary depending on a number of factors, including the frequency of administration, the size and tolerance of the individual, the severity of the condition, the risk of side effects, the route of administration, and the imaging modality of the detectable moiety (if any). Those skilled in the art will recognize that the dose may be modified based on the above factors or based on the progress of the treatment. The term "dosage form" refers to the specific form of a drug and depends on the route of administration. For example, the dosage form may be liquid, such as a saline solution for injection.

[0154] The terms "subject", "patient", "individual" and similar terms are used interchangeably and, unless otherwise indicated, refer to mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a specific disease, but generally refers to an individual under medical supervision. A patient may be an individual seeking treatment, monitoring, adjustment or modification of an existing treatment regimen, etc.

[0155] Unless otherwise indicated, the following meanings apply to the terms used herein. The word "may" is used in a permissive sense (i.e., meaning having the potential), rather than in a mandatory sense (i.e., meaning must). The word "comprising" and other variants mean including but not limited to. The singular forms "a", "an" and "the" include plural referents. Thus, for example, reference to "an element" includes combinations of two or more elements, even though other terms and phrases such as "one or more" are used with respect to one or more elements. Unless otherwise indicated, the term "or" is non-exclusive, i.e., includes both "and" and "or". The term "any one" between a modifier and a sequence means that the modifier modifies each member of the sequence. Thus, for example, the phrase "at least any one of 1, 2 or 3" means "at least 1, at least 2 or at least 3". The phrase "at least one" includes "more than one".

[0156] Definitions of commonly used terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); 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.

[0157] The term “native form” corresponds to a polypeptide encoded by the genome of an infectious agent, as understood. The term “exosome form” corresponds to any derivative in which all or part of the protein is fused to an exosome-associated protein. The term “cytoplasmic form” corresponds to any derivative in which all or part of the protein is configured or designed to be expressed within the cytoplasm of a cell rather than enter the typical secretory pathway.

[0158] The statement that a certain protein is “configured or designed to be expressed in a certain way” means that its nucleotide sequence encodes a certain specific amino acid sequence such that when the protein is expressed in a cell, the protein will be in its native form, exosome form, or cytoplasmic form by virtue of that specific amino acid sequence. For example, if the spike protein (S) is expressed in its native form, it is configured or designed to induce a humoral response or a cellular immune response by virtue of the fact that it is a transmembrane protein with an extracellular domain.

[0159] The term "extracellular vesicle" (EV) refers to lipid bilayer-delimited particles that are naturally released from cells. EVs range in diameter from approximately 20 - 30 nanometers to approximately 10 micrometers or larger. EVs can contain proteins, nucleic acids, lipids, and metabolites from the cells that produce them. EVs include exosomes (approximately 50 nm to approximately 200 nm), microvesicles (approximately 100 nm to approximately 300 nm), ectosomes (approximately 50 nm to approximately 1000 nm), apoptotic bodies (approximately 50 nm to approximately 5000 nm), and lipid - protein aggregates of the same size.

[0160] The term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and chemically modified nucleic acids such as morpholino nucleic acids (PMO), peptide nucleic acids (PNA), 2’O - methyl, 2’ methoxy - ethyl, 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 linear or covalently circular closed molecules. Nucleic acids can be, for example, in the form of RNA (which can be prepared by in vitro transcription from a DNA template) for introduction into cells, e.g., transfection of cells. In addition, RNA can be modified by stabilizing sequences, capping, and polyadenylation prior to 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).

[0161] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein and refer to any chain of at least two amino acids linked by covalent chemical bonds. As used herein, a peptide can refer to the complete amino acid sequence encoding an entire protein or a portion thereof. A "protein-coding sequence" or a sequence that "encodes" a particular polypeptide or peptide is a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. The coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. Transcription termination sequences will generally be located 3' of the coding sequence.

[0162] As used herein, the phrase "protein polypeptide" means a polypeptide sequence that is of or derived from a protein. For example, a CD9 protein polypeptide can be any polypeptide of the CD9 protein, such as, for example, the full-length CD9 protein, the transmembrane domain polypeptide of the CD9 protein, the N-terminal segment of the CD9 protein, the extracellular loop region of the CD9 protein, the intracellular (luminal) loop region of the CD9 protein, the C-terminal segment of the CD9 protein, combinations thereof, and the like. Here, the protein polypeptide can be at least 10 amino acids in length.

[0163] As used herein, the term "spike protein" includes any SARS-CoV-2 spike glycoprotein, a fragment of the SARS-CoV-2 spike glycoprotein, a monomer of the SARS-CoV-2 spike glycoprotein, a trimer of the SARS-CoV-2 spike glycoprotein monomer, a variant of the SARS-CoV-2 spike glycoprotein, a fusion protein or chimeric protein containing a SARS-CoV-2 spike glycoprotein sequence and another non-SARS-CoV-2 spike glycoprotein sequence, a SARS-CoV-2 spike glycoprotein having one or more deletions, additions, or substitutions of one or more amino acids, and a conservative substitution variant of the SARS-CoV-2 spike glycoprotein having at least 80% amino acid sequence identity, such as, for example, at least the stalk region of the S2 subunit, the membrane-proximal stalk helix region, or the receptor-binding domain or other similar domain.

[0164] Fragments of the SARS-CoV-2 spike glycoprotein include peptides or polypeptides that encompass, include, consist of, or overlap with the following: for example, antigenic epitopes, specific domains such as the receptor-binding domain (RBD) in an up or down conformational state, the receptor-binding fragment S1, the fusion fragment S2, the N-terminal domain (NTD), the C-terminal domain 1 (CTD1) of the receptor-binding domain (RBD), the C-terminal domain 2 (CTD2), the fusion peptide (FP), the fusion peptide proximal region (FPPR), the heptad repeat 1 (HR1), the central helix (CH), the linker domain (CD), the heptad repeat 2 (HR2), the transmembrane segment (TM), the cytoplasmic tail (CT), etc.

[0165] Variants of the SARS-CoV-2 spike glycoprotein include any known or yet-to-be-discovered spike protein sequences, including alpha, beta, gamma, delta, epsilon, eta, iota, kappa, 1.617.3, mu, zeta, Omicron or its subvariants, lineages, and conservative substitutions.

[0166] The SARS-CoV-2 spike glycoprotein can have additions, deletions, substitutions, point mutations. For example, the spike protein can have a deletion of several (2 - 20) amino acids from its C-terminus (see Johnson et al., 2020 and Xiong et al., 2020), or an altered furin cleavage site (see, e.g., Johnson et al., 2020).

[0167] As used herein, the term "nucleocapsid protein" refers to a soluble coronavirus structural protein that binds to RNA and the viral membrane protein (M) and forms a complex and is essential for viral genome packaging. The nucleocapsid protein contains (from the amino terminus to the carboxyl terminus) an intrinsically disordered region (IDR) and an N-terminal domain (NTD) containing an RNA-binding domain, a serine-arginine-rich linker region (LKR), a C-terminal domain containing an RNA-binding and dimerization domain and a nuclear localization signal, followed by a C-terminal IDR. 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 Vol. 12, Article number: 1936 (2021); and the references cited therein.

[0168] A fusion protein or chimeric protein of a SARS-CoV-2 spike glycoprotein or a SARS-CoV-2 nucleocapsid protein comprises a fusion of a spike protein sequence or a nucleocapsid protein sequence with a sequence of another protein to achieve a specific outcome, such as improving sorting or targeting to endosomes and the resulting extracellular vesicles such as exosomes. For example, the spike protein can be a fusion of a spike protein sequence or a nucleocapsid protein sequence with another glycoprotein known to be sorted to exosomes or useful for generating pseudovirions (such as VSV glycoprotein, lentiviral glycoprotein, etc.). The spike protein or nucleocapsid protein can be a fusion of a spike protein sequence and other proteins known to be sorted to exosomes (such as various tetraspanins (CD9, CD63, and CD81)). The fusion protein can mainly contain a SARS-CoV-2 sequence with a short (i.e., a dipeptide to a peptide of 100 amino acids) sequence of another protein.

[0169] As used herein, the term "tetraspanin" or "tetraspanin protein" means any member (or chimera thereof) of a family of proteins that have four transmembrane domains and in some cases are present in the exosome membrane. Tetraspanin proteins are known to regulate trafficking as well as cellular and membrane compartmentalization. Tetraspanins include, in particular, CD9, CD37, CD63, CD81, CD82, CD151, TSPAN7, TSPAN8, TSPAN12, TSPAN33, peripherin, UP1a / 1b, TSP-15, TSP-12, TSP3A, TSP86D, TSP26D, TSP-2 and their analogs, orthologs and homologs. Useful tetraspanins are contemplated to include chimeras of any one or more of the canonical tetraspanins, such as, for example, a CD9 / CD81 chimera and the like. Tetraspanins are generally described in: Charrin et al., "Tetraspanins at a glance," J Cell Sci (2014) 127(17):3641-3648; Kummer et al., "Tetraspanins: integrating cell surface receptors to functional microdomains in homeostasis and disease," Med Microbiol Immunol. 2020;209(4):397-405; and the 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 the references cited therein.

[0170] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percentage identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). Then the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage 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 x 100%). In one embodiment, the two sequences are of the same length. The determination of the percentage identity between two sequences can also be accomplished using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm for comparing two sequences is the following algorithm: Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., 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 the XBLAST program parameters set, e.g., 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 can be utilized, as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Optionally, PSI-BLAST can be used to perform iterative searches for detecting distant relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs can be used (e.g., the default parameters of XBLAST and NBLAST) (see, e.g., the NCBI website).

[0171] Another preferred non-limiting example of a mathematical algorithm for sequence comparison 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 comparing amino acid sequences using the ALIGN program, the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0172] The percent identity between two sequences can be determined using techniques that allow gaps or do not allow gaps similar to those described above. When calculating the percent identity, usually only exact matches are counted.

[0173] For antibodies, the percent sequence identity can be determined when the antibody sequences are maximally aligned by IMGT. After alignment, if the test antibody region (e.g., the entire mature variable region of the heavy or light chain) is compared to the same region of a reference antibody, the percent sequence identity between the test antibody region and the reference antibody region is the number of positions occupied by the same amino acids in both the test antibody region and the reference antibody region divided by the total number of aligned positions in the two regions, multiplied by 100 to convert to a percentage.

[0174] The percent amino acid sequence identity can also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be obtained from the National Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search parameters, all of which are set to default values, including, for example, unmasked = yes, strand = all, expect = 10, minimum low complexity length = 15 / 5, multi-pass e-value = 0.01, multi-pass constant = 25, final gap alignment dropoff = 25, and scoring matrix = BLOSUM62.

[0175] In the case of amino acid sequence comparison using NCBI-BLAST2, the percent amino acid sequence identity of a particular amino acid sequence A to, with, or relative to a particular amino acid sequence B (which can also alternatively be phrased as a particular amino acid sequence A has or contains a certain percent amino acid sequence identity to, with, or relative to a particular amino acid sequence B) is calculated as follows:

[0176] 100 times the fraction X / Y,

[0177] Where X is the number of amino acid residues in the alignment of A and B that are scored as identical matches by the sequence alignment program NCBI - BLAST2, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not be equal to the percent amino acid sequence identity of B to A. As used herein, the term "nucleic acid sequence" refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars, and inter - sugar (backbone) linkages, and includes 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 can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA), and can include naturally occurring bases, including adenine, guanine, cytosine, thymine, and uracil. The sequences can also contain modified bases. Examples of such modified bases include azido and deazido adenine, guanine, cytosine, thymine, and uracil; and xanthine and hypoxanthine. It should be understood that polynucleotides containing non - transcribable nucleobases can be used as probes, for example, in hybridization assays. The nucleic acid can be double - stranded or single - stranded, and represents the sense or antisense strand. In addition, the term "nucleic acid" includes complementary nucleic acid sequences as well as codon - optimized or synonymous codon equivalents.

[0178] As used herein, the term "antibody" refers to an immunoglobulin (Ig) molecule and the immunologically active portions of an immunoglobulin molecule (i.e., a molecule that contains an antigen-binding site that specifically binds an antigen). Antibodies are typically heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. The "light chains" from any vertebrate species can be assigned to one of two distinct types based on the amino acid sequence of their constant domains, called kappa (κ) and lambda (λ). Depending on the amino acid sequence of the constant domain of the heavy chain of the immunoglobulin, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. An antibody can have one or more effector functions, which refers to those biological activities that can be attributed to the Fc region of the antibody (native sequence Fc region or 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 cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor (BCR)); and cross-presentation of antigen by antigen-presenting cells or dendritic cells.

[0179] The term "neutralizing antibody" (Nab) refers to an antibody that protects cells from a pathogen or infectious particle by neutralizing any effect that the pathogen or infectious particle biologically has. Neutralization renders the particle no longer infectious or pathogenic. Neutralizing antibodies are part of the humoral response of the adaptive immune system against viruses, intracellular bacteria, and microbial toxins. By specifically binding to surface antigens on the infectious particle, neutralizing antibodies prevent the particle from interacting with the host cells that it might infect and destroy.

[0180] The immunity caused by neutralizing antibodies is also called sterilizing immunity because the immune system eliminates the infectious particles before any infection occurs.

[0181] The term "antigen" refers to any substance that will elicit an immune response. For example, an antigen refers to any substance that specifically reacts with an antibody or a T lymphocyte (T cell), preferably a peptide or a protein. As used herein, the term "antigen" includes any molecule that contains at least one epitope. For example, an antigen is a molecule that optionally induces an immune response after processing. For example, any suitable antigen that is a candidate for an immune response can be used, wherein the immune response can be a cellular immune response. For example, an antigen can be presented by a cell, which results in an immune response against the antigen. For example, an antigen is a product corresponding to or derived from a naturally occurring antigen. Such antigens include, but are not limited to, the SARS-CoV-2 structural proteins S, N, M, and E and any variants or mutants thereof.

[0182] The term "pharmaceutical composition" refers to a preparation that contains an active ingredient and optionally a pharmaceutically acceptable carrier, diluent, or excipient. The term "active ingredient" can be interchangeably referred to as "effective ingredient" and means any agent that is capable of inducing the sought-after effect upon administration. "Pharmaceutically acceptable" means that the carrier, diluent, or excipient must be compatible with the other ingredients of the preparation and not harmful to its recipient, nor harmful to the activity of the active ingredient of the preparation. Pharmaceutically acceptable carriers, excipients, or stabilizers are well known in the art, such as Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations employed and can include buffers such as phosphate, citrate, and other organic acid buffers; antioxidants, including ascorbic acid and methionine; preservatives (such as cetyltrimethylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, cresol, or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, including 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 nonionic surfactants, such as TWEEN™, PLURONIC™, or polyethylene glycol (PEG). Examples of carriers include, but are not limited to, liposomes, nanoparticles, ointments, micelles, microspheres, microparticles, creams, emulsions, and gels.

[0183] Examples of excipients include, but are not limited to, anti-adhesives 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, alcohols, saline solutions, glycols, mineral oil, and dimethyl sulfoxide (DMSO).

[0184] The term "vaccine" or "immunogenic composition" refers to a pharmaceutical product (pharmaceutical composition) or product that, upon administration, induces an immune response that recognizes and attacks pathogens or diseased cells, such as a cellular immune response. The term "immune response" refers to the overall bodily response to an antigen and refers to a cellular immune response and / or a humoral immune response. The immune response can be protective / preventive / prophylactic and / or therapeutic.

[0185] The term "cellular immune response" or "cell-mediated immune response" describes any adaptive immune response in which antigen-specific T cells play a major role. It is operationally defined as all adaptive immunity that cannot be transferred to naive recipients by serum antibodies. In contrast, the term "humoral immune response" describes immunity due to antibodies.

[0186] Cellular responses involve cells called T cells or T lymphocytes that act as "helpers" or "killers". Helper T cells (also called CD4+ T cells) play a central role by regulating the immune response, and killer cells (also called cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill diseased cells such as cancer cells to prevent the production of more diseased cells.

[0187] The term "immunoreactive cell", "immune cell", or "immune effector cell" refers to a cell that plays an effector function during an immune response. An "immunoreactive cell" is preferably a cell that is capable of binding an antigen or is characterized by presenting an antigen or an antigenic peptide derived from an antigen and mediating an immune response. For example, such cells secrete cytokines and / or chemokines, secrete antibodies, recognize cancer cells, and optionally eliminate such cells. For example, immunoreactive cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells.

[0188] The term "adjuvant" refers to a pharmacological or immunological agent that modifies the action of other agents. Adjuvants can be added to the vaccine compositions of the present invention to enhance the immune response to produce more antibodies and a more persistent immunity, thereby minimizing the required antigen dose. Adjuvants can also be used to enhance the potency of a vaccine by helping to direct the immune response to specific types of immune system cells: for example, by activating T cells rather than antibody-secreting B cells, depending on the purpose of the vaccine. Immunological adjuvants are added to vaccines to stimulate the immune system's response to the target antigen, but do not themselves confer immunity. Examples of adjuvants include, but are not limited to, adjuvants for pain relief; inorganic compounds such as alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide; mineral oils such as paraffin oil; bacterial products, such as killed bacteria (Bordetella pertussis, Mycobacterium bovis, toxoids); non-bacterial organic substances such as squalene; delivery systems such as detergents (Quil A); plant saponins from Quillaja saponaria, soybeans or Polygala senega; cytokines such as IL-1, IL-2, IL-12; combinations such as Freund's complete adjuvant, Freund's incomplete adjuvant; food-based oils such as Adjuvant 65, which is based on peanut oil.

[0189] Embodiment

[0190] The provided embodiments include:

[0191] 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 exosome polypeptide, and the second fusion protein comprises a second viral polypeptide and an exosome polypeptide.

[0192] 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 exosome polypeptide, the second fusion protein comprises a second viral polypeptide and an exosome polypeptide, and the third fusion protein comprises a third viral polypeptide and an exosome polypeptide.

[0193] Embodiment 3. The immunogenic composition according to Embodiment 1 or Embodiment 2, further comprising an excipient.

[0194] Embodiment 4. The immunogenic composition according to Embodiment 3, wherein the excipient comprises a buffer.

[0195] Embodiment 5. The immunogenic composition according to Embodiment 3 or 4, wherein the excipient comprises a cryoprotectant.

[0196] Embodiment 6. The immunogenic composition according to any one of Embodiments 1-5, which does not contain an adjuvant.

[0197] Embodiment 7. The immunogenic composition according to any one of Embodiments 1-4, wherein the first fusion protein and the second fusion protein are present in the membranes of their respective vesicles.

[0198] Embodiment 8. The immunogenic composition according to any one of Embodiments 1-7, wherein a part or all of each viral polypeptide is present at or on the outer surface of the vesicle.

[0199] Embodiment 9. The immunogenic composition according to any one of Embodiments 1-8, wherein each fusion protein is present in the composition at a concentration of about 1 ng / 100 μL to about 50 ng / 100 μL.

[0200] Embodiment 10. The immunogenic composition according to any one of Embodiments 1-9, wherein the first viral polypeptide is a SARS-CoV-2 polypeptide and the second viral polypeptide is an influenza polypeptide.

[0201] Embodiment 11. The immunogenic composition according to any one of Embodiments 1-10, wherein the first viral polypeptide is a SARS-CoV-2 spike protein polypeptide.

[0202] Embodiment 12. The immunogenic composition according to any one of Embodiments 1-11, wherein the first viral polypeptide is a SARS-CoV-2 Delta variant spike protein polypeptide.

[0203] Embodiment 13. The immunogenic composition according to any one of Embodiments 1-12, wherein the first viral polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO:1 or identical to SEQ ID NO:1.

[0204] Embodiment 14. The immunogenic composition according to any one of Embodiments 1-10, wherein the first viral polypeptide is a SARS-CoV-2 nucleocapsid protein polypeptide.

[0205] Embodiment 15. The immunogenic composition according to Embodiment 14, wherein the first viral polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO:5 or identical to SEQ ID NO:5.

[0206] Embodiment 16. The immunogenic composition according to any one of Embodiments 1-15, wherein the second viral polypeptide is an influenza hemagglutinin protein polypeptide.

[0207] Embodiment 17. The immunogenic composition according to any one of Embodiments 1-16, wherein the second viral polypeptide is an influenza hemagglutinin 3 (H3) protein polypeptide.

[0208] Embodiment 18. The immunogenic composition according to any one of Embodiments 1-17, wherein the second viral polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO: 14 or identical to SEQ ID NO: 14.

[0209] Embodiment 19. The immunogenic composition according to any one of Embodiments 2-18, wherein the third viral polypeptide is a respiratory syncytial virus (RSV) protein polypeptide.

[0210] Embodiment 20. The immunogenic composition according to any one of Embodiments 2-19, wherein the third viral polypeptide is an RSV fusion (RSV F) protein polypeptide.

[0211] Embodiment 21. The immunogenic composition according to any one of Embodiments 2-20, wherein the third viral polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO: 18 or identical to SEQ ID NO: 18.

[0212] Embodiment 22. The immunogenic composition according to any one of Embodiments 1-21, wherein the exosome polypeptide is a tetraspanin protein polypeptide.

[0213] Embodiment 23. The immunogenic composition according to any one of Embodiments 1-22, wherein the exosome polypeptide is a CD9 protein polypeptide.

[0214] Embodiment 24. The immunogenic composition according to any one of Embodiments 1-23, wherein the exosome polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO: 10 or identical to SEQ ID NO: 10.

[0215] Embodiment 25. The immunogenic composition according to any one of Embodiments 1-13 and 17-24, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: 2 or identical to SEQ ID NO: 2.

[0216] Embodiment 26. The immunogenic composition according to any one of Embodiments 1-13 and 17-24, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:3 or identical to SEQ ID NO:3.

[0217] Embodiment 27. The immunogenic composition according to any one of Embodiments 1-10 and 14-23, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:6 or identical to SEQ ID NO:6.

[0218] Embodiment 28. The immunogenic composition according to any one of Embodiments 1-10, 14-23 and 27, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:7 or identical to SEQ ID NO:7.

[0219] Embodiment 29. The immunogenic composition according to any one of Embodiments 1-28, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:15 or identical to SEQ ID NO:15.

[0220] Embodiment 30. The immunogenic composition according to any one of Embodiments 1-29, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:16 or identical to SEQ ID NO:16.

[0221] Embodiment 31. The immunogenic composition according to any one of Embodiments 2-30, wherein the third fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:19 or identical to SEQ ID NO:19.

[0222] Embodiment 32. The immunogenic composition according to any one of Embodiments 2-31, wherein the third fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO:20 or identical to SEQ ID NO:20.

[0223] Embodiment 33. The immunogenic composition according to any one of Embodiments 1-32, wherein the 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.

[0224] Embodiment 34. The immunogenic composition according to any one of Embodiments 2-33, wherein the immunogenic dose of the composition comprises about 1 ng to about 50 ng of the third fusion protein or the third viral polypeptide.

[0225] Embodiment 35. A method of immunizing a subject against a viral infection, the method comprising administering to the subject an immunogenic effective dose of an immunogenic composition according to any one of Embodiments 1-34.

[0226] Embodiment 36. The method according to Embodiment 35, wherein the immunogenic effective dose elicits protective immunity against a first virus and a second virus in the subject.

[0227] Embodiment 37. The method according to Embodiment 35 or 36, wherein the immunogenic effective dose elicits protective immunity against a first virus, a second virus and a third virus in the subject.

[0228] Embodiment 38. The method according to Embodiment 35 or 36, wherein the immunogenic composition comprises a SARS-CoV-2 spike protein polypeptide and an influenza hemagglutinin polypeptide, and the first virus is SARS-CoV2 and the second virus is an influenza virus.

[0229] Embodiment 39. The method according to any one of Embodiments 35-38, wherein the immunogenic composition comprises a SARS-CoV-2 spike protein polypeptide, an influenza hemagglutinin polypeptide and a respiratory syncytial virus (RSV) fusion protein polypeptide, and the first virus is SARS-CoV2, the second virus is an influenza virus, and the third virus is RSV.

[0230] Embodiment 40. The method according to any one of Embodiments 35-39, wherein the immunogenic effective dose elicits protective immunity against a SARS-CoV-2 Delta variant and a SARS-CoV-2 Omicron variant in the subject.

[0231] Embodiment 41. The method according to any one of Embodiments 35-40, wherein the immunogenic effective dose comprises from about 1 ng to about 50 ng of the first fusion protein, from about 1 ng to about 50 ng of the second fusion protein and from about 1 ng to about 50 ng of the third fusion protein.

[0232] Embodiment 42. The method according to any one of Embodiments 35-41, further comprising administering to the subject a second effective dose of the immunogenic composition according to any one of Embodiments 1-34.

[0233] Embodiment 43. The method according to any one of embodiments 35-42, wherein the induced protective immunity comprises (a) high antibody titers against the first virus, the second virus, and the third virus, (b) a CD4+ T cell response against the first virus, the second virus, and the third virus, and (c) a CD8+ cytotoxic T cell response against the first virus, the second virus, and the third virus.

[0234] Embodiment 44. A synthetic fusion protein comprising a viral polypeptide and an exosome polypeptide.

[0235] Embodiment 45. The synthetic fusion protein according to embodiment 44, further comprising a linker polypeptide located between the viral polypeptide and the exosome polypeptide.

[0236] Embodiment 46. The synthetic fusion protein according to embodiment 44 or 45, further comprising a hinge polypeptide located between the viral polypeptide and the exosome polypeptide.

[0237] Embodiment 47. The synthetic fusion protein according to any one of embodiments 44-46, further comprising a transmembrane domain polypeptide located between the viral polypeptide and the exosome polypeptide.

[0238] Embodiment 48. The synthetic fusion protein according to any one of embodiments 44-47, wherein the exosome polypeptide is a tetraspanin polypeptide.

[0239] Embodiment 49. The synthetic fusion protein according to any one of embodiments 44-48, wherein the exosome polypeptide is a CD9 polypeptide.

[0240] Embodiment 50. The synthetic fusion protein according to any one of embodiments 44-49, wherein the viral polypeptide is a SARS-CoV-2 structural protein polypeptide.

[0241] Embodiment 51. The synthetic fusion protein according to any one of embodiments 44-50, wherein the viral polypeptide is a SARS-CoV-2 spike protein polypeptide.

[0242] Embodiment 52. The synthetic fusion protein according to embodiment 51, wherein the SARS-CoV-2 spike protein polypeptide comprises one or more of a furin protease cleavage site mutation (CSM[682RRAR685-to-682GSAG685]) and a diproline substitution (2P[986KV987-to-986PP987]).

[0243] Embodiment 53. The synthetic fusion protein according to any one of embodiments 44-52, wherein the fusion protein comprises a SARS-CoV-2 spike protein polypeptide, a linker polypeptide, and a CD9 polypeptide in order from the amino terminus to the carboxyl terminus.

[0244] Embodiment 54. The synthetic fusion protein according to any one of embodiments 44-53, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:3.

[0245] Embodiment 55. The synthetic fusion protein according to any one of embodiments 44-54, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:3.

[0246] Embodiment 56. The synthetic fusion protein according to any one of embodiments 44-50, wherein the viral polypeptide is a SARS-CoV-2 nucleocapsid protein polypeptide.

[0247] Embodiment 57. The synthetic fusion protein according to any one of embodiments 44-50 and 56, wherein the fusion protein comprises a signal peptide, a SARS-CoV-2 nucleocapsid protein polypeptide, a hinge region, a transmembrane domain polypeptide, a linker polypeptide, and a CD9 polypeptide in order from the amino terminus to the carboxyl terminus.

[0248] Embodiment 58. The synthetic fusion protein according to any one of embodiments 44-50, 56, and 57, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:7.

[0249] Embodiment 59. The synthetic fusion protein according to any one of embodiments 44-50 and 56-58, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:7.

[0250] Embodiment 60. The synthetic fusion protein according to any one of embodiments 44-49, wherein the viral polypeptide is an influenza protein polypeptide.

[0251] Embodiment 61. The synthetic fusion protein according to embodiment 60, wherein the influenza protein polypeptide comprises hemagglutinin.

[0252] Embodiment 62. The synthetic fusion protein according to embodiment 60 or 61, wherein the influenza protein polypeptide comprises hemagglutinin 3 (H3).

[0253] Embodiment 63. The synthetic fusion protein according to any one of embodiments 60-62, wherein the fusion protein comprises a hemagglutinin protein polypeptide, a linker polypeptide, and a CD9 polypeptide in order from the amino terminus to the carboxyl terminus.

[0254] Embodiment 64. The synthetic fusion protein according to any one of Embodiments 60-63, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16.

[0255] Embodiment 65. The synthetic fusion protein according to any one of Embodiments 60-64, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0256] Embodiment 66. The synthetic fusion protein according to any one of Embodiments 44-49, wherein the viral polypeptide is a respiratory syncytial virus (RSV) protein polypeptide.

[0257] Embodiment 67. The synthetic fusion protein according to Embodiment 66, wherein the RSV protein polypeptide comprises the RSV fusion (RSV F) protein.

[0258] Embodiment 68. The synthetic fusion protein according to Embodiment 66 or 67, wherein the fusion protein comprises, in order from the amino terminus to the carboxyl terminus, an RSV F protein polypeptide, a linker polypeptide, and a CD9 polypeptide.

[0259] Embodiment 69. The synthetic fusion protein according to any one of Embodiments 66-68, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 20.

[0260] Embodiment 70. The synthetic fusion protein according to any one of Embodiments 60-64, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 20.

[0261] Embodiment 71. A synthetic polynucleotide encoding the synthetic fusion protein according to any one of Embodiments 44-70.

[0262] Embodiment 72. The synthetic polynucleotide according to Embodiment 71, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 13.

[0263] Embodiment 73. The synthetic polynucleotide according to Embodiment 71 or 72, comprising the nucleic acid sequence set forth in SEQ ID NO: 13.

[0264] Embodiment 74. The synthetic polynucleotide according to any one of Embodiments 71-73, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 11.

[0265] Embodiment 75. The synthetic polynucleotide according to any one of Embodiments 71-74, comprising the nucleic acid sequence set forth in SEQ ID NO: 11.

[0266] Embodiment 76. The synthetic polynucleotide according to any one of embodiments 71-75, comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO: 4.

[0267] Embodiment 77. The synthetic polynucleotide according to any one of embodiments 71-76, comprising the nucleic acid sequence set forth in SEQ ID NO: 4.

[0268] Embodiment 78. The synthetic polynucleotide according to any one of embodiments 71-73, comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO: 12.

[0269] Embodiment 79. The synthetic polynucleotide according to any one of embodiments 71-73 and 78, comprising the nucleic acid sequence set forth in SEQ ID NO: 12.

[0270] Embodiment 80. The synthetic polynucleotide according to any one of embodiments 71-73, 78 and 79, comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO: 8.

[0271] Embodiment 81. The synthetic polynucleotide according to any one of embodiments 71-73 and 78-80, comprising the nucleic acid sequence set forth in SEQ ID NO: 8.

[0272] Embodiment 82. The synthetic polynucleotide according to any one of embodiments 71-73, comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO: 22.

[0273] Embodiment 83. The synthetic polynucleotide according to any one of embodiments 71-73 and 82, comprising the nucleic acid sequence set forth in SEQ ID NO: 22.

[0274] Embodiment 84. The synthetic polynucleotide according to any one of embodiments 71-73, 82 and 83, comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO: 17.

[0275] Embodiment 85. The synthetic polynucleotide according to any one of embodiments 71-73 and 82-84, comprising the nucleic acid sequence set forth in SEQ ID NO: 17.

[0276] Embodiment 86. The synthetic polynucleotide according to any one of embodiments 71-73, comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO: 23.

[0277] Embodiment 87. The synthetic polynucleotide according to any one of Embodiments 71-73 and 86 comprises the nucleic acid sequence set forth in SEQ ID NO: 23.

[0278] Embodiment 88. The synthetic polynucleotide according to any one of Embodiments 71-73, 86 and 87 comprises a nucleic acid sequence having at least 80% identity with SEQ ID NO: 21.

[0279] Embodiment 89. The synthetic polynucleotide according to any one of Embodiments 71-73 and 86-88 comprises the nucleic acid sequence set forth in SEQ ID NO: 21.

[0280] Embodiment 90. A cell comprising the synthetic polynucleotide according to any one of Embodiments 71-89.

[0281] Embodiment 91. The cell according to Embodiment 90, wherein the cell is a metazoan cell.

[0282] Embodiment 92. The cell according to Embodiment 90 or 91, wherein the cell is a vertebrate cell.

[0283] Embodiment 93. The cell according to any one of Embodiments 90-92, wherein the cell is a mammalian cell.

[0284] Embodiment 94. The cell according to any one of Embodiments 90-93, wherein the cell is a primate cell.

[0285] Embodiment 95. The cell according to any one of Embodiments 90-94, wherein the cell is a human cell.

[0286] Embodiment 96. The cell according to any one of Embodiments 90-95, wherein the cell is a primary cell.

[0287] Embodiment 97. The cell according to any one of Embodiments 90-95, wherein the cell is a human embryonic kidney cell.

[0288] Embodiment 98. The cell according to Embodiment 97, wherein the cell is a 293 cell.

[0289] Embodiment 99. The cell according to any one of Embodiments 90-98, wherein the cell is produced by transducing the cell with a lentivirus containing the synthetic polynucleotide.

[0290] Embodiment 100. The cell according to any one of Embodiments 90-99, wherein the cell comprises the synthetic fusion protein according to any one of Embodiments 44-70.

[0291] Embodiment 101. A vesicle comprising the synthetic fusion protein of any one of embodiments 44-70.

[0292] Embodiment 102. The vesicle according to embodiment 101, wherein the vesicle is an exosome.

[0293] Embodiment 103. The vesicle according to embodiment 101 or 102, wherein the vesicle has a diameter of about 50-500 nm.

[0294] Embodiment 104. The vesicle according to any one of embodiments 101-103, wherein the SARS-CoV-2 spike protein polypeptide is expressed on the outer surface of the vesicle.

[0295] Embodiment 105. The vesicle according to any one of embodiments 101-103, wherein the vesicle expresses the SARS-CoV-2 nucleocapsid protein polypeptide on its surface.

[0296] Embodiment 106. The vesicle according to any one of embodiments 101-103, wherein the vesicle expresses the influenza hemagglutinin protein polypeptide on its surface.

[0297] Embodiment 107. The vesicle according to any one of embodiments 101-103, wherein the vesicle expresses the respiratory syncytial virus fusion protein polypeptide on its surface.

[0298] Embodiment 108. A method for preparing the vesicle of any one of embodiments 101-107, comprising culturing the cells of any one of embodiments 90-100 in a cell culture medium, collecting the cell culture medium, and purifying more than one vesicle comprising the vesicle from the cell culture medium.

[0299] Embodiment 109. The method according to embodiment 108, further comprising inducing the expression of the synthetic polynucleotide of any one of embodiments 71-89 to produce the synthetic fusion protein of any one of embodiments 44-70.

[0300] Embodiment 110. The method according to embodiment 105, wherein the induction comprises contacting the cells with tetracycline, doxycycline or an analogue thereof.

[0301] Embodiment 111. The method according to embodiment 105, wherein the induction comprises removing tetracycline, doxycycline or an analogue thereof from the cells.

[0302] Embodiment 112. A method of eliciting an immune response in a subject, the method comprising administering to the subject a first dose of an immunogenic composition, the immunogenic composition comprising more than one vesicle, the more than one vesicle comprising the vesicle of any one of Embodiments 101-107 or the vesicle produced by the method of any one of Embodiments 108-111, wherein the synthetic fusion protein of any one of Embodiments 44-70 is expressed on the outer surface of the vesicle.

[0303] Embodiment 113. The method according to Embodiment 112, further comprising administering to the subject a second dose of the immunogenic composition at a certain time period after administering the first dose.

[0304] Embodiment 114. The method according to Embodiment 113, wherein the time period is 14 days to 1 year.

[0305] Embodiment 115. The method according to any one of Embodiments 112-114, wherein the first dose or the second dose comprises about 100 μL - 1 mL of the immunogenic composition, wherein the immunogenic composition comprises about 0.3 ng / mL - 3 μg / mL of the synthetic fusion protein.

[0306] Embodiment 116. The method according to any one of Embodiments 112-115, wherein the first dose or the second dose comprises about 100 μL - 1 mL of the immunogenic composition, the immunogenic composition comprising about 2E9 vesicles / mL - 3E13 vesicles / mL.

[0307] Embodiment 117. The method according to any one of Embodiments 112-115, wherein the synthetic fusion protein comprises a SARS-CoV-2 spike protein polypeptide.

[0308] Embodiment 118. The method according to any one of Embodiments 112-117, wherein the synthetic fusion protein comprises a SARS-CoV-2 nucleocapsid protein polypeptide.

[0309] Embodiment 119. The method according to any one of Embodiments 112-117, wherein the synthetic fusion protein comprises an influenza hemagglutinin protein polypeptide.

[0310] Embodiment 120. The method according to any one of Embodiments 112-117, wherein the synthetic fusion protein comprises a respiratory syncytial virus fusion (RSV F) protein polypeptide.

[0311] Embodiment 121. The method according to any one of embodiments 112-120, 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 an RSV F protein polypeptide on the outer surface.

[0312] Embodiment 122. The method according to any one of embodiments 112-121, wherein the elicited immune response comprises the production of neutralizing antibodies against the antigens present in the synthetic fusion protein.

[0313] Embodiment 123. The method according to any one of embodiments 112-123, wherein the elicited immune response comprises the production of anti-spike antibodies.

[0314] Embodiment 124. The method according to any one of embodiments 112-123, wherein the elicited immune response comprises a spike-specific T cell response.

[0315] Embodiment 125. The method according to any one of embodiments 112-124, wherein the elicited immune response comprises the production of anti-nucleocapsid antibodies.

[0316] Embodiment 126. The method according to any one of embodiments 112-125, wherein the elicited immune response comprises a nucleocapsid-specific T cell response.

[0317] Embodiment 127. The method according to any one of embodiments 112-126, wherein the elicited immune response comprises the production of anti-hemagglutinin antibodies.

[0318] Embodiment 128. The method according to any one of embodiments 112-127, wherein the elicited immune response comprises a hemagglutinin-specific T cell response.

[0319] Embodiment 129. The method according to any one of embodiments 112-128, wherein the elicited immune response comprises the production of anti-RSV F antibodies.

[0320] Embodiment 130. The method according to any one of embodiments 112-129, wherein the elicited immune response comprises an RSV F-specific T cell response.

[0321] Embodiment 131. The method according to any one of embodiments 112-130, wherein the immune response persists in the subject for up to nine months.

[0322] Embodiment 132. The method according to any one of embodiments 112-130, wherein the immune response persists in the subject for at least nine months.

[0323] Extracellular Vesicles and Exosomes

[0324] 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 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 (E. coli), Bacillus spp., Streptomyces spp., Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SJ9, CHO, COS (e.g., COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H, or 293F. See, e.g., Portolano et al., “Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach,” Journal of Visualized Experiments, October 2014, 92, e51897, pp. 1-8, for a description of recombinant proteins in 293 cells in suspension culture.

[0325] Extracellular vesicles (EVs) are lipid-bound vesicles secreted by cells into the extracellular space. The three main subtypes of EVs are microvesicles (MVs), exosomes, and apoptotic bodies, which are distinguished based on EV biogenesis, release pathway, size, content, and function. For a review of extracellular vesicles, see, e.g., Doyle and Wang, “Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis,” Cells, v. 8(7), 2019 Jul., and references therein.

[0326] Exosomes include small secreted vesicles with a diameter of about 20 - 200 nm, which are particularly released by mammalian cells and are prepared by budding into endosomes or by budding from the plasma membrane. In some cases, exosomes have a characteristic buoyant density of about 1.1 - 1.2 g / mL and a characteristic lipid composition. Their lipid membranes are typically rich in cholesterol and contain sphingomyelin, ceramides, lipid rafts, and exposed phosphatidylserine. Exosomes express certain marker proteins, such as integrins and cell adhesion molecules, but usually lack markers of lysosomes, mitochondria, or caveolae. In some embodiments, exosomes contain cell-derived components, such as but not limited to proteins, DNA, and RNA (e.g., microRNA [miR] and non-coding RNA). In some embodiments, exosomes can be obtained from cells obtained from an allogeneic, autologous, xenogeneic, or syngeneic source relative to the recipient of the exosomes.

[0327] Certain types of RNA, such as microRNA (miRNA), are known to be carried by exosomes. miRNAs typically act as post-transcriptional regulators by binding to complementary sequences on target messenger RNA transcripts (mRNAs), resulting in translational inhibition, target mRNA degradation, and / or gene silencing.

[0328] Useful exosomes can be obtained from any cell source, including prokaryotes, plants, fungi, metazoans, vertebrates, mammals, primates, humans, autologous cells, and allogeneic cells. See, e.g., Kim et al., “Platform technologies and human cell lines for the production of therapeutic exosomes,” Extracell Vesicles Circ Nucleic Acids 2021;2:3-17. For example, exosomes can be derived from mesenchymal stem cells, embryonic stem cells, iPS cells, immune cells, PBMCs, neural stem cells, HEK293 cells (which are described, e.g., in Dumont et al., “Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives,” Crit Rev Biotechnol 2016;36:1110-22), HEK293T cells (which are described, e.g., in Li et al., “Identification and characterization of 293T cell-derived exosomes by profiling the protein, mRNA and MicroRNA components,” PLoS One 2016;11:e0163043), 293F cells (which are described in Stenkamp et al., “Exosomes represent a novel mechanism of regulatory T cell suppression (P1079),” J Immunol May 1, 2013, 190(1 Suppl) 121.11), amniotic cells, CAR-T cells, cardiospheres, and cardiospherederived cells (CDC) (which are described, e.g., in WO2014028493, WO2022006178A1, US20210032598A1, US9828603B2, EP2914273A1, US20200316226A1, US20120315252A1, US20170360842A1, and the references therein).

[0329] Briefly, methods for preparing exosomes can include the following steps: culturing cells in a culture medium, separating the cells from the culture medium, purifying the exosomes by, for example, sequential centrifugation, and optionally, clarifying the exosomes on a density gradient such as a sucrose density gradient. In some cases, the isolated and purified exosomes are substantially free of non-exosomal components such as cellular components or whole cells. The exosomes can be resuspended in a buffer such as sterile PBS buffer containing 0.01%-1% human serum albumin. The exosomes can be frozen and stored for future use.

[0330] Exosomes can be collected, concentrated, and / or purified using methods known in the art. For example, differential centrifugation has emerged as the leading technique, where secreted exosomes are isolated from the supernatant of cultured cells. This method allows the separation of exosomes from larger extracellular vesicles and most non-granular contaminants by taking advantage of the size of exosomes. Exosomes can be prepared as described in a variety of papers, including but not limited to Fordjour et al. at https: / / www.biorxiv.org / content / 10.1101 / 545228vl, “A shared pathway of exosome biogenesis operates at plasma and endosome membranes”, bioRxiv, preprint posted February 11, 2019; 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 are using commercial kits such as but not limited to ExoSpin TM Exosome Purification Kit, Total Exosome Purification Kit, Exosome Isolation Kit and ExoCap TMExosome isolation kit. Methods for isolating exosomes from stem cells are described, 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 cells derived from cardiospheres are described, for example, in Ibrahim et al., “Exosomes as critical agents of cardiac regeneration triggered by cell therapy,” Stem Cell Reports, 2014. Specific methods include ultracentrifugation, density gradient, HPLC, affinity-based substrate adhesion, or size-exclusion-based filtration.

[0331] Size exclusion allows their separation from biochemically similar but biophysically distinct microvesicles with larger diameters of up to 1,000 nm. Differences in flotation velocity further allow the separation of exosomes of different sizes. Typically, exosome sizes will have a diameter in the range of 30 - 200 nm, including sizes of 40 - 100 nm. Further purification can rely on the specific properties of the particular exosomes of interest. This includes, for example, using immunoadsorption of the protein of interest to select specific vesicles with an ectoplasm or outward orientation.

[0332] In current methods, such as differential centrifugation, discontinuous density gradient, immunoaffinity, ultrafiltration, and high-performance liquid chromatography (HPLC), differential ultracentrifugation is most commonly used for exosome isolation. This technique uses centrifugal forces increasing from 2,000×g to 10,000×g and 100,000×g to separate medium and larger-sized particles and cell debris from the exosome pellet. Centrifugation alone allows significant isolation / collection of exosomes from conditioned media, although it may not be sufficient to remove various protein aggregates, genetic material, particles, and cell debris, which are common contaminants. Enhanced specificity of exosome purification can be achieved by deploying sequential centrifugation in combination with ultrafiltration, or equilibrium density gradient centrifugation in a sucrose density gradient to provide a higher purity exosome preparation (flotation density 1.1 - 1.2 g / mL) or by applying discrete sugar pads in the preparation.

[0333] Ultrafiltration can be used to purify exosomes without compromising their biological activity. Membranes with different pore sizes—such as 100 kDa cut-off molecular weight (MWCO) and gel filtration for eliminating smaller particles—have been used to avoid using non-neutral pH or non-physiological salt concentrations. Current available tangential flow filtration (TFF) systems are scalable (to >10,000 L), allowing not only purification but also concentration of the exosome fraction, and such methods are more time-saving than differential centrifugation. HPLC can also be used to purify exosomes into a more uniform size particle preparation and maintain their biological activity while the preparation is maintained at physiological pH and salt concentration. Other chemical methods have utilized the different solubility of exosomes for precipitation techniques, in addition to size-excluding polymers (e.g., polyethylene glycol (PEG)), possibly combined with additional rounds of centrifugation or filtration. For example, a precipitation reagent can be added to conditioned cell culture medium to rapidly and promptly precipitate a population of exosomes, although the precipitate prepared via this technique may be difficult to resuspend. Flow field-flow fractionation (FlFFF) is an elution-based technique for separating and characterizing 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 medium.

[0334] In addition to these techniques that rely on general biochemical and biophysical characteristics, focusing techniques can be applied to isolate specific exosomes of interest. This includes relying on antibody immunaffinity to recognize certain exosome-associated antigens. As described, exosomes also express the extracellular domain of membrane-bound receptors at the membrane surface. This provides an opportunity to isolate and separate exosomes associated with their parental cell origin based on a common antigen profile. Conjugation with magnetic beads (e.g., such as anti-CD81 magnetic beads), chromatographic matrices, plates, or microfluidic devices allows the isolation of specific exosome populations of interest, which may be related to their production from the parental cells of interest or related cellular regulatory states. Other affinity capture methods use lectins that bind to specific sugar residues on the exosome surface.

[0335] For example, exosomes (and other extracellular vesicles) can be produced via 293F cells. 293F cells can be transfected with a polynucleotide encoding a spike protein or a nucleocapsid protein or an influenza hemagglutinin protein or a chimeric fusion thereof (or transduced with a lentivirus carrying the polynucleotide), as described herein (see Figure 4A ), and express the spike protein or the nucleocapsid protein such that the spike protein or the nucleocapsid is sorted into and displayed in or on exosomes isolated from 293F cells. Exemplary procedures for preparing exosomes from 293F cells can include the following steps: 293F cells (Gibco TM, catalog number 51-0029, ThermoFisher Scientific, Waltham, MA), 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) contaminants. The cells can be maintained in FreeStyle TM 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 can be seeded in a shake flask at a density of 1.5E6 cells / ml at a volume of approximately 1 / 4 of the shake flask volume and grown at a shaking speed of approximately 110 rpm. HEK293 cells can be grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum.

[0336] To purify exosomes, 293F cells can be cultured continuously in a shake flask for a period of three days. Cells and large cell debris can be removed by centrifugation at 300 × g for 5 minutes, followed by centrifugation at 3,000 × g for 15 minutes. The resulting supernatant can be passed through a 0.22 μm sterile filtration unit (Thermo Fisher, catalog number 566-0020) to produce a clarified tissue culture supernatant (CTCS). The 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), concentrating approximately 120 mL of CTCS to approximately 0.5 mL. The concentrated CTCS can then be purified by size exclusion chromatography (SEC) in 1×PBS (qEV original column / 35 nm: Izon Science, catalog number SP5), where exosomes present in each 0.5 ml starting sample are eluted in three 0.5 ml fractions. The purified exosomes can be re-concentrated using an Ultra-4 100 kDa cut-off spin column (catalog number UFC810024). This method can produce a population of exosomes / small EVs with the expected ultrastructure and size distribution profile of human exosomes and containing the exosome marker proteins CD9 and CD63, with a concentration effect of approximately 500-fold and 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).

[0337] SARS-CoV-2 Protein

[0338] Membrane-bound vesicles containing one or more populations of SARS-CoV-2 structural proteins are disclosed. "Containing" means that the proteins contained can be within the lumen of the vesicle, displayed on the surface of the vesicle, or both within the lumen and on the surface. Here, those fusion proteins containing the polypeptide sequence of a tetraspanin protein are mostly displayed on the surface of the vesicle. Those proteins displayed on the surface of the vesicle can have a protein portion within the lumen, a protein portion spanning the vesicle membrane (i.e., a transmembrane region or domain), and a protein portion extending outside the vesicle. In one embodiment, the SARS-CoV-2 structural protein is the spike glycoprotein (S), nucleocapsid (N) protein, membrane (M) protein, 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 Figure 1A , Figure 1B , Figure 2A and FIG. 2C, which depict the spike / CD9 and nucleocapsid / CD fusion proteins for expressing spike or nucleocapsid antigens on the surface of exosomes.

[0339] In one embodiment, the antigen protein is the SARS-CoV-2 spike glycoprotein (also known as the spike protein or simply "spike"). The spike protein can be any variant of SARS-CoV-2, such as, for example, the Wuhan-1 strain, Omicron variants (e.g., BA.2 variant), Delta variants (e.g., B.1.617.2, AY.3, AY.103, AY.44, AY.43 variants, etc.), and epsilon variants (e.g., B.1.427 or B.1.429 variants), or any variant now known or not yet discovered. As used herein, unless otherwise specified, the term spike refers to any SARS-CoV-2 spike glycoprotein, chimera, or fragment thereof.

[0340] In some embodiments, the SARS-CoV-2 spike protein is the Wuhan-1 strain SARS-CoV-2 spike protein or the Delta variant SARS-CoV-2 spike protein; the furin protease-blocked trimer-stabilized form of the Wuhan-1 strain SARS-CoV-2 spike protein; the Wuhan-1 strain SARS-CoV-2 spike protein with the D614G amino acid change; the Wuhan-1 strain SARS-CoV-2 spike protein with the diproline substitution of 986KV987-to-986PP987 (S-2P); and / or the Wuhan-1 strain SARS-CoV-2 spike protein or equivalent (S-CSM) with the cleavage site mutation of 682RRAR685-to-682GSAG685.

[0341] Extracellular Vesicles Exhibiting Viral Proteins

[0342] In one embodiment, the present invention provides extracellular vesicles that express (also referred to as "display") spike proteins or nucleocapsids on their surfaces, and these extracellular vesicles can be used as vaccines against various variants of SARS-CoV-2. In one embodiment, the present invention provides extracellular vesicles that express (also known as "display") hemagglutinin proteins or neuraminidase proteins on their surfaces, and these extracellular vesicles can be used as vaccines against various 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 trimer stabilization mutations, pre-fusion conformation stabilization mutations (e.g., diproline stabilization mutations), and furin cleavage site mutations.See, 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,” Science 367, 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 20200; and references therein.

[0343] Here, a C-terminal tetraspanin protein and a SARS-CoV-2 spike protein ( Figure 1A and Figure 1B ) and a SARS-CoV-2 nucleocapsid protein ( Figure 2A and Figure 2B) or a synthetic fusion protein of the influenza hemagglutinin protein. It is important to note that the nucleocapsid protein is a soluble protein that is not expressed at the surface of the virus, so engineered fusion proteins containing a four-transmembrane protein and other transmembrane domains can place the nucleocapsid protein on the surface of the vesicle to provide an accessible antigen for immunization (see Figure 2B ).

[0344] In one embodiment, exosomes expressing the spike protein or the nucleocapsid protein on their surface are prepared from 293F cells expressing the spike / nucleocapsid protein. Moving on to Figure 4A , in a specific exemplary embodiment, the packaging cell (300) is transfected with a plasmid (301a) encoding a spike-four transmembrane protein or a nucleocapsid-four transmembrane protein or a hemagglutinin-four transmembrane protein fusion protein and plasmids (302a and 303a) encoding lentiviral structural proteins. Lentiviral proteins (302b and 303b) are produced and incorporated into the fusion protein RNA to form a lentiviral vector (304) containing the fusion protein RNA. The host cell (311) is transduced with the lentivirus (304) carrying the fusion protein RNA, enabling the production of the SARS-CoV-2 spike or nucleocapsid-four transmembrane protein fusion protein (307a), which is sorted to the plasma membrane (307b)( Figure 4B ) and the surface of exosomes produced by the transduced host cell (Figure 4C, Figure 5A , Figure 5B and Figure 6C).

[0345] Moving on to Figures 5A - 5C and Figure 6A and Figure 6B , exosomes are isolated from 293F cells carrying the spike-CD9 construct ( Figure 1A and Figure 1B ). Figure 5A Shows the size distribution of those exosomes from about 50 nm to about 270 nm, with a median of about 100 - 150 nm. Figure 5B Shows the expression of the spike-containing fusion protein in the transduced 293F cells (lane 4), and the enriched expression / display of the spike-containing fusion protein in exosomes derived from the transduced 293F cells (lane 5). Figure 5C Shows significant expression of the spike-containing fusion protein on those exosomes as determined by spike flow cytometry.

[0346] Transmission electron microscopy confirmed that the spike protein is expressed on the surface of exosomes derived from spike-CD9 transduced 293F cells ( Figure 5A and Figure 5B arrows point to spikes at the vesicle surface).

[0347] Moving on to Figures 7A - 7C, exosomes were isolated from 293F cells carrying the nucleocapsid-CD9 construct ( Figure 2A and Figure 2B ). Figure 7A shows the size distribution of those exosomes from about 50 nm to about 270 nm, with a median of about 100 - 150 nm. Figure 7B shows 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 on those exosomes as determined by spike flow cytometry. In one embodiment, the expression of the nucleocapsid-CD9 fusion protein is placed under the control of a tet-inducible promoter. Here, exosomes derived from uninduced but transduced host cells do not express nucleocapsid (see Figure 7C the second curve from the left).

[0348] Moving on to Figures 8A - 8D , 293F cells were transfected with a hemagglutinin (e.g., H3)-CD9 construct ( Figure 3A and Figure 3B ) or transduced with virions encoding the construct. Figure 8A and Figure 8B show significant expression of the influenza H3-containing fusion protein on / in adherent 293F cells and 293F cells in suspension, respectively (~90% - 96% in this example), as determined by anti-H3 flow cytometry. Exosomes produced by 293F cells expressing the H3 fusion protein were purified and H3 and H3-CD9 were evaluated by Western blot ( Figure 8C , inset 1 and inset 2). Dual detection of both H3 and CD90 at slightly below 180 kDa in lane 6 of inset 1 and inset 2 shows a significant loading of the H3-CD9 fusion protein in 293F-derived exosomes. Additionally, Figure 8D shows significant expression of the spike-containing fusion protein on those exosomes as determined by spike flow cytometry. In one embodiment, exosomes produced by 293F cells expressing the influenza H3-CD9 fusion protein were combined with micron-sized magnetic beads conjugated to anti-human CD81 (an exosome marker) and flow cytometry was performed. Here, approximately 54% of those exosomes expressing CD81 express the influenza H3 antigen ( Figure 8D ).

[0349] Moving on to Figure 9, on day 1, administer (710) to subject mice by intramuscular injection with 1× dose (i.e., 3E10 vesicles) or 10× dose (i.e., 3E11 vesicles) or vesicles containing approximately 0.3 ng, approximately 3 ng, or approximately 10 ng of a fusion protein (comprising spike-CD9, nucleocapsid-CD9, or influenza H3-CD9 fusion protein). On day 14, collect blood from the administered mice and evaluate the early humoral immune response (720) (see Figure 10A and Figures 12A - 12F ). On day 21, the subject mice receive a second dose of exosomes expressing the subject spike or nucleocapsid (730). On day 35, collect blood from the administered mice and evaluate the humoral immune response, including antibody production (see Figure 10B and Figures 11A - 11C ) and neutralizing antibody production (740). On day 40, collect splenocytes from the administered mice and evaluate the cellular immune response by ELISpot assay (750) (see Figure 10C , Figure 10D , Figure 13C and Figure 13D ).

[0350] Go to Figure 10A and Figure 10B , as early as two weeks after the first injection ( Figure 10A ) and continuing for at least until day 35 after injection ( Figure 10B ), an immune response to exosomes expressing the COVID-19 spike antigen (STX-S) is induced in the subject at the humoral level. Additionally, using nanogram amounts of spike protein expressed on exosomes and without the inclusion of an adjuvant, a robust and persistent anti-spike antibody response is elicited. Here, for example, as measured on day 14 and day 35, in the absence of an adjuvant, as little as 10 ng of exosomes expressing the spike protein elicits a significant and useful antibody response. Further, compared to 32 ng of purified spike protein (without exosomes) with an adjuvant, 32 ng of exosomes expressing the spike protein without an adjuvant elicits a significantly ≥30-fold higher antibody response. It is generally known in the art that prior art spike protein vaccines are administered in microgram amounts and with an adjuvant, producing an antibody response similar to that observed with the nanogram amounts of exosomes expressing the spike protein antigen of the present invention.

[0351] Go to Figure 10C and Figure 10D, as shown by the ELISpot assay performed on splenocytes obtained on days 35 - 40, an immune response against STX - S was induced at the cellular level in the subject. Here, a dose of as little as 10 ng of exosomes expressing the spike antigen without adjuvant elicited a significant CD4+ T - cell response against the spike protein in the subject, as shown in the IL4 ELISpot assay of the subject's splenocytes (Figure 810C). Also here, 10 ng and 32 ng doses of exosomes expressing the spike antigen without adjuvant elicited a significant CD8+ cytotoxic T - cell response against the spike protein in the subject, as shown in the IFNγ ELISpot assay of the subject's splenocytes( Figure 10D ).

[0352] Go to Figures 11A - 11C , administration of nanogram amounts of STX - S to the subject elicited effective neutralization of both the Delta and Omicron variants of SARS - CoV - 2 induced by STX - S exosome injection. In one embodiment, sera from day 40 of subjects administered approximately 3.2 ng STX - S per injection (dose 2, Figure 11A ) and sera from days 14 and 40 of subjects administered approximately 9.8 ng STX - S per injection (dose 4, Figure 11A ) were tested for neutralizing antibodies against the SARS - CoV - 2 Delta variant. Here, STX - S in each test sample elicited effective neutralizing activity( Figure 11A ), comparable to SARS - CoV - 2 Delta - positive vaccine sera. STX - S demonstrated dose - dependent neutralization of the virus, as estimated by the ability to protect infected cells from virus - induced cytopathic effects (compare dose 4 with dose 2 in day 40 sera, Figure 11A ).

[0353] Thus, according to one embodiment, STX - S engineered exosome vaccine induced neutralizing antibodies against the Delta spike in one or more subjects by day 14 (after a single i.m. injection). By day 40, i.e., ~4 weeks after STX - S boost, robust neutralization was observed in all subjects regardless of the dose( Figure 11A ). Here, a single injection of approximately 9 ng of STX - S spike (dose 4, Figure 11A ) produced approximately 65% - 75% neutralization of the Delta variant. In addition, at doses of approximately 3 - 9 ng of spike delivered by STX - S exosomes, full immunization (i.e., initial injection and at least one booster or two i.m. injections) resulted in approximately 80% - 85% neutralization against the Delta variant.

[0354] Neutralization induction against the Delta variant triggered by administration of STX-S in subjects elicited a response comparable to that of human control plasma (CoV02-Delta, plasma from patients with breakthrough Delta infections immunized with Moderna's mRNA vaccine), with complete neutralization response at higher dilutions (see, e.g., 1:320, STX-S dose 4, day 40)( Figure 11A ).

[0355] Additionally, turning to Figure 11B and Figure 11C , neutralizing antibodies against the SARS-CoV-2 Omicron variants (Omicron BA.1 and BA.5.2.1) were also tested in serum samples collected on day 40 from subjects administered a dose of approximately 9 - 10 ng of STX-S (here, the spike protein of STX-S is the Delta variant - STX-S δ ). As shown in Figure 11B and Figure 11C , after complete immunization with a dose of approximately 9 - 10 ng of Delta spike delivered by STX-S exosomes (i.e., at least 2 i.m. injections), strong cross-neutralization was observed for sera obtained from subjects treated with STX-S δ , achieving neutralization of approximately 84% of the Omicron BA1 variant( Figure 11B ) and 16% to 97% of the Omicron BA5 variant( Figure 11C ) in this embodiment. Thus, in some embodiments, administration of an immunogenic composition containing STX-S exosomes with a single variant spike provides a certain level of protective immunity against other SARS-CoV-2 variants.

[0356] Since such low amounts of antigen-expressing exosomes (nanogram amounts, which are 1,000 - fold less than standard protein or subunit vaccines) are required to elicit immunity as measured by neutralization, antibody titers, and IL4 / IFNγ, specific exosomes expressing different antigens can be combined to provide a multi-vaccine or immunogenic composition. For example, exosomes expressing SARS-CoV-2 antigens (e.g., STX-S) can be combined with exosomes expressing influenza antigens (e.g., hemagglutinin, e.g., H3).

[0357] Turning to Figures 12A - 12F , a vaccine combination containing both STX-S and exosomes expressing influenza hemagglutinin 3 antigen on their surface (STX-H3) was administered to murine subjects. Sera obtained from mice 14 days after injection with a single low dose of STX-H3( Figures 12A - 12C , the second and third from the left) showed significant levels of anti-H3 IgG. As in the case of STX-S dose sizes and STX-N dose sizes (below and Figures 13A - 13D), low doses (3E10 to 3E11 exosomes represent low nanogram amounts of H3 antigen) elicited a significant IgG response in mice.

[0358] Combine low nanogram amounts of STX-S and STX-H3 and administer to murine subjects. Serum obtained on day 14 from these mice showed significant elicitation of IgG against the H3 antigen ( Figures 12A - 12C , fourth bar from the left) and against the spike antigen ( Figures 12D - 12F , right bar). Thus, low nanogram doses of exosomes expressing viral antigens can be combined to prepare multivalent vaccines. Here, for example, nanogram amounts of STX-S and STX-H3 are combined to prepare an immunogenic composition capable of eliciting strong antibody responses against both SARS-CoV-2 and influenza.

[0359] Similar nanogram doses of adjuvant-free exosomes expressing the SARS-CoV-2 nucleocapsid protein also elicited useful, robust, and significant humoral and cell-mediated immune responses ( Figures 13A - 13D ). The inventors envision that any or most viral antigens expressed on the surface of exosomes according to the present disclosure (such as, for example, influenza hemagglutinin, influenza neuraminidase, respiratory syncytial virus, and / or other viral glycoproteins or other proteins or fragments thereof) will elicit humoral and / or cell-mediated immune responses in recipient subjects.

[0360] Thus, in some embodiments, a low-dose immunogenic composition or vaccine is provided that contains an immunogenic dose of: viral antigen (e.g., SARS-CoV-2 spike, SARS-CoV-2 nucleocapsid, or influenza hemagglutinin or combinations thereof) between 1 ng and 1 μg (including endpoints), 1 - 900 ng, 1 - 800 ng, 1 - 700 ng, 1 - 600 ng, 1 - 500 ng, 1 - 400 ng, 1 - 300 ng, 1 - 200 ng, 1 - 100 ng, 1 - 90 ng, 1 - 80 ng, 1 - 70 ng, 1 - 60 ng, 1 - 50 ng, 1 - 40 ng, 5 - 100 ng, 5 - 90 ng, 5 - 80 ng, 5 - 70 ng, 5 - 60 ng, 5 - 50 ng, 5 - 40 ng, ≤100 μg, ≤50 μg, ≤25 μg, ≤20 μg, ≤10 μg, ≤1 μg, ≤900 ng, ≤800 ng, ≤700 ng, ≤600 ng, ≤500 ng, ≤400 ng, ≤300 ng, ≤200 ng, ≤100 ng, ≤90 ng, ≤80 ng, ≤70 ng, ≤60 ng, ≤50 ng, ≤40 ng, about 1 ng, about 1.5 ng, about 2 ng, about 2.5 ng, about 3 ng, about 3.5 ng, about 4 ng, about 4.5 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about 18 ng, about 19 ng, about 20 ng, about 25 ng, about 30 ng, about 35 ng, about 40 ng, about 45 ng, or about 50 ng of viral antigen (e.g., SARS-CoV-2 spike or nucleocapsid).

[0361] Go to Figure 13A and Figure 13B , induce an immune response to exosomes expressing the COVID-19 nucleocapsid antigen (STX-N) at the humoral level in a subject, and detect it on day 35 after injection ( Figure 13A and Figure 13B ). In addition, using nanogram amounts of spike protein expressed on exosomes and without the inclusion of an adjuvant, a robust and persistent anti-spike antibody response is elicited. Here, for example, as measured on day 35, as little as 3.2 ng of exosomes expressing the nucleocapsid protein without an adjuvant elicit a significant and useful antibody response ( Figure 13A , STX-N dose 2). Thus, in one embodiment, for a dose of about 3 ng / injection (dose 2, Figure 9 A), the complete immunization cycle (two i.m. injections) induces a significant 3-fold increase in IgG against the SARS-CoV-2 nucleocapsid protein (N) relative to PBS, up to 10 ng / injection (dose 3,Figure 13B ) a 10-fold increase.

[0362] Go to Figure 13C and Figure 13D , to characterize the T cell response to STX-N, the antigen-specific T cell response to the nucleocapsid protein was measured by an ELISpot assay performed on splenocytes obtained on day 40. Here, vaccination of subjects with STX-N elicited a multifunctional, antigen-specific T cell response against the SARS-CoV-2 nucleocapsid protein on day 40 (after the booster (second) injection). Administration of STX-N resulted in an effective IFNγ response ( Figure 13C and Figure 13D ). Here, assessment of IFNγ-secreting cells in response to ex vivo nucleocapsid protein stimulation showed a 6-fold increase in spleens immunized with STX-N ( Figure 13C and Figure 13D ), indicating a Th1-biased CD8+ T cell response. Since the nucleocapsid protein is not a surface protein and would most likely be presented to immune cells only after the virus is processed within infected cells, this response is of inestimable importance for the development of exosomes expressing immunogenic compositions with the potential to protect against a broad range of SARS-CoV-2 variants.

[0363] As in the humoral antibody response against the nucleocapsid protein ( Figure 13A , Figure 13B ), in this particular embodiment, in the absence of an adjuvant, administration of STX-N in the range of 3 ng to 10 ng (this range is three orders of magnitude lower than that of prior art protein subunit vaccines) achieved a significant CD8+ / IFNγ response against the nucleocapsid protein in subjects.

[0364] In addition to their excellent immune response elicitation, the spike exosome vaccine, nucleocapsid exosome vaccine, hemagglutinin exosome vaccine, and RSV exosome vaccine each exhibit several other advantages over currently available vaccines. First, the exosome-based vaccines of the present invention deliver antigens through a fully endogenous lipid bilayer that can be easily integrated into the host cell membrane and promote engineered antigen presentation to immune cells. Membrane-bound antigens can be easily presented to circulating immune cells for rapid activation of the response, while free antigens contained within exosomes can additionally be processed by the lysosomal system and activate cytotoxic T lymphocyte responses. Thus, the utilization of a natural delivery system promotes effective delivery and response compared to synthetic lipid nanoparticle technology.

[0365] Second, spike, nucleocapsid, RSV, and hemagglutinin exosome vaccines are protein-based vaccines, making the antigen readily available to the subject's immune system without the need for translation in host cells as required for RNA-based vaccines. Incomplete translation and / or incorrect folding of the mRNA encoding the spike, nucleocapsid, or hemagglutinin protein limits the amount of antigen available after vaccination, making the immune response highly variable and reducing efficacy.

[0366] Third, the exosome-based spike, nucleocapsid, hemagglutinin, and RSV fusion protein vaccines of the present invention do not require adjuvants or synthetic lipid nanoparticles (LNPs) for delivery and immune response. Traditional protein-based vaccines require adjuvants (such as, for example, aluminum salts and squalene oil-in-water emulsion systems MF59 (Novartis) and AS03 (GlaxoSmithKline)) (see, for example, Wong, S.S. and R.J. Webby, “Traditional and new influenza vaccines,” ClinMicrobiol Rev, 2013.26(3): pp. 476-92) to enhance and coordinate the immune response and affect the affinity, specificity, magnitude, and functional profile of B and T cell responses. Current mRNA vaccines do not use adjuvants, which, in combination with the need for mRNA translation for antigen availability, may also reduce long-term efficacy. Currently approved COVID vaccines use LNPs to deliver the mRNA of the protein or adjuvants, and undesirable side effects have been reported.

[0367] Fourth, the exosomal spike fusion protein, nucleocapsid fusion protein, hemagglutinin fusion protein, and RSV fusion protein immunogenic compositions 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 5 μg to 25 μg of antigen in combination with an adjuvant to induce immunity (see, e.g., Formica et al., “Different dose regimens of a SARS-CoV-2 recombinant spike protein vaccine (NVX-CoV2373) in younger and older adults: A phase 2 randomized placebo-controlled trial,” PLoS Med, 2021.18(10): p.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 antibodies and CD4 T cell responses after a single immunization in mice. EBioMedicine, 2021.63: p. 103197; and the references therein). Here, immunological data from subjects immunized with approximately 1 / 1000 of the amount of protein antigen currently administered in currently clinically approved vaccines (i.e., nanogram amounts versus microgram amounts) with spike fusion exosomes or nucleocapsid fusion exosomes elicited complete immunity, which was associated with high antibody levels, strong virus neutralization, broad variant activity, and both B and T cell memory.

[0368] Fifth, an additional limitation of available vaccines is the need for refrigeration and the short shelf life of the product at higher temperatures. Due to the high stability of exosomes at physiological pH and temperature, exosomes, including display exosomes, can be stored at 4°C for longer periods. Exosomal compositions expressing spike fusions or nucleocapsid fusions can also be lyophilized for long-term storage at convenient temperatures (see, e.g., U.S. Patent Application No. 2017 / 0360842A1).

[0369] Thus, here, when the spike protein, nucleocapsid protein, or hemagglutinin protein is delivered via exosomes, a strong CD8+ T cell response and a strong B cell response are induced, as shown by IgG production and potentially neutralizing antibodies. While not wishing to be bound by theory, this result can be explained by the role of extracellular vesicles in cell-cell communication and antigen presentation. In particular, multiple copies of the spike protein ( Figure 6A and Figure 6B ), nucleocapsid protein, or hemagglutinin protein can be present on the surface of the vesicles, facilitating crosslinking with the B cell receptor. In addition, the spike protein in an extracellular vesicle-based vaccine can indirectly activate B cells and CD8(+) T cells through antigen cross-presentation.

[0370] It is also envisioned that exosomes expressing the fusion proteins of the present invention can be engineered to express antigens of interest to target new COVID variants and / or problematic influenza strains. The antigens of interest can be easily exchanged and adapted as needed. The antigen can be expressed as a fusion protein (e.g., a chimera), such as by fusing an exosome expression or display domain with an antigen domain, to enable the display of the antigen on the exosome for delivery to the host subject's immune system.

[0371] It is also envisioned that exosomes can be engineered to selectively target organs or tissues of interest and allow for the safe and targeted delivery of antigens to specific immune subsystems to elicit a specific type of response in a subject.

[0372] Characterization of STX-S and STX-H3 Cells and Exosomes

[0373] Moving on to Figure 14A , high expression of the spike was detected on the cell surface by flow cytometry (right curve). The parental non-engineered 293F cells do not express the SARS-CoV-2 spike protein, as expected.

[0374] Moving on to Figure 14B , enrichment of the spike protein in exosomes was confirmed by Jess automated Western blotting (lane 5: STX-S exosomes). Here, lane 1: marker, lane 2: non-engineered 293F cells, lane 3: non-engineered 293F exosomes, lane 4: STX-S cells, and lane 6: spike protein.

[0375] Moving on to Figure 15A , high expression of influenza hemagglutinin 3 (H3) was detected on the cell surface by flow cytometry (right curve). The parental non-engineered 293F cells do not express the influenza H3 protein (left curve), as expected.

[0376] Moving on to Figure 15B, the enrichment of H3 protein in exosomes was confirmed by Jess automated Western blotting (lane 5: STX-H3 exosomes). Here, lane 1: marker, lane 2: non-engineered 293F cells, lane 3: non-engineered 293F exosomes, lane 4: STX-H3 cells, lane 5: STX-H3 exosomes.

[0377] Go to Figure 16A , CD81 was detected on STX exosomes by flow cytometry using bead assays (right curve), compared to no signal from the isotype control antibody (left curve).

[0378] Go to Figure 16B , spike protein was detected on STX-S exosomes (right curve), but not on 293F parental exosomes (left curve).

[0379] Go to Figure 16C , H3 protein was detected on STX-H3 exosomes (right curve), but not on 293F parental exosomes (left curve).

[0380] Figures 17 - 19 show the immune responses to a combination (designated STX-S + H3) vaccine of intramuscularly injected spike-expressing exosomes (STX-S) and H3-expressing exosomes (STX-H3) in a mouse model. Here, mice were injected on day 1, blood was collected on day 14, mice received a booster injection (IM) on day 21, blood was collected on day 35, and splenocytes were obtained on day 40. As analyzed by ELISA, the STX-S + H3 vaccine induced robust expression of influenza H3 and SARS-CoV-2 spike antibodies in mice after 1 (day 14) and 2 (day 35) IM injections. In all studies, PBS was used as a vehicle control. It was also observed that the STX-S + H3 vaccine induced T cell responses against both H3 and spike after in vitro stimulation, as shown by IFNg ELISPOT on isolated splenocytes ( Figure 19A and Figure 19B ). All data presented in Figures 17 - 19 are shown as mean ± SEM. **** p < 0.0005, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant, one-way ANOVA. Here, N = 10 / experimental group.

[0381] Go to Figure 17A , IgG against H3 was observed on day 14 after a single IM injection.

[0382] Go to Figure 17B, IgG against the spike was observed on day 14 after a single IM injection.

[0383] Go to Figure 18A , IgG against H3 was observed on day 35 after full immunization (two IM injections).

[0384] Go to Figure 18B , IgG against the spike was observed on day 35 after full immunization (two IM injections).

[0385] Go to Figure 19A , an IFNg response to in vitro H3 stimulation was observed.

[0386] Go to Figure 19B , an IFNg response to in vitro spike stimulation was observed.

[0387] Multivalent Vaccine Against COVID-19, Influenza, and / or Respiratory Syncytial Virus

[0388] Vesicles expressing respiratory syncytial virus - F

[0389] Construct various constructs of the RSV F protein to discover the stable and native conformation of the F protein that confers robust and stable immunity in subjects, especially when presented on the surface of vesicles such as exosomes. Figure 20Depicts five construct forms of RSV F. RSV F form 1 (“V1”) is an engineered form of DS-Cav1 as described in: McLellan et al., “Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus,” Science, 1 Nov 2013, Vol. 342, No. 6158, pp. 592-598, doi:10.1126 / science.1243283. RSV F form 2 (“V2”) starts with the V1 DS-Cav1 engineered form with the polyadenylation signal removed across the F gene. RSV F form 3 (“V3”) starts with the V2 engineered form with two furin cleavage site mutations as described in: Brakel et al., “Coexpression of respiratory syncytial virus (RSV) fusion (F) protein and attachment glycoprotein (G) in a vesicular stomatitis virus (VSV) vector system provides synergistic effects against RSV infection in a cotton rat model,” Vaccine, 2021 Nov 16; 39(47):6817-6828. doi:10.1016 / J.vaccine.2021.10.042. Epub 2021 Oct 23. RSV F form 4 (“V4”) starts with the V2 engineered form with the deletion of most of the N-terminal furin cleavage sites as described in: Patel et al., “Flexible RSV Prefusogenic Fusion Glycoprotein Exposes Multiple Neutralizing Epitopes that May Collectively Contribute to Protective Immunity,” Vaccines 2020, 8(4), 607; doi.org / 10.3390 / vaccines8040607. A specific form of the V4 construct is exemplified in SEQ ID NO:20.

[0390] Various RSV engineered constructs were expressed in cells, and protein stability, expression levels, and exosome loading were determined. Here, the V1 construct was observed to show low RSV F protein expression in multiple transduced 293F cell lines generated. Cells that did show high RSV F expression (∼10%) were sorted by FACS, but 50% of those cells lost expression within one week. Here, the V2 construct, which had the polyadenylation signal spanning the F gene removed, was also poorly expressed in the transduced 293F cell line.

[0391] The V3 construct, which had two furin cleavage sites removed, showed a high percentage (∼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 the RSV F V3-CD9 chimera with the top 10% RSV F expression were sorted by FACS. CD9 and RSV F protein expression were measured 1 week and 2 weeks after FACS sorting. Both the unsorted pool and the sorted pool showed loss of RSV F protein expression (using anti-AM22), however, CD9 expression remained at a high level, indicating loss of V3 expression. Exosomes generated from the sorted pool by 293F showed very low to no RSV F on the exosomes.

[0392] In addition, the in vivo humoral immune response to exosomes expressing RSV F V3-CD9 was tested, and mice showed a low antibody response against the RSV F protein on day 35 after two injections (Figure 22, Figures A and B). Here, two batches of exosomes expressing the V3 RSV F-CD9 chimeric fusion protein, where batch #1 generated 2.0E12 exosomes / mL at a protein concentration of approximately 43 ng / mL, and batch #2 generated 1.8E12 exosomes / mL at a protein concentration of approximately 26 ng / mL. Batches #1 (2 mL) and #2 (1.3 mL) were combined to obtain 3.3 mL of combined batch #1.2 with a final concentration of 25.27 ng / mL. 10 mice received two injections (using 100 ul of the stock solution) at ∼3 ng / injection. Antibodies were detected on day 14 ( Figure 22A ) and day 35 ( Figure 22B ) after injection, but the immune response was insufficient to elicit a good humoral immunity.

[0393] The V4 construct was observed to have improved (relative to V3) expression levels and stability in transduced 293F cells, as determined by anti-RSV F flow cytometry using antigen site antibodies (AM22, D25) and two RSV neutralizing site II monoclonal antibodies (palivizumab, motavizumab) and JESS western blot.

[0394] Test the in vivo humoral immune response to exosomes expressing RSV F V4-CD9 and observe a strong immune response elicited on days 14 and 35 (Figure 23, Figures A and B), with a significant enhancement effect on day 35 ( Figure 23B ). Here, the RSV concentration on the exosomes is approximately 250 μg / mL, and the total protein is 990 μg / mL / 1.9E12 exosomes / mL.

[0395] Table 1

[0396] Exosomes / mL RSV (μg / mL) Dose Size (μg) Dose (ng / 100 mL) Dose (ng / 50 mL) 1.90E+12 250.0 25 1.00E+12 131.6 13.16 1.00E+11 13.2 1.32 1315.79 657.89 3.00E+10 4.0 0.40 398.72 199.36 1.00E+10 1.3 0.13 131.58 65.79 1.00E+9 0.65 0.065 6.5 3.25

[0397] In some embodiments, the RSV F protein is fused to a tetraspanin protein. In a particular embodiment, the RSV F protein is fused to CD9, i.e., as part of an engineered chimeric protein. Here, the engineered chimeric protein contains an RSV F polypeptide at the N-terminus, preferably the RSV F V4 construct (SEQ ID NO:18), followed by a linker, and then the CD9 protein at the C-terminus ( Figure 21A ), which allows the RSV F protein to be expressed on the outer surface of the vesicle ( Figure 21B ). In a particular embodiment, the RSV F-CD9 chimeric construct has the amino acid sequence of SEQ ID NO:20.

[0398] Combined vaccine

[0399] In one embodiment, the vaccine or immunogenic composition of the present invention contains a combination of vesicles expressing the SARS-CoV2 spike and vesicles expressing influenza hemagglutinin ( Figure 24 ). In another embodiment, the vaccine or immunogenic composition of the present invention contains a combination of vesicles expressing the SARS-CoV2 spike and vesicles expressing the respiratory syncytial virus fusion protein (RSV F) ( Figure 25 ). In another embodiment, the vaccine or immunogenic composition of the present invention contains a combination of vesicles expressing influenza hemagglutinin and vesicles expressing the respiratory syncytial virus fusion protein (RSV F) ( Figure 26 ). In another embodiment, the vaccine or immunogenic composition of the present invention contains a combination of vesicles expressing the SARS-CoV2 spike, vesicles expressing RSV F, and vesicles expressing influenza hemagglutinin ( Figure 27 ).

[0400] In one embodiment, a combination of exosomes expressing SARS-CoV2 spike (STX-S), exosomes expressing RSV F (STX-RSV), and exosomes expressing influenza hemagglutinin (STX-H3) was injected into mice either as a single formulation or in combination with each other to verify the induction of an antibody response. For spike and H3, 10 - 15 ng of protein was used for immunization. For RSV, 130 ng of RSV protein was used for immunization. Antibody responses were analyzed in all groups on day 14 (after 1 injection) (Figure 28) and day 35 (after a booster injection on day 21, full immunization) (Figure 29).

[0401] Here, it was observed that injection of STX-S, STX-H3, and STX-RSV induced the production of virus type-specific antibodies when injected as exosomes expressing a single viral antigen type and in combination with one or more other exosomes expressing another viral antigen. A distinct booster effect was also observed. In addition, no immune interference was observed when injecting two or more exosome virus species.

[0402] All patents and other publications cited throughout this application, including literature references, issued patents, published patent applications, and co-pending patent applications, such as methods described in such publications that can be used in combination with the techniques described herein, are hereby expressly incorporated by reference for the purposes of description and disclosure. These publications are provided only for their disclosure prior to the filing date of this application. In this regard, nothing should be construed as an admission by the present inventors that they are not entitled to rely on prior invention or for any other reason prior to such disclosure. With respect to dates or statements, all representations as to the content of these documents are based on information available to the applicant and do not constitute any admission as to the correctness of the dates or content of these documents.

[0403] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed. While specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications within the scope of the present disclosure are possible, as will be recognized by those skilled in the relevant art. For example, while method steps or functions are presented in a particular order, alternative embodiments may perform the functions in a different order or may perform the functions substantially concurrently. The teachings of the present disclosure provided herein may be applied to other programs 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 necessary to incorporate the features, functions, and concepts of the above references and applications to provide yet additional embodiments of the present disclosure. In addition, some changes to the protein structure may be made without affecting the biological or chemical action in terms of the type or amount, due to considerations of biological functional equivalence. These and other changes may be made to the present disclosure in accordance with the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0404] The specific elements of any of the above embodiments may be combined or replaced with elements in other embodiments. Additionally, while the advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments need to 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 exosome polypeptide, and the second fusion protein comprises a second viral polypeptide and an exosome 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 exosome polypeptide, the second fusion protein comprises a second viral polypeptide and an exosome polypeptide, and the third fusion protein comprises a third viral polypeptide and an exosome polypeptide.

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

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

5. The immunogenic composition according to claim 3, wherein the excipient comprises a cryoprotectant.

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

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

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

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

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

13. The immunogenic composition according to claim 11, wherein the first viral polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO:1 or identical to SEQ ID NO:

1.

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

15. The immunogenic composition according to claim 14, wherein the first viral polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO:5 or identical to SEQ ID NO:

5.

16. The immunogenic composition according to any one of claim 10, wherein the second viral polypeptide is an influenza hemagglutinin protein polypeptide.

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

18. The immunogenic composition according to claim 17, wherein the second viral polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO: 14 or identical to SEQ ID NO:

14.

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

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

21. The immunogenic composition according to claim 20, wherein the third viral polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO: 18 or identical to SEQ ID NO:

18.

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

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

24. The immunogenic composition according to claim 22, wherein the exosome polypeptide comprises an amino acid sequence that is 80% identical to SEQ ID NO: 10 or identical to SEQ ID NO:

10.

25. The immunogenic composition according to any one of claims 1-13 and 17-24, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: 2 or identical to SEQ ID NO:

2.

26. The immunogenic composition according to claim 22, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: 3 or identical to SEQ ID NO:

3.

27. The immunogenic composition according to claim 10, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: 6 or identical to SEQ ID NO:

6.

28. The immunogenic composition according to claim 10, wherein the first fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: 7 or identical to SEQ ID NO:

7.

29. The immunogenic composition according to claim 10, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: 15 or identical to SEQ ID NO:

15.

30. The immunogenic composition according to claim 10, wherein the second fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: 16 or identical to SEQ ID NO:

16.

31. The immunogenic composition according to claim 20, wherein the third fusion protein comprises an amino acid sequence that is 80% identical to SEQ ID NO: 19 or identical to SEQ ID NO:

19.

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

20.

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

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

36. The method according to claim 35, wherein the immunogenically effective dose elicits protective immunity against the first virus and the second virus in the subject.

37. The method according to claim 35, wherein the immunogenically effective dose elicits protective immunity against the first virus, the second virus and the third virus in the subject.

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

39. The method according to claim 35, wherein the immunogenic composition comprises a SARS-CoV-2 spike protein polypeptide, an influenza hemagglutinin polypeptide and a respiratory syncytial virus (RSV) fusion protein polypeptide, and the first virus is SARS-CoV2, the second virus is an influenza virus, and the third virus is RSV.

40. The method according to claim 35, wherein the immunogenically effective dose elicits protective immunity against the SARS-CoV-2 Delta variant and the SARS-CoV-2 Omicron variant in the subject.

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

42. The method according to claim 35, further comprising administering to the subject a second effective dose of the immunogenic composition of claim 1 or claim 2.

43. The method according to claim 35, wherein the protective immunity elicited comprises (a) high antibody titers against the first virus, the second virus and the third virus, (b) a CD4+ T cell response against the first virus, the second virus and the third virus, and (c) a CD8+ cytotoxic T cell response against the first virus, the second virus and the third virus.

44. A synthetic fusion protein, the synthetic fusion protein comprising a viral polypeptide and an exosome polypeptide.

45. The synthetic fusion protein according to claim 44, further comprising a linker polypeptide located between the viral polypeptide and the exosome polypeptide.

46. The synthetic fusion protein according to claim 45, further comprising a hinge polypeptide located between the viral polypeptide and the exosome polypeptide.

47. The synthetic fusion protein according to claim 46, the synthetic fusion protein further comprising a transmembrane domain polypeptide located between the viral polypeptide and the exosome polypeptide.

48. The synthetic fusion protein according to claim 44, wherein the exosome polypeptide is a tetraspanin polypeptide.

49. The synthetic fusion protein according to any one of claims 44 or 48, wherein the exosome polypeptide is a CD9 polypeptide.

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

51. The synthetic fusion protein according to any one of claims 44 or 50, wherein the viral polypeptide is a SARS-CoV-2 spike protein polypeptide.

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

53. The synthetic fusion protein according to claim 51, wherein the fusion protein comprises, in order from the amino terminus to the carboxyl terminus, a SARS-CoV-2 spike protein polypeptide, a linker polypeptide, and a CD9 polypeptide.

54. The synthetic fusion protein according to claim 53, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

3.

55. The synthetic fusion protein according to any one of claims 53 or 54, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:

3.

56. The synthetic fusion protein according to any one of claims 44 or 50, wherein the viral polypeptide is a SARS-CoV-2 nucleocapsid protein polypeptide.

57. The synthetic fusion protein according to claim 56, wherein the fusion protein comprises, in order from the amino terminus to the carboxyl terminus, a signal peptide, a SARS-CoV-2 nucleocapsid protein polypeptide, a hinge region, a transmembrane domain polypeptide, a linker polypeptide, and a CD9 polypeptide.

58. The synthetic fusion protein according to claim 57, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

7.

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

7.

60. The synthetic fusion protein according to any one of claims 44 or 45, wherein the viral polypeptide is an influenza protein polypeptide.

61. The synthetic fusion protein according to claim 60, wherein the influenza protein polypeptide comprises hemagglutinin.

62. The synthetic fusion protein according to claim 61, wherein the influenza protein polypeptide comprises hemagglutinin 3 (H3).

63. The synthetic fusion protein according to claim 61 or 62, wherein the fusion protein comprises a hemagglutinin protein polypeptide, a linker polypeptide, and a CD9 polypeptide in order from the amino terminus to the carboxy terminus.

64. The synthetic fusion protein according to claim 63, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

16.

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

16.

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

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

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

69. The synthetic fusion protein according to claim 68, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

20.

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

20.

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

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

13.

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

13.

74. The synthetic polynucleotide according to claim 71, comprising a nucleic acid sequence that has at least 80% identity to SEQ ID NO:

11.

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

11.

76. The synthetic polynucleotide according to claim 71, comprising a nucleic acid sequence that has at least 80% identity to SEQ ID NO:

4.

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

4.

78. The synthetic polynucleotide according to claim 71, comprising a nucleic acid sequence that has at least 80% identity to SEQ ID NO:

12.

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

12.

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

8.

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

8.

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

22.

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

22.

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

17.

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

17.

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

23.

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

23.

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

21.

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

21.

90. A cell, said cell comprising the synthetic polynucleotide of claim 71.

91. The cell according to claim 90, wherein said cell is a metazoan cell.

92. The cell according to claim 91, wherein said cell is a vertebrate cell.

93. The cell according to claim 92, wherein said cell is a mammalian cell.

94. The cell according to claim 93, wherein said cell is a primate cell.

95. The cell according to claim 94, wherein said cell is a human cell.

96. The cell according to claim 90 or 95, wherein said cell is a primary cell.

97. The cell according to claim 90 or 95, wherein said cell is a human embryonic kidney cell.

98. The cell according to claim 97, wherein said cell is a 293 cell.

99. The cell according to claim 90 or 95, wherein said cell is produced by transducing said cell with a lentivirus comprising said synthetic polynucleotide.

100. The cell according to claim 90, wherein said cell comprises the synthetic fusion protein according to claim 44.

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

102. The vesicle according to claim 101, wherein said vesicle is an exosome.

103. The vesicle according to claim 101 or 102, wherein said vesicle has a diameter of about 50 - 500 nm.

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

105. The vesicle according to claim 101 or 102, wherein the vesicle expresses the SARS-CoV-2 nucleocapsid protein polypeptide on its surface.

106. The vesicle according to claim 101 or 102, wherein the vesicle expresses the influenza hemagglutinin protein polypeptide on its surface.

107. The vesicle according to claim 101 or 102, wherein the vesicle expresses the respiratory syncytial virus fusion protein polypeptide on its surface.

108. A method for preparing the vesicle of claim 101, comprising culturing the cells of claim 90 in a cell culture medium, collecting the cell culture medium, and purifying more than one vesicle comprising the vesicle from the cell culture medium.

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

110. The method according to claim 105, wherein the induction comprises contacting the cells with tetracycline, doxycycline or an analogue thereof.

111. The method according to claim 105, wherein the induction comprises removing tetracycline, doxycycline or an analogue thereof from the cells.

112. A method for eliciting an immune response in a subject, comprising administering to the subject a first dose of an immunogenic composition, the immunogenic composition comprising more than one vesicle, the more than one vesicle comprising the vesicle of claim 101 or a vesicle produced by the method according to claim 108, wherein the synthetic fusion protein of claim 44 is expressed on the outer surface of the vesicle.

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

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

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

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

117. The method according to claim 112, wherein the synthetic fusion protein comprises the SARS-CoV-2 spike protein polypeptide.

118. The method according to claim 112, wherein the synthetic fusion protein comprises the SARS-CoV-2 nucleocapsid protein polypeptide.

119. The method according to claim 112, wherein the synthetic fusion protein comprises the influenza hemagglutinin protein polypeptide.

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

121. The method according to 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 an RSV F protein polypeptide on the outer surface.

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

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

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

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

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

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

128. The method according to claim 112 or 121, wherein the elicited immune response comprises a hemagglutinin-specific T cell response.

129. The method according to claim 112 or 121, wherein the elicited immune response comprises the production of anti-RSV F antibodies.

130. The method according to claim 112 or 121, wherein the elicited immune response comprises an RSV F-specific T cell response.

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

132. The method according to claim 112 or 121, wherein the immune response persists in the subject for at least nine months.

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