SARS-COV-2 Immunogenic Compositions and Methods

Abstract

The present disclosure relates to compositions and methods for vaccinating a subject against multiple SARS-CoV-2 variants, including the creation and delivery to the subject of extracellular vesicles that express engineered spike proteins and / or engineered nucleocapsid proteins on their surface. The present invention also relates to compositions and methods for the design, preparation, manufacturing, formulation, and / or use of spike- and nucleocapsid-displaying vesicle vaccines designed to elicit strong humoral and cellular immune responses against multiple SARS-CoV-2 variants. TIFF2025529904000023.tif80128

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the priority dates of U.S. Provisional Application No. 63 / 373,418, filed August 24, 2022, U.S. Provisional Application No. 63 / 411,090, filed September 28, 2022, U.S. Provisional Application No. 63 / 413,193, filed October 4, 2022, U.S. Provisional Application No. 63 / 437,710, filed January 8, 2023, and U.S. Provisional Application No. 63 / 456,380, filed March 31, 2023, the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated by reference in its entirety. The XML copy, created on August 18, 2023, is named 514PCT.xml and is 27,456 bytes in size. [Background technology]

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

[0004] Current major vaccines utilize mRNA-lipid nanoparticle or viral vector technology, while more recent vaccines utilize recombinant proteins (see, e.g., Krammer, F., SARS-CoV-2 vaccines in development. Nature, 2020. 586(7830): pp. 516-527). Compared with the major mRNA vaccines, recombinant protein vaccines or inactivated protein vaccines are safe and reliable approaches for immunization, but generally suffer from weak immunogenicity and therefore require formulation with appropriate adjuvants. All current mRNA vaccine candidates achieve strong initial immune responses against the virus, resulting in reduced hospitalization rates, but all lack long-term protection. It has been reported that antibody levels decline significantly over time, and therefore, their effectiveness in protecting against infection also declines significantly (see, for example, Xiang, T., et al., Declining Levels of Neutralizing Antibodies Against SARS-CoV-2 in Convalescent COVID-19 Patients One Year Post-Symptom Onset. Front Immunol, 2021.12:p.708523). Furthermore, mRNA vaccines to date lack cross-reactivity to emerging variants of concern and require multiple booster injections to maintain protection against the virus. Therefore, there remains a significant medical and public health need for a better, globally applicable vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to provide improved, broader, and more durable neutralization of SARS-CoV-2, more robust T cell responses, and an enhanced safety profile.

[0005] Briefly, the problem to which the present invention may be directed includes the emergence of SARS-CoV-2 variants of concern that have greater infectivity and lower immunogenicity than some variants of less concern, creating an ongoing public health and medical problem of vaccine ineffectiveness against new strains and new strains with reduced immunogenicity (long-term and short-term immunogenicity).

[0006] Solutions to this problem are disclosed, including safe (LNP-free, adjuvant-free) and effective vaccines that confer long-lasting humoral and cell-mediated immunity against multiple variants, emerging variants, and refractory variants of SARS-CoV-2, comprising extracellular vesicles displaying spike proteins from a single variant of SARS-CoV-2 that confer robust humoral and / or cell-mediated immunity against several variants of concern or of note of SARS-CoV-2, and optionally comprising extracellular vesicles displaying nucleocapsid proteins from a single variant of SARS-CoV-2 that also confer robust humoral and / or cell-mediated immunity against several variants of concern or of note of SARS-CoV-2. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Krammer, F., SARS-CoV-2 vaccines in development. Nature, 2020.586(7830):p.516-527 [Non-patent document 2] Xiang,T.,et al.,Declining Levels of Neutralizing Antibodies Against SARS-CoV-2 in Convalescent COVID-19 Patients One Year Post Symptom Onset.Front Immunol,2021.12:p.708523 Summary of the Invention

[0008] overview In one embodiment, a synthetic fusion protein is provided that contains a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosomal tetraspanin protein, the fusion protein being designed and engineered such that the SARS-CoV-2 protein antigen can be expressed on the surface of exosomes, enabling an immune response to be elicited when administered to a subject.

[0009] In one aspect, the SARS-CoV-2 protein is a spike protein. In another aspect, the SARS-CoV-2 protein is a nucleocapsid protein. In one aspect, the exosomal tetraspanin protein is a CD9 protein.

[0010] In some aspects where the SARS-CoV-2 protein is a spike protein and the exosomal tetraspanin protein is a CD9 protein, the spike protein polypeptide is located N-terminal to the CD9 protein polypeptide. In some cases, a linker peptide is located between the spike protein polypeptide and the CD9 protein polypeptide, as shown in Figure 1A.

[0011] In some aspects where the SARS-CoV-2 protein is a nucleocapsid protein and the exosomal tetraspanin protein is a CD9 protein, the nucleocapsid protein polypeptide is located N-terminally of the CD9 protein polypeptide. In some cases, a signal peptide is located N-terminally of the nucleocapsid protein polypeptide. In some cases, as shown in Figure 2A, a hinge peptide, a transmembrane domain peptide, and a linker peptide are located between the spike protein polypeptide and the CD9 protein polypeptide.

[0012] In some aspects where the SARS-CoV-2 protein is a spike protein, the SARS-CoV-2 spike protein polypeptide contains one or more mutations, for example, a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and / or a diproline substitution (2P [986KV987-to-986PP987]).

[0013] In one embodiment, a polynucleotide is provided that encodes a synthetic fusion protein containing a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosomal tetraspanin protein, the encoded fusion protein being designed and engineered such that the SARS-CoV-2 protein antigen can be expressed intracellularly and sorted onto the surface of exosomes, enabling an immune response to be elicited when the exosomes are administered to a subject.

[0014] In one aspect, the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide. In another aspect, the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide. In one aspect, the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide.

[0015] In some aspects where the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded spike protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some cases, a linker peptide is encoded by the polynucleotide such that it is positioned between the spike protein polypeptide and the CD9 protein polypeptide in the encoded synthetic fusion protein, as shown in Figure 1A.

[0016] In some aspects where the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded nucleocapsid protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide. In some cases, a signal peptide is positioned N-terminal to the nucleocapsid protein polypeptide. In some cases, as shown in Figure 2A, a hinge peptide, a transmembrane domain peptide, and a linker peptide are encoded by the polynucleotide such that they are positioned between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein.

[0017] In some aspects where the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide, the encoded SARS-CoV-2 spike protein polypeptide contains one or more mutations, e.g., a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and / or a diproline substitution (2P [986KV987-to-986PP987]).

[0018] In one embodiment, a cell is provided that contains a polynucleotide encoding a synthetic fusion protein containing a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosomal tetraspanin protein, the encoded fusion protein being designed and engineered such that the SARS-CoV-2 protein antigen is expressed intracellularly and sorted onto the surface of exosomes, enabling an immune response to be elicited when the exosomes are administered to a subject.

[0019] In some aspects, the cell is a metazoan cell. In some aspects, the cell is a vertebrate cell. In some aspects, the cell is a mammalian cell. In some aspects, the cell is a primate cell. In some aspects, the cell is a human cell. In some aspects, the cell is a primary cell. In some aspects, the cell is an established cell line. In some aspects, the cell is a human embryonic kidney cell. In some aspects, the cell is a HEK293 cell. In some aspects, the cell is a 293F cell.

[0020] In one aspect, the polynucleotide in the cell encodes a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide. In another aspect, the polynucleotide in the cell encodes a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein polypeptide. In one aspect, the polynucleotide in the cell encodes an exosomal tetraspanin protein polypeptide that is a CD9 protein polypeptide.

[0021] In some aspects, where a polynucleotide in a cell encodes a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide and an exosomal tetraspanin protein polypeptide that is a CD9 protein polypeptide, the encoded spike protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some cases, the polynucleotide encodes a linker peptide sequence positioned between the spike protein polypeptide and the CD9 protein polypeptide in the encoded synthetic fusion protein, as shown in Figure 1A.

[0022] In some aspects where a polynucleotide in a cell encodes a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein polypeptide and an exosomal tetraspanin protein polypeptide that is a CD9 protein polypeptide, the encoded nucleocapsid protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide. In some cases, the polynucleotide encodes a signal peptide positioned N-terminal to the nucleocapsid protein polypeptide. In some cases, as shown in Figure 2A, the polynucleotide encodes a hinge peptide, a transmembrane domain peptide, and a linker peptide positioned between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein.

[0023] In some aspects, where the cell contains a polynucleotide encoding a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide, the encoded SARS-CoV-2 spike protein polypeptide contains one or more mutations, e.g., a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and / or a diproline substitution (2P [986KV987-to-986PP987]).

[0024] In one embodiment, a cell is provided that contains a synthetic fusion protein containing a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosomal tetraspanin protein, the synthetic fusion protein being designed and engineered such that the SARS-CoV-2 protein antigen is expressed intracellularly and sorted onto the surface of exosomes produced by the cell, enabling an immune response to be elicited when the exosomes are administered to a subject.

[0025] In some aspects, the cell is a metazoan cell. In some aspects, the cell is a vertebrate cell. In some aspects, the cell is a mammalian cell. In some aspects, the cell is a primate cell. In some aspects, the cell is a human cell. In some aspects, the cell is a primary cell. In some aspects, the cell is an established cell line. In some aspects, the cell is a human embryonic kidney cell. In some aspects, the cell is a HEK293 cell. In some aspects, the cell is a 293F cell.

[0026] In one aspect, the cell contains a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide. In another aspect, the cell contains a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein polypeptide. In one aspect, the cell contains an exosomal tetraspanin protein polypeptide that is a CD9 protein polypeptide.

[0027] In some aspects where the cell contains a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide and the exosomal tetraspanin protein is a CD9 protein, the spike protein polypeptide is located N-terminal to the CD9 protein polypeptide of the included synthetic fusion protein. In some cases, a linker peptide is located between the spike protein polypeptide and the CD9 protein polypeptide of the included synthetic fusion protein, as shown in Figure 1A.

[0028] In some aspects, where a cell contains a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein polypeptide and an exosomal tetraspanin protein polypeptide that is a CD9 protein polypeptide, the nucleocapsid protein polypeptide is positioned N-terminal to the CD9 protein polypeptide. In some cases, a signal peptide is positioned N-terminal to the nucleocapsid protein polypeptide of the contained synthetic fusion protein. In some cases, as shown in Figure 2A, a hinge peptide, a transmembrane domain peptide, and a linker peptide are positioned between the spike protein polypeptide and the CD9 protein polypeptide of the contained synthetic fusion protein.

[0029] In some aspects where the cell contains a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide, the SARS-CoV-2 spike protein polypeptide contains one or more mutations, for example, a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and / or a diproline substitution (2P [986KV987-to-986PP987]).

[0030] In one embodiment, a cell is provided that is produced by transducing a cell with a lentivirus containing a synthetic polynucleotide encoding a synthetic fusion protein containing a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosomal tetraspanin. The encoded fusion protein is designed and engineered such that the SARS-CoV-2 protein antigen is expressed intracellularly and sorted onto the surface of exosomes, enabling an immune response to be elicited when the exosomes are administered to a subject.

[0031] In some aspects, the cell is a metazoan cell. In some aspects, the cell is a vertebrate cell. In some aspects, the cell is a mammalian cell. In some aspects, the cell is a primate cell. In some aspects, the cell is a human cell. In some aspects, the cell is a primary cell. In some aspects, the cell is an established cell line. In some aspects, the cell is a human embryonic kidney cell. In some aspects, the cell is a HEK293 cell. In some aspects, the cell is a 293F cell.

[0032] In one aspect, the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide. In another aspect, the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide. In one aspect, the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide.

[0033] In some aspects where the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded spike protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some cases, a linker peptide is encoded by the polynucleotide such that it is positioned between the spike protein polypeptide and the CD9 protein polypeptide in the encoded synthetic fusion protein, as shown in Figure 1A.

[0034] In some aspects where the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide and the encoded exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide, the encoded nucleocapsid protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide. In some cases, a signal peptide is positioned N-terminal to the nucleocapsid protein polypeptide. In some cases, as shown in Figure 2A, a hinge peptide, a transmembrane domain peptide, and a linker peptide are encoded by the polynucleotide such that they are positioned between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein.

[0035] In some aspects where the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide, the encoded SARS-CoV-2 spike protein polypeptide contains one or more mutations, e.g., a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and / or a diproline substitution (2P [986KV987-to-986PP987]).

[0036] In one embodiment, vesicles are provided that contain a synthetic fusion protein containing a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosomal tetraspanin protein, the fusion protein being designed and engineered such that the SARS-CoV-2 protein antigen can be expressed on the surface of exosomes, enabling an immune response to be elicited when administered to a subject.

[0037] In one aspect, the vesicles contain a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide. In another aspect, the vesicles contain a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein polypeptide. In one aspect, the vesicles contain an exosomal tetraspanin protein polypeptide that is a CD9 protein.

[0038] In some aspects where the vesicle contains a SARS-CoV-2 spike protein polypeptide and an exosomal tetraspanin CD9 protein polypeptide, the spike protein polypeptide is located N-terminal to the CD9 protein polypeptide. In some cases, a linker peptide is located between the spike protein polypeptide and the CD9 protein polypeptide, as shown in Figure 1A.

[0039] In some aspects, in which the vesicles contain a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein and an exosomal tetraspanin protein polypeptide that is a CD9 protein, the nucleocapsid protein polypeptide is located N-terminal to the CD9 protein polypeptide. In some cases, a signal peptide is located N-terminal to the nucleocapsid protein polypeptide. In some cases, as shown in Figure 2A, a hinge peptide, a transmembrane domain peptide, and a linker peptide are located between the spike protein polypeptide and the CD9 protein polypeptide.

[0040] In some aspects where the vesicles contain a SARS-CoV-2 protein polypeptide that is a spike protein, the SARS-CoV-2 spike protein polypeptide contains one or more mutations, for example, a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and / or a diproline substitution (2P [986KV987-to-986PP987]).

[0041] In one aspect, the vesicles have a diameter of approximately 50-500 nm. In one aspect, the vesicles are synthetic vesicles. In one aspect, the vesicles are produced by cells. In one aspect, the vesicles are extracellular vesicles. In one aspect, the vesicles are microvesicles. In one aspect, the vesicles are exosomes. In one aspect, the vesicles are apoptotic bodies. In one aspect, the vesicles express the CD81 protein on their surface.

[0042] In some aspects, the lumen of the vesicle contains a cargo molecule. In some cases, the surface-displayed protein or polypeptide is a targeting ligand that functions to target the vesicle to a receptive target and deliver the cargo molecule. In some aspects, the cargo molecule is a nucleic acid, e.g., a PMO, an antisense oligonucleotide, a peptide, a polypeptide, or a protein, a hydrophobic small molecule drug, a hydrophilic small molecule drug, an imaging agent, an aptamer, a capture molecule, a nanobody, an antibody of a fragment thereof, a receptor tyrosine kinase, or the like. For example, spike-expressing vesicles can contain immune effector molecule cargo.

[0043] In one embodiment, a method is provided for producing vesicles containing a synthetic fusion protein containing a polypeptide sequence of a SARS-CoV-2 protein fused with a polypeptide sequence of an exosomal tetraspanin protein. The fusion protein is designed and engineered to express a SARS-CoV-2 protein antigen on the surface of exosomes, enabling an immune response to be elicited when administered to a subject. Here, cells containing a polynucleotide encoding the synthetic fusion protein are cultured in cell culture medium so that the vesicles are secreted into the medium by the cells, and the cell culture medium is then collected, after which the vesicles are purified.

[0044] In some aspects, the cells cultured to produce vesicles are metazoan cells. In some aspects, the cells are vertebrate cells. In some aspects, the cells are mammalian cells. In some aspects, the cells are primate cells. In some aspects, the cells are human cells. In some aspects, the cells are primary cells. In some aspects, the cells are established cell lines. In some aspects, the cells are human embryonic kidney cells. In some aspects, the cells are HEK293 cells. In some aspects, the cells are 293F cells.

[0045] In some aspects, expression of the polynucleotide to generate the synthetic fusion protein is constitutive. In other aspects, expression of the polynucleotide to generate the synthetic fusion protein is induced in the cell. In one aspect, induction is carried out by contacting the cell with tetracycline, doxycycline, or an analog thereof. In another aspect, induction is carried out by removing tetracycline, doxycycline, or an analog thereof from the cell or cell culture medium.

[0046] In one aspect, the polynucleotide encodes and is expressed in the cell a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide. In another aspect, the polynucleotide encodes and is expressed in the cell a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein polypeptide. In one aspect, the polynucleotide encodes and is expressed in the cell a SARS-CoV-2 protein polypeptide that is a CD9 protein polypeptide.

[0047] In some aspects where a polynucleotide in a cell encodes a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide and an exosomal tetraspanin protein polypeptide that is a CD9 protein polypeptide, the spike protein polypeptide is positioned N-terminal to the CD9 protein polypeptide in the encoded and expressed synthetic fusion protein. In some cases, the polynucleotide encodes a linker peptide sequence positioned between the spike protein polypeptide and the CD9 protein polypeptide in the encoded and expressed synthetic fusion protein, as shown in Figure 1A.

[0048] In some aspects, where a polynucleotide in a cell encodes a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein polypeptide and an exosomal tetraspanin protein polypeptide that is a CD9 protein polypeptide, the encoded nucleocapsid protein polypeptide is positioned N-terminal to the encoded CD9 protein polypeptide in the encoded and expressed fusion protein. In some cases, the polynucleotide encodes a signal peptide positioned N-terminal to the nucleocapsid protein polypeptide. In some cases, as shown in Figure 2A, the polynucleotide encodes a hinge peptide, a transmembrane domain peptide, and a linker peptide positioned between the spike protein polypeptide and the CD9 protein polypeptide in the encoded and expressed synthetic fusion protein.

[0049] In some aspects in which the cell contains a polynucleotide that encodes and expresses a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide as part of an expressed fusion protein, the encoded and expressed SARS-CoV-2 spike protein polypeptide contains one or more mutations, e.g., a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and / or a diproline substitution (2P [986KV987-to-986PP987]).

[0050] In one embodiment, an immunogenic composition is provided that contains a plurality of vesicles containing a synthetic fusion protein, wherein the synthetic fusion protein contains a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosomal tetraspanin protein. The fusion protein is designed and engineered such that the SARS-CoV-2 protein antigen can be expressed on the surface of exosomes, enabling an immune response to be elicited when administered to a subject.

[0051] In some aspects, the immunogenic composition comprises (i) a first plurality of vesicles containing a first synthetic fusion protein containing the polypeptide sequence of a first SARS-CoV-2 protein fused to the polypeptide sequence of an exosomal tetraspanin protein, and (ii) a second plurality of vesicles containing a second synthetic fusion protein containing the polypeptide sequence of a second SARS-CoV-2 protein fused to the polypeptide sequence of an exosomal tetraspanin protein. For example, in one aspect, the polypeptide sequence of the first SARS-CoV-2 protein is a spike protein polypeptide sequence, and the polypeptide sequence of the second SARS-CoV-2 protein is a nucleocapsid protein polypeptide sequence.

[0052] In one aspect, the immunogenic composition contains a plurality of vesicles containing a synthetic fusion protein in which the SARS-CoV-2 protein polypeptide is a spike protein polypeptide. In another aspect, the immunogenic composition contains a plurality of vesicles containing a synthetic fusion protein in which the SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide. In another aspect, the immunogenic composition contains a plurality of vesicles containing a combination of vesicles, some of which contain a synthetic fusion protein in which the SARS-CoV-2 protein polypeptide is a spike protein polypeptide and other of which contain a synthetic fusion protein in which the SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide. In one aspect, the immunogenic composition contains a plurality of vesicles containing a synthetic fusion protein in which the exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide.

[0053] In some aspects, where the immunogenic composition contains a plurality of vesicles containing a synthetic fusion protein in which the SARS-CoV-2 protein polypeptide is a spike protein polypeptide and the exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide, the spike protein polypeptide is positioned N-terminal to the CD9 protein polypeptide. In some cases, a linker peptide is positioned between the spike protein polypeptide and the CD9 protein polypeptide, as shown in Figure 1A.

[0054] In some aspects, the immunogenic composition contains a plurality of vesicles containing a synthetic fusion protein in which the SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide and the exosomal tetraspanin protein polypeptide is a CD9 protein. In some aspects, the nucleocapsid protein polypeptide is positioned N-terminal to the CD9 protein polypeptide. In some cases, a signal peptide is positioned N-terminal to the nucleocapsid protein polypeptide. In some cases, as shown in Figure 2A, a hinge peptide, a transmembrane domain peptide, and a linker peptide are positioned between the spike protein polypeptide and the CD9 protein polypeptide.

[0055] In some aspects where the immunogenic composition contains a plurality of vesicles containing a synthetic fusion protein in which the SARS-CoV-2 protein polypeptide is a spike protein polypeptide, the SARS-CoV-2 spike protein polypeptide contains one or more mutations, for example, a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and / or a di-proline substitution (2P [986KV987-to-986PP987]).

[0056] In some aspects, the immunogenic composition contains a plurality of vesicles containing a synthetic fusion protein at a concentration of about 2E9 to 3E13 vesicles / mL. In aspects where the immunogenic composition contains two types of vesicles, one type containing a first synthetic fusion protein and another type containing a second synthetic fusion protein, each vesicle type can be provided at a concentration of about 1E9 to 2E13 vesicles / mL or 2E9 to 3E13 vesicles / mL.

[0057] In some aspects, the immunogenic composition contains a plurality of vesicles containing a synthetic fusion protein at a concentration of about 0.3 ng / mL to 3 μg / mL of synthetic fusion protein. In aspects where the immunogenic composition contains two types of vesicles, one type containing a first synthetic fusion protein and another type containing a second synthetic fusion protein, each vesicle type can be provided at a fusion protein concentration of about 0.1 ng / mL to 1.5 μg / mL or about 0.3 ng / mL to 3 μg / mL.

[0058] In some aspects, the immunogenic composition further comprises one or more pharmaceutically acceptable excipients. In one aspect, the immunogenic composition does not comprise an adjuvant.

[0059] In some aspects, the vesicles of the plurality of vesicles of the immunogenic composition have an average diameter of about 50-500 nm. In one aspect, the vesicles are synthetic vesicles. In one aspect, the vesicles are produced by cells. In one aspect, the vesicles are extracellular vesicles. In one aspect, the vesicles are microvesicles. In one aspect, the vesicles are exosomes. In one aspect, the vesicles are apoptotic bodies. In one aspect, the vesicles express CD81 protein on their surface.

[0060] In one embodiment, a method for inducing an immune response in a subject is provided by administering to the subject a dose of the immunogenic composition described in the above embodiment. In some aspects, the subject is administered multiple doses, for example, a second dose administered a period of time after the first dose and / or a subsequent booster dose. In some embodiments, the period (e.g., between doses) is 14 days to 1 year.

[0061] In one embodiment, a dose contains about 100 μL to 1 mL of the immunogenic composition. In some aspects, the immunogenic composition contains about 0.1 ng / mL to 3 μg / mL of the synthetic fusion protein expressed on the surface of vesicles. In some aspects, the immunogenic composition contains about 2.81E9 to 2.81E13 vesicles / mL expressing the synthetic fusion protein on their surface.

[0062] In some aspects, the immune response induced in the subject is the production of neutralizing antibodies against a virus, e.g., SARS-CoV-2, influenza, etc. In some aspects, the immune response induced in the subject is the production of neutralizing antibodies against two or more SARS-CoV-2 variants, e.g., the delta variant, the omicron variant, or other currently known or undiscovered variants of note or concern. In some aspects, the immune response induced in the subject is the production of anti-spike antibodies. In some aspects, the immune response induced in the subject is the production of anti-nucleocapsid antibodies. In some aspects, the immune response induced in the subject is a spike-specific T cell response, e.g., a CD4+ response and / or a CD8+ response. In some aspects, the immune response induced in the subject is a nucleocapsid-specific T cell response, e.g., a CD4+ response and / or a CD8+ response. [Brief explanation of the drawings]

[0063] [Figure 1]Figure 1A shows a linear representation of a SARS-CoV-2 spike-CD9 fusion protein with the amino terminus on the left and the carboxy terminus on the right, with a linker sequence positioned between the spike and CD9 protein polypeptides. Figure 1B shows the relationship of the SARS-CoV-2 spike-CD9 fusion protein of Figure 1A to a vesicle membrane with the amino terminus on the left and the carboxy terminus on the right. The spike protein polypeptide spans the membrane once, with its amino terminus facing the exterior of the vesicle. The linker is positioned on the luminal side of the membrane. The CD9 protein polypeptide spans the membrane four times, with its carboxy terminus facing the lumen of the vesicle. [Figure 2] Figure 2A shows a linear diagram of a SARS-CoV-2 nucleocapsid CD9 fusion protein, with the amino terminus on the left and the carboxy terminus on the right, in which an amino-terminal signal peptide is fused to a nucleocapsid protein polypeptide, which is fused to a hinge region peptide, which is fused to a transmembrane domain peptide, which is fused to a linker peptide, which is fused to a CD9 protein polypeptide. Figure 2B shows a diagram of the SARS-CoV-2 nucleocapsid CD9 fusion protein of Figure 2A, with the amino terminus on the left and the carboxy terminus on the right, in relation to a vesicle membrane. The nucleocapsid protein polypeptide with the amino-terminal signal sequence is positioned externally (cytoplasmically or externally), and a linker sequence connects the nucleocapsid protein polypeptide to the transmembrane domain peptide, which spans the membrane and is then connected to a lumenally positioned linker, which connects the carboxy terminus to the CD9 protein polypeptide, which spans the membrane four times toward the lumen of the vesicle. [Figure 3] Figure 3A is a flow chart showing the elements and steps for lentiviral vector (304)-mediated generation of cells expressing spike protein polypeptide fusion proteins using packaging cells (301) and host cells (311). Figure 3B is a histogram showing relative fluorescence intensity flow analysis of host cells expressing spike protein on their surface. [Figure 4]Figure 4A is a graph showing the concentration of spike-expressing exosomes per milliliter as a function of exosome diameter in nanometers. Figure 4B is a Western blot stained for SARS-CoV-2 spike protein. From left to right, the first column represents lanes loaded with size markers, the second column represents lanes loaded with untransduced 293F (host cell) proteins, the third column represents lanes loaded with proteins derived from exosomes from untransduced 293F cells, the fourth column represents lanes loaded with proteins derived from 293F cells constitutively expressing the spike fusion protein, the fifth column represents lanes loaded with proteins derived from exosomes from 293F cells constitutively expressing the spike fusion protein, and the sixth column represents lanes loaded with the spike fusion protein. Figure 4C is a histogram showing the relative fluorescence intensity flow analysis of exosomes expressing the spike on their surface. The left curve represents exosomes derived from 293F cells that do not express the spike-CD9 fusion protein, and the right curve represents exosomes derived from 293F cells that do express the spike-CD9 fusion protein. [Figure 5] Figure 5A shows a transmission electron micrograph of exosomes expressing the SARS-CoV-2 spike protein. The inset is a magnified image showing exosomes decorated with spike protein. The arrows point to the SARS-CoV-2 spikes around the exosomes. Figure 5B shows a higher magnification transmission electron micrograph of exosomes expressing the SARS-CoV-2 spike protein. The arrows point to the SARS-CoV-2 spikes around the exosomes. [Figure 6]Figure 6A is a graph showing the concentration of nucleocapsid-expressing exosomes per milliliter as a function of exosome diameter in nanometers. Figure 6B is a Western blot stained for SARS-CoV-2 nucleocapsid protein. From left to right, the first column represents lanes loaded with size markers, the second column represents lanes loaded with nucleocapsid protein, the third column represents lanes loaded with protein from exosomes derived from non-transduced 293F cells, the fourth column represents lanes loaded with protein from non-transduced 293F cells, and the fifth column represents lanes loaded with protein from exosomes derived from 293F cells expressing the nucleocapsid fusion protein. Figure 6C is a histogram showing the relative fluorescence intensity flow analysis of exosomes with and without nucleocapsid protein expressed on their surface. From left to right, the first curve represents exosomes derived from unmodified 293F cells, the second curve represents exosomes derived from uninduced 293F cells harboring a polynucleotide encoding a nucleocapsid-CD9 fusion protein, the third curve represents exosomes derived from 293F cells expressing a nucleocapsid-CD9 fusion protein, the third smaller curve represents exosomes derived from 293F cells expressing a nucleocapsid-CD9 fusion protein under tet induction, and the third larger curve represents exosomes derived from 293F cells harboring a CD9 knockout and expressing a nucleocapsid-CD9 fusion protein. [Figure 7A] Figure 7 is a timeline showing the process of generating an immune response by administering an immunogenic composition containing exosomes expressing a SARS-CoV-2 fusion protein on their surface. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 8A]Figures 8A-8D. STX-S exosomes elicited robust immune responses using significantly lower antigen concentrations than recombinant protein vaccines. Data are shown as mean ± SEM. ****p<0.0005, ***p<0.001, **p<0.01, *p<0.05, ns=non-significant, *one-way ANOVA; ##p<0.01, #p<0.05, #-two-tailed t-test. Figure 8A is a histogram showing the fold change in antibody titers against the spike protein in 1:100 diluted serum from mice on day 14 as a function of the dose in nanograms of spike-expressing exosomes. The leftmost bar represents the PBS control; the second bar represents a 10 ng dose of spike-expressing exosomes (STX-S); the third bar represents a 32 ng dose of STX-S; and the rightmost bar represents serum from mice injected with a 32 ng dose of spike protein combined with adjuvant. Figure 8B is a histogram showing the fold change in antibody titer against spike protein in 1:100 diluted serum from mice on day 35 as a function of the dose in nanograms of spike-expressing exosomes. The leftmost bar represents the PBS control; the second bar represents a 10 ng dose of spike-expressing exosomes (STX-S); the third bar represents a 32 ng dose of STX-S; and the rightmost bar represents serum from mice injected with a 32 ng dose of spike protein combined with adjuvant. FIG. 8C is a bar graph showing an ELISpot assay showing the number of spots producing IL4 in response to spike protein as a function of antigen dose.From left to right on the x-axis, the first bar represents non-spike-containing wells derived from PBS control splenocytes; the second bar represents reactivity to spike protein derived from PBS control splenocytes; the third bar represents non-spike-containing wells derived from splenocytes of a subject administered 10 ng STX-S; the fourth bar represents reactivity to spike protein derived from splenocytes of a subject administered 10 ng STX-S; the fifth bar represents non-spike-containing wells derived from splenocytes of a subject administered 32 ng STX-S; the sixth bar represents reactivity to spike protein derived from splenocytes of a subject administered 32 ng STX-S; the seventh bar represents non-spike-containing wells derived from splenocytes of a subject administered 32 ng spike protein with adjuvant; and the eighth bar represents reactivity to spike protein derived from splenocytes of a subject administered 32 ng spike protein with adjuvant. FIG. 8D is a bar graph showing an ELISpot assay showing the number of spots producing IFNγ in response to spike protein as a function of antigen dose. From left to right on the x-axis, the first bar represents non-spike-containing wells derived from PBS control splenocytes; the second bar represents reactivity to spike protein derived from PBS control splenocytes; the third bar represents non-spike-containing wells derived from splenocytes of a subject administered 10 ng STX-S; the fourth bar represents reactivity to spike protein derived from splenocytes of a subject administered 10 ng STX-S; the fifth bar represents non-spike-containing wells derived from splenocytes of a subject administered 32 ng STX-S; the sixth bar represents reactivity to spike protein derived from splenocytes of a subject administered 32 ng STX-S; the seventh bar represents non-spike-containing wells derived from splenocytes of a subject administered 32 ng spike protein with adjuvant; and the eighth bar represents reactivity to spike protein derived from splenocytes of a subject administered 32 ng spike protein with adjuvant. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 9A] Figure 9, Panels A-D. The STX-N vaccine induces polyvalent immune responses. Data are shown as mean ± SEM. *p<0.05, ***p<0.005, ****p<0.001, analysis of variance, corrected for multiple comparisons; ns=non-significant. Dose 1 = 0.32 ng N per injection; Dose 2 = 3.2 ng N per injection; Dose 3 = 10 ng N per injection. Panels 9A and 9B: Histograms showing anti-nucleocapsid antibody titers in serum at day 35 post-immunization as a function of dose. The STX-N vaccine induced moderate expression of SARS-CoV-2 nucleocapsid antibodies in two sample bins (N1 and N2). PBS was used as a vehicle control. Panels 9C and 9D: Histograms showing anti-nucleocapsid IFNγ ELISpot-positive wells of splenocytes at day 40 post-immunization as a function of dose. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 10A] Figure 10, Panels A-C: Line graphs showing % SARS-CoV-2 neutralization as a function of immune serum at increasing serum dilutions. The graph shows the production of neutralizing antibodies after STX-S injection. Data are presented as mean ± SEM. COV-02-Delta = Plasma from a patient immunized with Moderna's mRNA vaccine who developed a breakthrough SARS-CoV-2 delta spike infection. Dose 4 = 9.8 ng S per injection; Dose 2 = 3.2 ng S per injection. Panel 10A. The STX-S vaccine produced potent neutralization against the SARS-CoV-2 delta spike (B.1.617.2). Panel 10B. The STX-S vaccine resulted in neutralization of the SARS-CoV-2 spike omicron BA.1. Panel 10C. The STX-S vaccine resulted in neutralization of the SARS-CoV-2 spike omicron BA.5.2.1. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 11]Figure 11 is a histogram showing the fold change in anti-spike antibody titers from mice as a function of time and dose. Data are shown as mean ± SEM. *p<0.05, ***p<0.005, ****p<0.001, analysis of variance, corrected multiple comparisons; ns=non-significant.From left to right, the first histogram represents serum from mice that received PBS, the second histogram represents day 14 serum from mice that received a single dose of 10 ng STX-S (exosomes expressing CD9-spike fusions), the third histogram represents day 35 serum from mice that received the first dose of 10 ng STX-S followed by a 7 ng booster on day 21, the fourth histogram represents day 61 serum from mice that received the first dose of 10 ng STX-S followed by a 7 ng booster on day 21, the fifth histogram represents day 89 serum from mice that received the first dose of 10 ng STX-S followed by a 7 ng booster on day 21, and the sixth histogram represents day 89 serum from mice that received the first dose of 10 ng STX-S followed by a 7 ng booster on day 21. The seventh histogram represents day 123 serum from mice that received the first dose of STX-S followed by a 7 ng booster on day 21; the seventh histogram represents day 151 serum from mice that received the first dose of 10 ng STX-S followed by a 7 ng booster on day 21; the eighth histogram represents day 278 serum from mice that received the first dose of 10 ng STX-S followed by a 7 ng booster on day 21; the ninth histogram represents day 166 serum from mice that received the first dose of 10 ng STX-S followed by a 7 ng booster on day 21; the tenth histogram represents day 166 and 4-month follow-up serum from mice that received the first dose of 10 ng STX-S followed by a 7 ng booster on day 21; and the eleventh histogram represents day 166 and 4-month follow-up serum from mice that received the first dose of 10 ng STX-S followed by a 7 ng booster on day 21. The first histogram represents day 166 serum from mice that received the first dose of STX-S followed by a 3 ng booster on day 21, and the 12th histogram represents day 166 and 4 month follow-up serum from mice that received the first dose of 10 ng STX-S followed by a 3 ng booster on day 21. [Figure 12]Figure 12 is a line graph showing the % uptake of exosomes by HEK293-hACE2 cells as a function of exosome concentration, expressed as exosomes per mL. Series 1 (blue) represents the uptake of 293F exosomes that do not express the spike protein by HEK293-hACE2 cells. Series 2 (orange) represents the uptake of spike-expressing exosomes by HEK293-hACE2 cells. [Figure 13] Figure 13 shows histograms showing the relative fluorescence intensity flow analysis of exosomes expressing spike (Panel 13A) or nucleocapsid (Panel 13B). For each panel, the left curve represents exosomes derived from 293F cells that do not express spike (A) or nucleocapsid (B). For each panel, the right curve represents exosomes derived from 293F cells that express spike (A) or nucleocapsid (B). [Figure 14] Figure 14 is a line graph showing the size distribution of STX-S and STX-N exosomes as a function of exosome concentration by ZetaView nanoparticle tracking analysis (NTA). The blue line represents spike-expressing exosomes. The green line represents nucleocapsid-expressing exosomes. The gray line represents unmodified 293F-derived exosomes. [Figure 15] FIG. 15 shows TEM images of purified STX-S exosomes (Panel A; bar = 200 nm) and STX-N exosomes (Panel B; bar = 500 nm). [Figure 16]Figure 16 shows a JESS Western blot stained for SARS-CoV-2 spike protein. From left to right, the first column indicates lanes loaded with size markers, the second column represents lanes loaded with protein from non-transduced 293F cells, the third column represents lanes loaded with protein from exosomes from non-transduced 293F cells, the fourth column represents 293F cells expressing spike fusion protein, and the fifth column represents lanes loaded with protein from exosomes from 293F cells expressing nucleocapsid fusion protein. 0.8 μg of protein, as calculated by BCA assay, was loaded in each lane. [Figure 17] Figure 17 shows a JESS Western blot stained for SARS-CoV-2 nucleocapsid protein. From left to right, the first column indicates lanes loaded with size markers, the second column represents lanes loaded with protein from non-transduced 293F cells, the third column represents lanes loaded with protein from exosomes from non-transduced 293F cells, the fourth column represents 293F cells expressing the spike fusion protein, and the fifth column represents lanes loaded with protein from exosomes from 293F cells expressing the nucleocapsid fusion protein. 0.8 μg of protein was loaded in each lane, as calculated by BCA assay. [Figure 18]Figure 18A is a histogram showing the fold change in antibody titer against the spike protein in serum from mice on day 14 after a single im injection as a function of spiked dose formulation. The leftmost bar represents the PBS control; the second bar represents the dose of spike-expressing exosomes (STX S); and the third bar represents an equal dose of spike combined with adjuvant (S). N=10 per experimental group. Data are presented as mean ± SEM. ****p<0.0005, ***p<0.001, **p<0.01, ns=non-significant, one-way ANOVA. Figure 18B is a histogram showing the fold change in antibody titer against the spike protein in 1:100 diluted serum from mice on day 35 after two im injections as a function of spiked dose formulation. The leftmost bar represents the PBS control; the second bar represents the dose of spike-expressing exosomes (STX S); and the third bar represents an equal dose of spike combined with adjuvant (S). N=10 / experimental group. Data are shown as mean ± SEM. ****p<0.0005, ***p<0.001, **p<0.01, ns=non-significant, one-way ANOVA. [Figure 19]Figure 19A is a histogram showing the fold change in antibody titer against the nucleocapsid protein in the serum of mice on day 14 after a single im injection as a function of nucleocapsid dose formulation. The leftmost bar represents the PBS control; the second bar represents the dose of nucleocapsid-expressing exosomes (STX S); and the third bar represents an equal dose of nucleocapsid combined with adjuvant (N). N = 10 per experimental group. Data are presented as mean ± SEM. ****p < 0.0005, ***p < 0.001, **p < 0.01, ns = not significant, one-way ANOVA. Figure 19B is a histogram showing the fold change in antibody titer against the spike protein in the serum of mice on day 35 after two im injections as a function of spike dose formulation. The leftmost bar represents the PBS control; the second bar represents the dose of spike-expressing exosomes (STX S); and the third bar represents an equal dose of spike combined with adjuvant (S). N=10 / experimental group. Data are shown as mean ± SEM. ****p<0.0005, ***p<0.001, **p<0.01, ns=non-significant, one-way ANOVA. [Figure 20] Figure 20 shows histograms demonstrating the induction of strong antibody levels in mice by the STX-S+N combination vaccine. Panel A represents IgG levels against the spike on day 14. Panel B represents IgG levels against the spike on day 35. For both panels A and B, the first column shows the fold change in IgG with PBS. Column 2 shows the fold change in IgG with S+N at dose 1 (25 ng S, 2.5 ng N). Column 3 shows the fold change in IgG with S+N at dose 2 (10 ng S, 4 ng N). Column 4 shows the fold change in IgG with S+N at dose 3 (3 ng S, 9 ng N). Data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001; ns=non-significant, one-way ANOVA corrected for multiple comparisons. N=10 per experimental group. [Figure 21]Figure 21 shows histograms demonstrating the induction of strong antibody levels in mice by the STX-S+N combination vaccine. Panel A shows IgG levels against nucleocapsids on day 14. Panel B shows IgG levels against nucleocapsids on day 35. For both panels A and B, the first column shows the fold change in IgG with PBS. Column 2 shows the fold change in IgG with S+N at dose 1 (25 ng S, 2.5 ng N). Column 3 shows the fold change in IgG with S+N at dose 2 (10 ng S, 4 ng N). Column 4 shows the fold change in IgG with S+N at dose 3 (3 ng S, 9 ng N). Data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001; ns=non-significant, one-way ANOVA corrected for multiple comparisons. N=10 per experimental group. [Figure 22] Figure 22 shows histograms showing anti-spike IFNγ ELISpot-positive wells (Panel A) and anti-nucleocapsid IFNγ ELISpot-positive wells (Panel B) of splenocytes at day 40 post-immunization as a function of the dose of spike-expressing exosomes (STX-S) plus nucleocapsid-expressing exosomes (STX-N). Here, S = spike protein; N = nucleocapsid protein; Dose 1 = 25 ng S, 2.5 ng N; Dose 2 = 10 ng S, 4 ng N; Dose 3 = 3 ng S, 9 ng N. Data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001; ns = non-significant; one-way ANOVA corrected for multiple comparisons. N = 10 animals per experimental group. [Figure 23]Figure 23 shows histograms showing the change in anti-spike antibody titers (Panel A) or anti-nucleocapsid antibody titers (Panel B) as a function of dose of the combination vaccine STX-S+N, where Dose 1 is 125 ng spike + 10 ng nucleocapsid and Dose 2 is 50 ng spike + 20 ng nucleocapsid. For each antibody reading and dose regimen, from left to right, the first bar represents serum from blood collected on day 7 after the first dose; the second bar represents serum from blood collected on day 14 after the first dose; the third bar represents serum from blood collected on day 7 after the second dose and day 21 after the first dose; and the fourth bar represents serum from blood collected on day 14 after the second dose and day 28 after the first dose. Data are shown as mean ± SEM. N = 8 animals per experimental group. [Figure 24] Figure 24, Panels A-C: Line graphs showing % SARS-CoV-2 neutralization as a function of increasing serum dilutions of immune serum. The graph shows the production of neutralizing antibodies after STX-S+N (combination vaccine) injection. Data are presented as mean ± SEM. Data are presented as mean ± SEM. COV-02-delta = plasma from a patient immunized with Moderna's mRNA vaccine who developed a breakthrough SARS-CoV-2 delta spike infection; CoV-01 = plasma from a patient with no prior SARS-CoV-2 infection who received two doses of the Moderna covid-19 vaccine. N = 8 animals per experimental group. Panel 24A. The STX-S vaccine produced potent neutralization against the SARS-CoV-2 delta spike (B.1.617.2). Panel 24B. The STX-S vaccine resulted in neutralization of the SARS-CoV-2 spike omicron BA.1. Panel 24C. The STX-S vaccine resulted in neutralization of the SARS-CoV-2 spike omicron BA.5.2.1. [Figure 25]Figure 25 shows histograms showing anti-spike IFNγ ELISpot-positive wells (Panel A) and anti-nucleocapsid IFNγ ELISpot-positive wells (Panel B) of rabbit splenocytes at day 28 post-immunization as a function of dose of combination vaccine containing spike-expressing exosomes (STX-S) and nucleocapsid-expressing exosomes (STX-N). Here, S = spike protein; N = nucleocapsid protein; Dose 1 = 125 ng S, 10 ng N; Dose 2 = 50 ng S, 20 ng N. Data are presented as mean ± SEM. N = 8 animals per experimental group. DETAILED DESCRIPTION OF THE INVENTION

[0064] Detailed Description of the Embodiments definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, and / or steps of the type described herein that will become apparent to those skilled in the art upon review of this disclosure, and so forth.

[0065] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, and also refers to and includes the absence of a combination when interpreted as alternatives (or).

[0066] The terms "about" and "approximately," as used herein, when referring to a measurable value, e.g., amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, location, length, etc., are meant to encompass a variation of ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, location, length, etc. When the terms "about" and "approximately" are used in reference to the location or position of a region within a reference polypeptide, these terms encompass a variation of up to ±20 amino acid residues, up to ±15 amino acid residues, up to ±10 amino acid residues, up to ±5 amino acid residues, up to ±4 amino acid residues, up to ±3 amino acid residues, up to ±2 amino acid residues, or even ±1 amino acid residue.

[0067] The term "derived from" means that "A is derived from B" means that A is obtained from B in such a way that A is not identical to B.

[0068] The terms "treat," "therapeutic," "prophylactic," and "prevent" are not intended to be absolute terms. Treatment, prevention, and prophylaxis can refer to delaying onset, ameliorating symptoms, improving patient survival, increasing survival time or survival rate, etc. Treatment, prevention, and prophylaxis can be complete or partial. The term "prophylactic" refers not only to "preventing" but also to minimizing disease and illness. For example, a "prophylactic" agent can be administered to a subject, e.g., a human subject, to prevent infection or minimize the extent of disease and illness caused by such infection. The effect of treatment can be compared to an untreated individual or pool of individuals, or the same patient at different time points before or during treatment. In some aspects, the severity of the disease is reduced by at least 10%, for example, compared to the individual before administration or to an untreated control individual. In some aspects, the severity of the disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, becomes undetectable using standard diagnostic techniques.

[0069] As used herein, a treatment can be considered "effective" if, following treatment with the methods described herein, one or more of the signs or symptoms of a condition described herein are beneficially altered, other clinically accepted symptoms are improved or even ameliorated, or the desired response is induced, for example, by at least 2%, 3%, 4%, 5%, 10%, or more. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or prevalence of a condition treated according to the methods described herein, or any other suitable measurable parameter. Efficacy can also be measured by the lack of progression (e.g., cessation of disease progression) of an individual, as assessed by the need for hospitalization or medical intervention. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or animals), including (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) alleviating the severity of the disease, e.g., causing regression of symptoms. The effective amount for the treatment of a disease means that when administered to a subject in need thereof, it is sufficient to bring about an effective treatment (as this term is defined herein) for that disease.The effectiveness of an active substance can be determined by assessing the physical indicators of the condition or the desired response.Those skilled in the art can monitor the effectiveness of administration and / or treatment by measuring any one of these parameters or any combination of parameters.

[0070] The term "effective amount," as used herein, refers to the amount of a composition or agent necessary to alleviate at least one or more symptoms of a disease or disorder, and relates to the amount of a therapeutic composition sufficient to provide a desired effect. The term "therapeutically effective amount" refers to the amount of a composition or therapeutic agent sufficient to provide a particular effect when administered to a typical subject. As used herein, an effective amount can include, in various circumstances, an amount sufficient to delay the onset of disease symptoms, alter the course of a disease condition (e.g., but not limited to, slowing the progression of disease symptoms), or reverse disease symptoms. For example, for a given parameter, a therapeutically effective amount indicates an increase or decrease in therapeutic effect of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy may also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount may have an effect of at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more compared to a control. A therapeutically effective amount may be administered in one or more doses of a therapeutic agent. A therapeutically effective amount may be administered in a single dose or in multiple doses administered over a period of time.

[0071] "Administration," as used herein, can include any suitable route of administration of a therapeutic agent or composition disclosed herein. Suitable routes of administration include, but are not limited to, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, dermal, injection, or topical administration. Administration can be local or systemic.

[0072] As used herein, the term "pharmaceutically acceptable" refers to a carrier that is compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. This term is used interchangeably with "physiologically acceptable" and "pharmacologically acceptable." Pharmaceutical compositions generally include agents for buffering and preservation during storage, and may include buffers and carriers for appropriate delivery, depending on the route of administration. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0073] The terms "dose" and "administration" are used interchangeably herein. Dose refers to the amount of active ingredient given to an individual at each administration. For the present invention, dose can refer to the concentration of extracellular vesicles or related components, such as the amount of therapeutic agent or the amount of radiolabel administered. Dose varies depending on many factors, including the frequency of administration; the size and tolerance of the individual; the severity of the condition; the risk of side effects; the administration route; and the imaging modality of the detectable moiety (if any). Those skilled in the art will recognize that dose can be modified depending on the above factors or based on the progress of treatment. The term "dosage form" refers to the specific form of the medicine and depends on the administration route. For example, the dosage form can be a liquid for injection, such as a saline solution.

[0074] The terms "subject," "patient," "individual," and the like are used interchangeably and, unless otherwise indicated, refer to mammals, e.g., humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision. A patient may be an individual seeking treatment, monitoring, adjustment or modification of an existing treatment regimen, and the like.

[0075] As used herein, the following meanings apply unless otherwise specified: The word "may" is used in its permissive sense (i.e., having the potential to), rather than its obligatory sense (i.e., meaning must). Words such as "include," "including," and "includes" mean including, but not limited to. The singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "an element" includes a combination of two or more elements, despite the use of other terms and phrases, e.g., "one or more," that mean one or more elements. The term "or" is non-exclusive, i.e., includes both "and" and "or," unless otherwise indicated. The term "any of" between a modifier and a series of words means that the modifier modifies every member of the series. Thus, for example, the phrase "any of at least 1, 2, or 3" means "at least 1, at least 2, or at least 3." The phrase "at least 1" includes "a plurality."

[0076] Definitions of common terms in cell and molecular biology can be found in "The Merck Manual of Diagnosis and Therapy," 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-91 1910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8), and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

[0077] The term "native form" refers to a polypeptide as understood to be encoded by the genome of an infectious pathogen. The term "exosomal form" refers to any derivative of a protein that is fused in whole or in part to an exosome-associated protein. The term "cytoplasmic form" refers to any derivative of a protein that is configured or designed in whole or in part to be expressed in the cytoplasm of a cell rather than entering the standard secretory pathway.

[0078] The phrase "configured, configured, or designed to be expressed" in a particular manner means that the nucleotide sequence encodes a particular amino acid sequence such that when the protein is expressed in a cell, the protein is expressed in a native, exosomal, or cytosolic form. For example, when the spike protein (S) is expressed in its native form, it is configured or designed to induce a humoral or cellular immune response by virtue of being a transmembrane protein that includes an extracellular domain.

[0079] The term "extracellular vesicles" (EVs) refers to lipid bilayer-bound particles naturally released from cells. EV diameters range from approximately 20-30 nanometers to approximately 10 microns or larger. EVs can contain proteins, nucleic acids, lipids, and metabolites derived from the cells that produced them. EVs include exosomes (approximately 50-200 nm), microvesicles (approximately 100-300 nm), ectosomes (approximately 50-1000 nm), apoptotic bodies (approximately 50-5000 nm), and lipid-protein aggregates of similar dimensions.

[0080] The term "nucleic acid" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), as well as chemically modified nucleic acids, such as morpholino (PMO), peptide nucleic acid (PNA), 2'O-methyl, 2'methoxyethyl, phosphoramidate, methylphosphonate, and phosphorothioate. Nucleic acids can be of any size. Nucleic acids include, but are not limited to, genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules, such as aptamers, plasmids, antisense DNA strands, shRNA, ribozymes, conjugated nucleic acids, and oligonucleotides. According to the present invention, nucleic acids can exist as single-stranded or double-stranded, and can exist as linear or covalently circularly closed molecules. Nucleic acids can be utilized for introduction into cells, e.g., transfection of cells, in the form of RNA, which can be prepared, for example, by in vitro transcription from a DNA template. RNA can be further modified by stabilizing sequences, capping, and polyadenylation before application. Generally, nucleic acids can be extracted, isolated, amplified, or analyzed by a variety of techniques, such as those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY, 2,028 pages (2012).

[0081] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to any chain of at least two amino acids linked by a covalent chemical bond. As used herein, a peptide can refer to a complete amino acid sequence encoding an entire protein or a portion thereof. A "protein coding sequence," or a sequence "encoding" a particular polypeptide or peptide, is a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, either in vitro or in vivo, when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, cDNA derived from prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence is typically located 3' to the coding sequence.

[0082] As used herein, the phrase "protein polypeptide" refers to a polypeptide sequence of a protein or a polypeptide sequence derived from a protein. For example, a CD9 protein polypeptide can be any polypeptide of a CD9 protein, such as a full-length CD9 protein, a transmembrane domain polypeptide of a CD9 protein, a C-terminal stretch of a CD9 protein, an extracellular loop region of a CD9 protein, an intracellular (intraluminal) loop region of a CD9 protein, a C-terminal stretch of a CD9 protein, a combination thereof, etc. Here, a protein polypeptide can be at least 10 amino acids in length.

[0083] As used herein, the term "spike protein" includes any SARS-CoV-2 spike glycoprotein, fragments of SARS-CoV-2 spike glycoprotein, SARS-CoV-2 spike glycoprotein monomers, trimers of SARS-CoV-2 spike glycoprotein monomers, variants of SARS-CoV-2 spike glycoprotein, fusion or chimeric proteins containing a SARS-CoV-2 spike glycoprotein sequence and another non-SARS-CoV-2 spike glycoprotein sequence, SARS-CoV-2 spike glycoproteins with one or more deletions, additions, or substitutions of one or more amino acids, and conservatively substituted variations of SARS-CoV-2 spike glycoproteins with at least 80% amino acid sequence identity, for example, to at least the stem region, membrane-proximal stem helix region, or receptor-binding domain of the S2 subunit, or other similar domain.

[0084] Fragments of the SARS-CoV-2 spike glycoprotein include, for example, peptides or polypeptides that encompass, comprise, consist of, or overlap with antigenic epitopes, specific domains, such as the receptor binding domain (RBD) in the up or down conformational state, receptor binding fragment S1, fusion fragment S2, N-terminal domain (NTD), receptor binding domain (RBD) C-terminal domain 1 (CTD1), C-terminal domain 2 (CTD2), fusion peptide (FP), fusion peptide proximal region (FPPR), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), heptad repeat 2 (HR2), transmembrane segment (TM), cytoplasmic tail (CT), etc.

[0085] Variants of the SARS-CoV-2 spike glycoprotein include any known or yet to be discovered, such as alpha, beta, gamma, delta, epsilon, eta, iota, kappa, 1.617.3, mu, zeta, omicron, or their subspecies, strains, and conservatively substituted spike protein sequences.

[0086] The SARS-CoV-2 spike glycoprotein can have additions, deletions, substitutions, or point mutations. For example, the spike protein can have a deletion of several (2-20) amino acids from the C-terminus (see Johnson et al., 2020 and Xiong et al., 2020) or a furin cleavage site alteration (see Johnson et al., 2020).

[0087] As used herein, the term "nucleocapsid protein" refers to a soluble coronavirus structural protein that binds to and forms a complex with RNA and the viral membrane protein (M), and is important for packaging the viral genome. The nucleocapsid protein contains (from the amino terminus to the carboxy terminus) an N-terminal domain containing an intrinsically disordered region (IDR) and an RNA-binding domain (NTD), a serine-arginine-rich linker region (LKR), a C-terminal domain containing an RNA-binding dimerization domain and a nuclear localization signal, followed by the C-terminal IDR. The nucleocapsid protein is generally described in, for example, McBride et al., "The Coronavirus Nucleocapsid Is a Multifunctional Protein," Viruses. 2014 Aug;6(8):2991-3018; Cubuk et al., "The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA," Nature Communications volume 12, Article number: 1936 (2021); and references cited therein.

[0088] Fusion or chimeric proteins of the SARS-CoV-2 spike glycoprotein or SARS-CoV-2 nucleocapsid protein include fusions of the spike protein or nucleocapsid protein sequence with the sequence of another protein that provides a particular outcome, such as improved sorting or targeting to endosomes and the resulting extracellular vesicles, e.g., exosomes. For example, the spike protein can be a fusion of the spike protein or nucleocapsid protein sequence with another glycoprotein known to sort into exosomes or useful in the production of pseudovirions, such as VSV glycoprotein or lentiviral glycoprotein. The spike protein or nucleocapsid protein can be a fusion of the spike protein sequence with another protein known to sort into exosomes, such as various tetraspanins (CD9, CD63, and CD81). The fusion protein may contain primarily SARS-CoV-2 sequences along with short sequences of other proteins (i.e., from dipeptides to peptides of 100 amino acids).

[0089] As used herein, the term "tetraspanin" or "tetraspanin protein" refers to any member of a family of proteins (or chimeras thereof) that have four transmembrane domains and, in some cases, are present in exosomal membranes. Tetraspanin proteins are known to regulate trafficking and cellular and membrane compartmentalization. Tetraspanins include, among others, CD9, CD37, CD63, CD81, CD82, CD151, TSPAN7, TSPAN8, TSPAN12, TSPAN33, peripherin, UP1a / 1b, TSP-15, TSP-12, TSP3A, TSP86D, TSP26D, TSP-2, and analogs, orthologs, and homologs thereof. It is contemplated that useful tetraspanins may include chimeras of any one or more standard tetraspanins, such as, for example, CD9 / CD81 chimeras. Tetraspanins are generally described in Charrin et al., "Tetraspanins at a glance," J Cell Sci (2014) 127(17):3641-3648; Kummer et al., "Tetraspanins: integrating cell surface receptors to functional microdomains in homeostasis and disease," Med Microbiol Immunol. 2020;209(4):397-405; and references cited therein. The CD9 member of the tetraspanin superfamily is generally described in Umeda et al., "Structural insights into tetraspanin CD9 function," Nature Communications volume 11, Article number: 1606 (2020) and references cited therein.

[0090] The term "sequence identity," as used herein, refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are the same length. Determining the percent identity between two sequences may be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed, for example, using the NBLAST nucleotide program parameters set at score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application. BLAST protein searches can be performed with, for example, the XBLAST program parameters set to score-50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules described herein.To obtain gapped alignments for comparison purposes, Gapped BLAST, as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402, can be utilized. Alternatively, PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules (supra). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the NCBI website).

[0091] Another preferred, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0092] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0093] The percentage sequence identity for an antibody can be determined when the antibody sequences are maximally aligned by IMGT. After alignment, when a region of a subject antibody (e.g., the entire mature variable region of a heavy or light chain) is compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody regions, divided by the total number of aligned positions in the two regions, multiplied by 100, and converted to a percentage.

[0094] The percent amino acid sequence identity may be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program is available from the National Institutes of Health (Bethesda, Md). NCBI-BLAST2 uses several search parameters, all of which are set to default values, e.g., unmask=Yes, strand=All, expected occurrences=10, minimum low complexity length=15 / 5, multipass e-value=0.01, multipass constant=25, dropoff for final gap alignment=25, and scoring matrix=BLOSUM62.

[0095] In situations where NCBI-BLAST2 is utilized for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B is calculated as follows (alternatively, it may be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B): 100 x fraction X / Y where X is the number of amino acid residues scored as an identical match by the sequence alignment program NCBI-BLAST2 in an alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. The term "nucleic acid sequence," as used herein, refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages, including cDNA. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may contain naturally occurring bases, including adenine, guanine, cytosine, thymidine, and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza- and deaza-forms of adenine, guanine, cytosine, thymidine, and uracil; and xanthine and hypoxanthine. It is understood that polynucleotides containing non-transcribeable nucleotide bases can be useful as probes, for example, in hybridization assays. Nucleic acids can be either double-stranded or single-stranded, representing sense or antisense strands. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences, including codon-optimized equivalents or synonymous codon equivalents.

[0096] As used herein, the term "antibody" refers to immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that specifically binds an antigen. Antibodies are typically heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Light chains from vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Antibodies can have one or more effector functions, which refer to the biological activities attributable to the Fc region of an antibody (a native sequence Fc region, an amino acid sequence variant Fc region, or any other modified Fc region). Non-limiting examples of antibody effector functions include Clq binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors, such as B cell receptors (BCR); and cross-presentation of antigens by antigen-presenting cells or dendritic cells.

[0097] The term "neutralizing antibodies" (Nab) refers to antibodies that protect cells from pathogens or infectious particles by neutralizing their biological effects. Neutralization renders particles non-infectious or non-pathogenic. Neutralizing antibodies are part of the adaptive immune system's humoral response to viruses, intracellular bacteria, and microbial toxins. By specifically binding to surface antigens on infectious particles, neutralizing antibodies prevent the particles from interacting with host cells that they could infect and destroy.

[0098] Neutralizing antibody immunity is also known as sterilizing immunity because the immune system eliminates infectious particles before infection can occur.

[0099] The term "antigen" refers to any substance that induces an immune response. For example, an antigen relates to any substance, preferably a peptide or protein, that specifically reacts with antibodies or T lymphocytes (T cells). As used herein, the term "antigen" includes any molecule that contains at least one epitope. For example, an antigen is a molecule that, optionally after processing, induces an immune response. For example, any suitable antigen that is a candidate for an immune response can be used, where the immune response can be a cellular immune response. For example, the antigen can be presented by cells, which results in an immune response against the antigen. For example, the antigen corresponds to a naturally occurring antigen or is a product derived from a naturally occurring antigen. Such antigens include, but are not limited to, SARS-CoV-2 structural proteins S, N, M, and E, and any variants or mutants thereof.

[0100] The term "pharmaceutical composition" refers to a formulation containing an active ingredient and, optionally, a pharmaceutically acceptable carrier, diluent, or excipient. The term "active ingredient" can refer interchangeably to "active ingredient" and refers to any agent capable of inducing a desired effect upon administration. "Pharmaceutically acceptable" means that the carrier, diluent, or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof and to the activity of the active ingredient of the formulation. Pharmaceutically acceptable carriers, excipients, or stabilizers are well known in the art (e.g., Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers, e.g., phosphate, citric acid, and other organic acids; antioxidants, e.g., ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, e.g., methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins. , such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).Examples of carriers include, but are not limited to, liposomes, nanoparticles, ointments, micelles, microspheres, microparticles, creams, emulsions, and gels.

[0101] Examples of excipients include, but are not limited to, anti-adherents such as magnesium stearate, binders such as sugars and their derivatives (sucrose, lactose, starch, cellulose, sugar alcohols, etc.), proteins such as gelatin and synthetic polymers, lubricants such as talc and silica, and preservatives such as antioxidants, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate, and parabens. Examples of diluents include, but are not limited to, water, alcohol, saline solution, glycol, mineral oil, and dimethyl sulfoxide (DMSO).

[0102] The term "vaccine" or "immunogenic composition" relates to a pharmaceutical preparation (pharmaceutical composition) or product that, upon administration, induces an immune response, e.g., a cellular immune response, that recognizes and attacks pathogens or disease cells. The term "immune response" refers to the body's orchestrated response to an antigen, and refers to a cellular immune response and / or a humoral immune response. The immune response can be protective / preventative / prophylactic and / or therapeutic.

[0103] The term "cellular immune response" or "cell-mediated immune response" refers to any adaptive immune response in which antigen-specific T cells play a major role. This is practically defined as any adaptive immunity that cannot be transferred to a native recipient by serum antibodies. In contrast, the term "humoral immune response" refers to antibody-mediated immunity.

[0104] The cellular response involves cells called T cells or T lymphocytes that function as either "helpers" or "killers." Helper T cells (also called CD4+ T cells) play a central role by controlling the immune response, while killer cells (also called cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill diseased cells, such as cancer cells, and prevent the production of more diseased cells.

[0105] The terms "immunoreactive cell," "immune cell," or "immune effector cell" refer to cells that perform effector functions in immune responses. "Immune reactive cells" are preferably cells characterized by antigen presentation, or antigenic peptides derived from antigens, and can mediate immune responses. For example, such cells secrete cytokines and / or chemokines, secrete antibodies, recognize cancer cells, and optionally eliminate such cells. For example, immune reactive cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells.

[0106] The term "adjuvant" refers to a pharmacological or immunological agent that modifies the effect of another agent. Adjuvants can be added to the vaccine compositions of the present invention to boost the immune response so that more antibodies and longer-lasting immunity are produced, thereby minimizing the dose of antigen required. Depending on the purpose of the vaccine, adjuvants can also be used to enhance the effectiveness of vaccines by helping to modify the immune response to specific types of immune system cells, for example, by activating T cells rather than antibody-secreting B cells. Immunological adjuvants are added to vaccines to stimulate the immune system's response to the target antigen, but do not themselves provide immunity. Examples of adjuvants include, but are not limited to, analgesic adjuvants; inorganic compounds such as alum, aluminum hydroxide, aluminum phosphate, and calcium hydroxide phosphate; mineral oils such as paraffin oil; bacterial products such as killed bacteria (Bordetella pertussis, Mycobacterium bovis, and toxoids); non-bacterial organics such as squalene; delivery systems such as surfactants (Quil A); plant saponins derived from Quillaja, soybean, or Polygala senega; cytokines such as IL-1, IL-2, and IL-12; combinations such as Freund's complete and incomplete adjuvants; and food-based oils such as peanut oil-based adjuvants.

[0107] Detailed aspects Embodiment 1. In one embodiment, an immunogenic composition is provided comprising a vesicle and a fusion protein, wherein the fusion protein comprises a viral polypeptide and an exosomal polypeptide.

[0108] Embodiment 2. The immunogenic composition of embodiment 1 is provided, further comprising an excipient.

[0109] Embodiment 3. The immunogenic composition of embodiment 2 is provided, wherein the excipient is a buffer.

[0110] Aspect 4. There is provided an immunogenic composition according to any one of Aspects 1 to 3, which does not include an adjuvant.

[0111] Embodiment 5. An immunogenic composition according to any one of embodiments 1 to 4 is provided, wherein the fusion protein is present in the membrane of a vesicle.

[0112] Embodiment 6. The immunogenic composition of any one of embodiments 1 to 5 is provided, wherein some or all of the viral polypeptides are present on the outer surface of the vesicle.

[0113] Embodiment 7. An immunogenic composition according to any one of Embodiments 1 to 6 is provided, wherein the fusion protein is present in the composition at a concentration of from about 1 ng / 100 μL to about 150 ng / 100 μL.

[0114] Embodiment 8. The immunogenic composition of any one of Embodiments 1 to 7 is provided, wherein the exosomal polypeptide is a tetraspanin protein polypeptide.

[0115] Embodiment 9. The immunogenic composition of any one of Embodiments 1 to 8 is provided, wherein the exosomal polypeptide is a CD9 protein polypeptide.

[0116] Embodiment 10 The immunogenic composition of any one of Embodiments 1 to 9 is provided, wherein the exosomal polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:10.

[0117] Embodiment 11 The immunogenic composition of any one of Embodiments 1 to 10 is provided, wherein the exosomal polypeptide comprises the amino acid sequence of SEQ ID NO:10.

[0118] Embodiment 12. The immunogenic composition of any one of embodiments 1 to 11 is provided, wherein the viral polypeptide is a SARS-CoV-2 polypeptide.

[0119] Embodiment 13 The immunogenic composition of any one of embodiments 1 to 12 is provided, wherein the viral polypeptide is a SARS-CoV-2 spike protein polypeptide.

[0120] Embodiment 14 The immunogenic composition of any one of embodiments 1 to 13 is provided, wherein the viral polypeptide is a SARS-CoV-2 delta variant spike protein polypeptide.

[0121] Embodiment 15. The immunogenic composition of any one of embodiments 1 to 14 is provided, wherein the viral polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1.

[0122] Embodiment 16 The immunogenic composition of any one of embodiments 1 to 15 is provided, wherein the viral polypeptide comprises the amino acid sequence of SEQ ID NO:1.

[0123] Embodiment 17 The immunogenic composition of any one of embodiments 1 to 11 is provided, wherein the viral polypeptide is a SARS-CoV-2 nucleocapsid protein polypeptide.

[0124] Embodiment 18 The immunogenic composition of any one of embodiments 1 to 11 and 17 is provided, wherein the viral polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:5.

[0125] Embodiment 19. The immunogenic composition of any one of embodiments 1 to 11, 17, and 18 is provided, wherein the viral polypeptide comprises the amino acid sequence of SEQ ID NO:5.

[0126] Embodiment 20 The immunogenic composition of any one of embodiments 1 to 16 is provided, wherein the fusion protein comprises a SARS-CoV-2 spike protein polypeptide and a CD9 protein polypeptide.

[0127] Embodiment 21 The immunogenic composition of any one of embodiments 1 to 16 and 20 is provided, wherein the fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:2.

[0128] Embodiment 22 The immunogenic composition of any one of embodiments 1 to 16, 20, and 21 is provided, wherein the fusion protein comprises an amino acid sequence having the sequence of SEQ ID NO:2.

[0129] Embodiment 23 The immunogenic composition of any one of embodiments 1 to 16 and 20 to 22 is provided, wherein the fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:3.

[0130] Embodiment 24. The immunogenic composition of any one of embodiments 1 to 16 and 20 to 23 is provided, wherein the fusion protein comprises an amino acid sequence having the sequence of SEQ ID NO:3.

[0131] Embodiment 25. The immunogenic composition of any one of embodiments 1 to 11 and 17 to 19 is provided, wherein the fusion protein comprises a SARS-CoV-2 nucleocapsid protein polypeptide and a CD9 protein polypeptide.

[0132] Embodiment 26 The immunogenic composition of any one of embodiments 1 to 11, 17 to 19, and 25 is provided, wherein the fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:6.

[0133] Embodiment 27 The immunogenic composition of any one of embodiments 1 to 11, 17 to 19, 25, and 26 is provided, wherein the fusion protein comprises an amino acid sequence having the sequence of SEQ ID NO:6.

[0134] Embodiment 28 The immunogenic composition of any one of embodiments 1 to 11, 17 to 19, and 25 to 27 is provided, wherein the fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:7.

[0135] Embodiment 29. The immunogenic composition of any one of embodiments 1 to 11, 17 to 19, and 25 to 28 is provided, wherein the fusion protein comprises an amino acid sequence having the sequence of SEQ ID NO:7.

[0136] Embodiment 30. The immunogenic composition of any one of embodiments 1 to 29 is provided, wherein an immunogenic dose of the composition comprises, in 0.5 mL, (i) from about 1 ng to about 300 ng of fusion protein or viral polypeptide, or (ii) from 10 to 200 ng of total fusion protein.

[0137] Embodiment 31. The immunogenic composition of any one of embodiments 1 to 30 is provided, comprising a second vesicle and a second fusion protein, wherein the second fusion protein comprises a second viral polypeptide and an exosomal polypeptide, and wherein the second fusion protein is present in a membrane of the second vesicle.

[0138] Embodiment 32 The immunogenic composition of embodiment 31 is provided, wherein the viral polypeptide is a SARS-CoV-2 spike protein polypeptide and the second viral polypeptide is a SARS-CoV-2 nucleocapsid protein.

[0139] Embodiment 33 The immunogenic composition of embodiment 31 or 32 is provided, wherein the fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:2, and the second fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:6.

[0140] Embodiment 34 The immunogenic composition of any one of embodiments 31 to 33 is provided, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:2 and the second fusion protein comprises the amino acid sequence of SEQ ID NO:6.

[0141] Embodiment 35. The immunogenic composition of any one of embodiments 31 to 34 is provided, wherein the fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:3, and the second fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:7.

[0142] Embodiment 36 The immunogenic composition of any one of embodiments 31 to 35 is provided, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:3 and the second fusion protein comprises the amino acid sequence of SEQ ID NO:7.

[0143] Embodiment 37 The immunogenic composition of any one of embodiments 1 to 36 is provided, wherein the vesicles are exosomes.

[0144] Embodiment 38. Provided is a method of immunizing a subject against viral infection, comprising administering to the subject an immunogenically effective dose of the immunogenic composition of any of Embodiments 1 to 37.

[0145] Embodiment 39 The method of embodiment 38 is provided, wherein the immunogenic effective dose induces protective immunity in the subject against multiple variants of a given virus.

[0146] Embodiment 40. The method of embodiment 38 or 39 is provided, wherein the immunogenic composition comprises a SARS-CoV-2 nucleocapsid protein polypeptide.

[0147] Embodiment 41 The method of any one of embodiments 38 to 40 is provided, wherein the immunogenic composition comprises a fusion protein having an amino acid sequence that is at least 80% identical to SEQ ID NO:6.

[0148] Embodiment 42 The method of any one of embodiments 38 to 41 is provided, wherein the immunogenic composition comprises a fusion protein having an amino acid sequence that is at least 80% identical to SEQ ID NO:7.

[0149] Embodiment 43 The method of any one of embodiments 38 to 42 is provided, wherein the immunogenic composition comprises a fusion protein having the amino acid sequence of SEQ ID NO:6.

[0150] Embodiment 44 The method of any one of embodiments 38 to 43 is provided, wherein the immunogenic composition comprises a fusion protein having the amino acid sequence of SEQ ID NO:7.

[0151] Embodiment 45. The method of embodiment 38 or 39 is provided, wherein the immunogenic composition comprises a SARS-CoV-2 delta variant spike protein polypeptide, and wherein the immunogenic effective dose induces protective immunity in the subject against SARS-CoV-2 delta variant and SARS-CoV-2 omicron variant.

[0152] Embodiment 46 The method of any one of embodiments 38, 39, and 45 is provided, wherein the immunogenic composition comprises a fusion protein having an amino acid sequence that is at least 80% identical to SEQ ID NO:2.

[0153] Embodiment 47 The method of any one of embodiments 38, 39, 45, and 46 is provided, wherein the immunogenic composition comprises a fusion protein having an amino acid sequence that is at least 80% identical to SEQ ID NO:3.

[0154] Embodiment 48 The method of any one of embodiments 38, 39, and 45-47 is provided, wherein the immunogenic composition comprises a fusion protein having the amino acid sequence of SEQ ID NO:2.

[0155] Embodiment 49 The method of any one of embodiments 38, 39, and 45-48 is provided, wherein the immunogenic composition comprises a fusion protein having the amino acid sequence of SEQ ID NO:3.

[0156] Embodiment 50. The method of embodiment 38 or 39 is provided, wherein the immunogenic composition comprises a first fusion protein comprising a SARS-CoV-2 nucleocapsid protein polypeptide located in the membrane of the first vesicle, and a second fusion protein comprising a SARS-CoV-2 spike protein polypeptide located in the membrane of the second vesicle.

[0157] Embodiment 51 The method of embodiment 50 is provided, wherein the first fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:6.

[0158] Embodiment 52. The method of embodiment 50 or 51 is provided, wherein the first fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:7.

[0159] Embodiment 53 The method of any one of embodiments 50 to 52 is provided, wherein the first fusion protein has the amino acid sequence of SEQ ID NO:6.

[0160] Embodiment 54 The method of any one of embodiments 50 to 53 is provided, wherein the first fusion protein has the amino acid sequence of SEQ ID NO:7.

[0161] Embodiment 55 The method of any one of embodiments 50 to 54 is provided, wherein the second fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:2.

[0162] Embodiment 56 The method of any one of embodiments 50 to 55 is provided, wherein the second fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:3.

[0163] Embodiment 57 The method of any one of embodiments 50 to 56 is provided, wherein the second fusion protein has the amino acid sequence of SEQ ID NO:2.

[0164] Embodiment 58 The method of any one of embodiments 50 to 57 is provided, wherein the second fusion protein has the amino acid sequence of SEQ ID NO:3.

[0165] Embodiment 59. The method of any one of embodiments 38 to 58 is provided, wherein the immunogenically effective dose comprises, in 0.5 mL, (i) about 1 ng to about 300 ng of fusion protein or viral polypeptide, or (ii) 10 to 200 ng of total fusion protein.

[0166] Embodiment 60. The method of any one of embodiments 38 to 59 is provided, further comprising administering to the subject a second effective dose of the immunogenic composition of any one of embodiments 1 to 37.

[0167] Embodiment 61. A synthetic fusion protein is provided comprising a viral polypeptide and an exosomal polypeptide.

[0168] Embodiment 62 The synthetic fusion protein of embodiment 61 is provided, further comprising a linker polypeptide positioned between the viral polypeptide and the exosomal polypeptide.

[0169] Embodiment 63 The synthetic fusion protein of embodiment 61 or 62 is provided, further comprising a hinge polypeptide positioned between the viral polypeptide and the exosomal polypeptide.

[0170] Embodiment 64. The synthetic fusion protein of any one of embodiments 61 to 63 is provided, further comprising a transmembrane domain polypeptide positioned between the viral polypeptide and the exosomal polypeptide.

[0171] Embodiment 65. The synthetic fusion protein of any one of embodiments 61 to 64 is provided, wherein the exosomal polypeptide is a tetraspanin polypeptide.

[0172] Embodiment 66 The synthetic fusion protein of any one of embodiments 61 to 65 is provided, wherein the exosomal polypeptide is a CD9 polypeptide.

[0173] Embodiment 67. The synthetic fusion protein of any one of embodiments 61 to 66 is provided, wherein the viral polypeptide is a SARS-CoV-2 structural protein polynucleotide.

[0174] Embodiment 68. The synthetic fusion protein of any one of embodiments 61 to 67 is provided, wherein the viral polypeptide is a SARS-CoV-2 spike protein polypeptide.

[0175] Embodiment 69. The synthetic fusion protein of embodiment 68 is provided, wherein the SARS-CoV-2 spike protein polypeptide comprises one or more of a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and a diproline substitution (2P [986KV987-to-986PP987]).

[0176] Embodiment 70. The synthetic fusion protein of any one of embodiments 61 to 69 is provided, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, a SARS-CoV-2 spike protein polypeptide, a linker polypeptide, and a CD9 polypeptide.

[0177] Embodiment 71. The synthetic fusion protein of any one of embodiments 61 to 70 is provided, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:3.

[0178] Embodiment 72. There is provided a synthetic fusion protein according to any one of embodiments 61 to 71, comprising the amino acid sequence set forth in SEQ ID NO:3.

[0179] Embodiment 73. A viral polypeptide in a SARS-CoV-2 nucleocapsid protein polypeptide, a synthetic fusion protein according to any one of embodiments 61 to 67 is provided.

[0180] Embodiment 74. The synthetic fusion protein of any one of embodiments 61 to 67 and 73 is provided, wherein the fusion protein comprises, in order from amino terminus to carboxy terminus, a signal peptide, a SARS-CoV-2 nucleocapsid protein polypeptide, a hinge region, a transmembrane domain polypeptide, a linker polypeptide, and a CD9 polypeptide.

[0181] Embodiment 75. There is provided a synthetic fusion protein of any one of embodiments 61 to 67, 73, and 74, comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:7.

[0182] Embodiment 76. There is provided a synthetic fusion protein according to any one of embodiments 61 to 67 and 73 to 75, comprising the amino acid sequence set forth in SEQ ID NO:7.

[0183] Embodiment 77. A synthetic polynucleotide encoding the synthetic fusion protein of any one of embodiments 61 to 76 is provided.

[0184] Embodiment 78. The synthetic polynucleotide of embodiment 77 is provided, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:13.

[0185] Embodiment 79. There is provided a synthetic polynucleotide of embodiment 77 or 78, comprising the nucleic acid sequence set forth in SEQ ID NO: 13.

[0186] Embodiment 80. There is provided a synthetic polynucleotide of any one of embodiments 77 to 79, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:11.

[0187] Embodiment 81. There is provided a synthetic polynucleotide according to any one of embodiments 77 to 80, comprising the nucleic acid sequence set forth in SEQ ID NO:11.

[0188] Embodiment 82. There is provided a synthetic polynucleotide of any one of embodiments 77 to 81, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:4.

[0189] Embodiment 83. There is provided a synthetic polynucleotide according to any one of embodiments 77 to 82, comprising the nucleic acid sequence set forth in SEQ ID NO:4.

[0190] Embodiment 84. There is provided a synthetic polynucleotide of any one of Embodiments 77 to 79, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO: 12.

[0191] Embodiment 85. There is provided a synthetic polynucleotide according to any one of embodiments 77 to 79 and 84, comprising the nucleic acid sequence set forth in SEQ ID NO: 12.

[0192] Embodiment 86. There is provided a synthetic polynucleotide of any one of embodiments 77 to 79, 84, and 85, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:8.

[0193] Embodiment 87. There is provided a synthetic polynucleotide according to any one of embodiments 77 to 79 and 84 to 86, comprising the nucleic acid sequence set forth in SEQ ID NO:8.

[0194] Embodiment 88. A cell is provided comprising the synthetic polynucleotide of any one of embodiments 77 to 87.

[0195] Embodiment 89. The cell of embodiment 88 is provided, wherein the cell is a metazoan cell.

[0196] Embodiment 90. The cell of embodiment 88 or 89 is provided, which is a vertebrate cell.

[0197] Embodiment 91. The cell of any one of embodiments 88 to 90 is provided, wherein the cell is a mammalian cell.

[0198] Embodiment 92 The cell of any one of embodiments 88 to 91 is provided, wherein the cell is a primate cell.

[0199] Embodiment 93. The cell of any one of embodiments 88 to 92 is provided, wherein the cell is a human cell.

[0200] Embodiment 94. The cell of any one of embodiments 88 to 93 is provided, wherein the cell is a primary cell.

[0201] Embodiment 95. The cell of any one of embodiments 88 to 93 is provided, wherein the cell is a human embryonic kidney cell.

[0202] Embodiment 96. The cell of embodiment 95 is provided, wherein the cell is a 293 cell.

[0203] Embodiment 97. A cell of any one of embodiments 88 to 96 is provided, wherein the cell is produced by transducing the cell with a lentivirus comprising the synthetic polynucleotide.

[0204] Embodiment 98. A cell of any one of embodiments 88 to 97 is provided, comprising the synthetic fusion protein of any one of embodiments 61 to 77.

[0205] Embodiment 99. A vesicle comprising the synthetic fusion protein of any one of embodiments 61 to 76 is provided.

[0206] Embodiment 100. The vesicle of embodiment 99 is provided, which is an exosome.

[0207] Embodiment 101. The vesicle of embodiment 99 or 100 is provided, having a diameter of about 50 to 500 nm.

[0208] Embodiment 102. A vesicle according to any one of embodiments 99 to 101 is provided, wherein a SARS-CoV-2 spike protein polypeptide is expressed on the outer surface of the vesicle.

[0209] Embodiment 103. A vesicle according to any one of embodiments 99 to 102 is provided, which expresses a SARS-CoV-2 nucleocapsid protein polypeptide on its surface.

[0210] Embodiment 104. Provided is a method for producing a vesicle of any one of embodiments 99-103, comprising culturing a cell of any one of embodiments 88-98 in a cell culture medium, collecting the cell culture medium, and purifying a plurality of vesicles comprising the vesicles from the cell culture medium.

[0211] Embodiment 105. There is provided a method of embodiment 104, further comprising inducing expression of the synthetic polynucleotide of any one of embodiments 77 to 87 to produce the synthetic fusion protein of any one of embodiments 61 to 76.

[0212] Embodiment 106 The method of embodiment 105, wherein the inducing comprises contacting the cells with tetracycline, doxycycline, or an analog thereof.

[0213] Embodiment 107. The method of embodiment 105, wherein the inducing comprises removing tetracycline, doxycycline, or an analog thereof from the cells.

[0214] Embodiment 108. Provided is a method of eliciting an immune response in a subject, comprising administering to the subject a first dose of an immunogenic composition comprising a plurality of vesicles, the vesicles comprising a vesicle according to any one of embodiments 99 to 103, or a vesicle made by a method according to any one of embodiments 104 to 107, wherein the synthetic fusion protein according to any one of embodiments 61 to 76 is expressed on the outer surface of the vesicle.

[0215] Embodiment 109. The method of embodiment 108 is provided, further comprising administering a second dose of the immunogenic composition to the subject a period of time after administration of the first dose.

[0216] Embodiment 110. The method of embodiment 109 is provided, wherein the period is 14 days to 1 year.

[0217] Embodiment 111. The method of any one of embodiments 108 to 110 is provided, wherein the first dose or the second dose comprises between about 100 μL and 1 mL of the immunogenic composition, and the immunogenic composition comprises between about 0.3 ng / mL and 3 μg / mL of the synthetic fusion protein.

[0218] Embodiment 112 The method of any one of embodiments 108 to 111 is provided, wherein the first dose or the second dose comprises about 100 μL to 1 mL of an immunogenic composition containing about 2E9 vesicles / mL to 3E13 vesicles / mL.

[0219] Embodiment 113 The method of any one of embodiments 108 to 112 is provided, wherein the synthetic fusion protein comprises a SARS-CoV-2 spike protein polypeptide.

[0220] Embodiment 114 The method of any one of embodiments 108 to 113 is provided, wherein the synthetic fusion protein comprises a SARS-CoV-2 nucleocapsid protein polypeptide.

[0221] Embodiment 115. The method of any one of embodiments 108 to 114 is provided, wherein the immunogenic composition comprises vesicles expressing a SARS-CoV-2 spike protein polypeptide on their outer surface and other vesicles expressing a SARS-CoV-2 nucleocapsid protein polypeptide on their outer surface.

[0222] Embodiment 116 The method of any one of embodiments 108 to 115 is provided, wherein the immune response elicited comprises the production of neutralizing antibodies against an antigen present in the synthetic fusion protein.

[0223] Embodiment 117. The method of any one of embodiments 108 to 116 is provided, wherein the elicited immune response comprises the production of neutralizing antibodies against two or more SARS-CoV-2 variants.

[0224] Embodiment 118 The method of any one of embodiments 108 to 117 is provided, wherein the elicited immune response comprises the production of anti-spike antibodies.

[0225] Embodiment 119 The method of any one of embodiments 108 to 118 is provided, wherein the elicited immune response comprises a spike-specific T cell response.

[0226] Embodiment 120 The method of any one of embodiments 108 to 119 is provided, wherein the elicited immune response comprises the production of anti-nucleocapsid antibodies.

[0227] Embodiment 121 The method of any one of embodiments 108 to 120 is provided, wherein the elicited immune response comprises a nucleocapsid-specific T cell response.

[0228] Embodiment 122 The method of any one of embodiments 108 to 121 is provided, wherein the immune response persists in the subject for up to 9 months.

[0229] Embodiment 123 The method of any one of embodiments 108 to 121 is provided, wherein the immune response persists in the subject for at least 9 months.

[0230] array SEQ ID NO:1 provides the amino acid sequence of the coronavirus spike protein polypeptide. TIFF2025529904000002.tif201117

[0231] SEQ ID NO:2 provides the amino acid sequence of the spike polypeptide-linker-CD9 proximal region chimera. TIFF2025529904000003.tif26128

[0232] SEQ ID NO:3 provides the amino acid sequence of the spike-CD9 fusion protein. TIFF2025529904000004.tif237117

[0233] SEQ ID NO:4 provides the nucleic acid sequence encoding the spike-CD9 fusion protein. TIFF2025529904000005.tif252117TIFF2025529904000006.tif252117TIFF2025529904000007.tif237117

[0234] SEQ ID NO:5 provides the amino acid sequence of a coronavirus nucleocapsid protein polypeptide. TIFF2025529904000008.tif70128

[0235] SEQ ID NO:6 provides the amino acid sequence of the nucleocapsid polypeptide-linker-transmembrane domain proximal region chimera. TIFF2025529904000009.tif40128

[0236] SEQ ID NO:7 provides the amino acid sequence of the nucleocapsid-CD9 fusion protein. TIFF2025529904000010.tif120128

[0237] SEQ ID NO:8 provides the nucleic acid sequence encoding the nucleocapsid-CD9 fusion protein. TIFF2025529904000011.tif193117TIFF2025529904000012.tif186128

[0238] SEQ ID NO:9 provides the amino acid sequence of the transmembrane domain polypeptide. TIFF2025529904000013.tif3128

[0239] SEQ ID NO:10 provides the amino acid sequence of the CD9 protein polypeptide. TIFF2025529904000014.tif33128

[0240] SEQ ID NO:11 provides the nucleic acid sequence encoding the coronavirus spike protein polypeptide. TIFF2025529904000015.tif215117TIFF2025529904000016.tif252117TIFF2025529904000017.tif157128

[0241] SEQ ID NO:12 provides a nucleic acid sequence encoding a coronavirus nucleocapsid protein polypeptide. TIFF2025529904000018.tif208117

[0242] SEQ ID NO:13 provides a nucleic acid sequence encoding a CD9 exosomal tetraspanin protein polypeptide. TIFF2025529904000019.tif208117

[0243] Extracellular vesicles and exosomes A variety of host cells are known in the art and are suitable for protein expression and extracellular vesicle production. Non-limiting examples of typical cells used for transfection include, but are not limited to, bacterial cells, eukaryotic cells, yeast cells, insect cells, or plant cells. For example, human embryonic kidney 293 (HEK293), Escherichia coli, Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SJ9, CHO, COS (e.g., COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H, or 293F. For a description of recombinant proteins in 293 cells in suspension culture, see, e.g., Portolano et al., "Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach," Journal of Visualized Experiments, October 2014, 92, e51897, pp. 1-8.

[0244] Extracellular vesicles (EVs) are lipid-bound vesicles secreted by cells into the extracellular space. Three major subtypes of EVs are microvesicles (MVs), exosomes, and apoptotic bodies, which are distinguished based on their biogenesis, release pathway, size, contents, and function. For a review of extracellular vesicles, see, for example, Doyle and Wang, "Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis," Cells, v. 8(7), 2019 Jul., and references therein.

[0245] Exosomes, in particular, include small secretory vesicles approximately 20-200 nm in diameter that are released by mammalian cells and are generated either by budding into endosomes or from the cell's plasma membrane. In some cases, exosomes have a characteristic buoyant density of approximately 1.1-1.2 g / mL and a characteristic lipid composition. The lipid membrane is typically cholesterol-rich and contains sphingomyelin, ceramide, lipid rafts, and exposed phosphatidylserine. Exosomes express certain marker proteins, such as integrins and cell adhesion molecules, but generally lack lysosomal, mitochondrial, or caveolar markers. In some embodiments, exosomes contain cell-derived components, including, but not limited to, proteins, DNA, and RNA (e.g., microRNAs [miRs] and non-coding RNAs). In some embodiments, exosomes can be obtained from cells obtained from sources that are allogeneic, autologous, xenogeneic, or syngeneic to the exosome recipient.

[0246] Certain types of RNA, such as microRNAs (miRNAs), are known to be transported by exosomes. miRNAs often function as post-transcriptional regulators by binding to complementary sequences on target messenger RNA transcripts (mRNAs), thereby resulting in translational repression, target mRNA degradation, and / or gene silencing.

[0247] Useful exosomes can be obtained from any cellular source, including prokaryotic, plant, fungal, metazoan, vertebrate, mammalian, primate, human, autologous, and allogeneic cells. See, e.g., Kim et al., "Platform technologies and human cell lines for the production of therapeutic exosomes," Extracell Vesicles Circ Nucleic Acids 2021;2:3-17. For example, exosomes can be derived from mesenchymal stem cells, embryonic stem cells, iPS cells, immune cells, PBMCs, neural stem cells, HEK293 cells, as described in, for example, Dumont et al., "Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives," Crit Rev Biotechnol 2016;36:1110-22, HEK293T cells, as described in, for example, Li et al., "Identification and characterization of 293T cell-derived exosomes by profiling the protein, mRNA, and MicroRNA components," PLoS One 2016;11:e0163043, and 293F cells (Stenkamp et al., "Exosomes represent a novel mechanism of regulatory T cell suppression (P1079)," J Immunol May 1, 2013, 190(1 Supplementary Note 121.11), amniocytes, CAR-T cells, e.g., cardiospheres and cardiosphere-derived cells (CDCs), etc., as described in WO2014028493, WO2022006178A1, US20210032598A1, US9828603B2, EP2914273A1, US20200316226A1, US20120315252A1, US20170360842A1, and references therein.

[0248] Briefly, methods for preparing exosomes can include culturing cells in a medium, isolating the cells from the medium, e.g., by serial centrifugation, and purifying the exosomes, optionally using a density gradient, e.g., a sucrose density gradient. In some cases, the isolated and purified exosomes are essentially free of non-exosome components, e.g., cellular components or whole cells. The exosomes can be resuspended in a buffer, e.g., sterile PBS buffer containing 0.01-1% human serum albumin. The exosomes can be frozen and stored for future use.

[0249] Exosomes can be collected, concentrated, and / or purified using methods known in the art. For example, differential centrifugation has become the primary technique for isolating secreted exosomes from the supernatant of cultured cells. This approach leverages size to separate exosomes from larger extracellular vesicles and most non-particulate contaminants. Exosomes can be prepared as described in numerous publications, including, but not limited to, Fordjour et al., "A shared pathway of exosome biogenesis operates at plasma and endosome membranes," bioRxiv, preprint posted February 11, 2019, at https: / / www.biorxiv.org / content / 10.1101 / 545228vl; Booth et al., "Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane," J Cell Biol., 172:923-935 (2006); and Fang et al., "Higher-order oligomerization targets plasma membrane proteins and HIV gag to exosomes," PLoS Biol., 5:el58 (2007). Exosomes can be isolated using commercially available kits, including, but not limited to, ExoSpin™ Exosome Purification Kit, Invitrogen® Total Exosome Purification Kit, PureExo® Exosome Isolation Kit, and ExoCap™ Exosome Isolation Kit. Methods for isolating exosomes from stem cells can be found, for example, in Tan et al., Journal of Extracellular Vesicles, 2:22614 (2013); Ono et al., Sci Signal, 7(332):ra63 (2014), and U.S. Application Publication Nos. 2012 / 0093885 and 2014 / 0004601.Methods for isolating exosomes from cardiosphere-derived cells can be found, for example, in Ibrahim et al., "Exosomes as critical agents of cardiac regeneration triggered by cell therapy," Stem Cell Reports, 2014. Specific methodologies include ultracentrifugation, density gradients, HPLC, affinity-based attachment to substrates, or size-exclusion-based filtration.

[0250] Size exclusion allows for the separation of biochemically similar but biophysically distinct microvesicles with relatively large diameters up to 1,000 nm. Differences in flotation velocity further allow for the separation of exosomes of different sizes. Typically, exosomes range in diameter from 30 to 200 nm, e.g., 40 to 100 nm. Further purification can rely on the specific properties of the particular exosome of interest. This includes, for example, the use of immunoadsorption with a protein of interest to select for specific vesicles with exoplasmic or outward orientation.

[0251] Among current methods, such as differential centrifugation, discontinuous density gradients, immunoaffinity, ultrafiltration, and high-performance liquid chromatography (HPLC), differential centrifugation is the most commonly used for exosome isolation. This technique utilizes increasing centrifugal forces from 2,000 x g to 10,000 x g to separate medium-sized particles and cellular debris from the exosome pellet at 100,000 x g. Centrifugation alone allows for substantial separation / collection of exosomes from conditioned medium, but may be insufficient to remove common contaminants such as various protein aggregates, genetic material, medium-derived particulates, and cellular debris. Enhanced specificity of exosome purification can be achieved by utilizing continuous centrifugation combined with ultrafiltration, or equilibrium density gradient centrifugation on a sucrose density gradient to provide higher purity of exosome preparations (buoyant density 1.1–1.2 g / mL), or by applying a discontinuous sugar cushion to the preparation.

[0252] Ultrafiltration can be used to purify exosomes without compromising biological activity. Membranes with different pore sizes, e.g., a 100 kDa molecular weight cutoff (MWCO), and gel filtration to exclude smaller particles have been used to avoid the use of non-neutral pH or non-physiological salt concentrations. Currently available tangential flow filtration (TFF) systems are scalable (up to over 10,000 L) and allow for the purification and concentration of exosome fractions, with such approaches requiring less time than differential centrifugation. HPLC can also be used to purify exosomes into more uniformly sized particle preparations and preserve biological activity, as the preparation is maintained at physiological pH and salt concentration. Other chemical methods utilize differences in exosome solubility for precipitation techniques, the addition of volume-exclusion polymers (e.g., polyethylene glycol (PEG)), and additional rounds of centrifugation or filtration, which may be combined. For example, the precipitation reagent ExoQuick® can be added to conditioned cell culture media to rapidly precipitate exosome populations, but resuspension of pellets prepared via this technique can be difficult.Flow field-flow fractionation (FlFFF) is an elution-based technique that has been used to separate and characterize macromolecules (e.g., proteins) and nano- to micro-sized particles (e.g., organelles and cells) and has been successfully applied to fractionate exosomes from culture media.

[0253] In addition to these techniques that rely on common biochemical and biophysical features, focused techniques can be applied to isolate specific exosomes of interest. These include relying on antibody immunoaffinity to recognize certain exosome-associated antigens. As noted, exosomes also express the extracellular domains of membrane-bound receptors on their surface. This provides an opportunity to isolate and separate exosomes based on shared antigen profiles and in relation to their parental cellular origin. Conjugation to magnetic beads (e.g., anti-CD81 magnetic beads), chromatography matrices, plates, or microfluidic devices allows for the isolation of specific exosome populations of interest, which may be related to their production from the parental cell of interest or to associated cellular regulatory states. Other affinity capture methods use lectins that bind to specific carbohydrate residues on the exosome surface.

[0254] For example, exosomes (and other extracellular vesicles) can be produced via 293F cells. The 293F cells can be transfected with (or transduced with a lentivirus carrying) a polynucleotide encoding the spike protein or nucleocapsid protein described herein, or a chimeric fusion thereof, and express the spike protein or nucleocapsid protein so that the spike protein or nucleocapsid is sorted and presented in exosomes isolated therefrom. An exemplary method for producing exosomes derived from 293F cells can include the following steps: 293F cells (Gibco™, Catalog No. 51-0029, ThermoFisher Scientific, Waltham, MA) can be tested for pathogens and found to be free of viral (cytomegalovirus, human immunodeficiency virus I and II, Epstein-Barr virus, hepatitis B virus, and parvovirus B19) and bacterial (mycoplasma) contamination. Cells can be maintained in FreeStyle™ 293 Expression Medium (Gibco, Catalog No. 12338-018, ThermoFisher Scientific, Waltham, MA) and incubated at 37°C in 8% CO2. For exosome production, 293F cells can be seeded into shaker flasks at a density of 1.5E6 cells / ml in approximately 1 / 4 of the flask volume and grown at a shaking speed of approximately 110 rpm. HEK293 cells can be grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum.

[0255] To purify exosomes, 293F cells can be cultured in shaker flasks for 3 days. Cells and large cell debris can be removed by centrifugation at 300 x g for 5 minutes, followed by 3,000 x g for 15 minutes. The resulting supernatant can be passed through a 0.22 μm sterile filter unit (Thermo Fisher, catalog number 566-0020) to produce clarified tissue culture supernatant (CTCS). CTCS can be concentrated by centrifugal filtration (Centricon Plus-70, Ultracel-PL Membrane, 100 kDa size exclusion, Millipore Sigma, catalog number UFC710008, St. Louis, MO) to reduce the volume of approximately 120 mL of CTCS to approximately 0.5 mL. The enriched CTCS can then be purified by size-exclusion chromatography (SEC) (qEV Original Column / 35 nm: Izon Science, Catalog No. SP5) in 1x PBS. The exosomes present in each 0.5 mL starting sample can be eluted into three 0.5 mL fractions. The purified exosomes can be re-enriched using an Amicon® Ultra-4 100 kDa cutoff spin column (Cat. No. UFC810024). This process yields a population of exosomes / small EVs with the expected ultrastructural and size distribution profile of human exosomes and containing the exosome marker proteins CD9 and CD63, resulting in a 500-fold enrichment effect at a final concentration of 1E10–2E12 exosomes / mL. The concentration and size of the isolated extracellular vesicles can be measured using a NANOSIGHT nanoparticle tracking analysis system (Malvern Panalytical, Malvern, UK).

[0256] SARS-CoV-2 Proteins Membrane-bound vesicles containing one or more populations of SARS-CoV-2 structural proteins are disclosed. By "containing," we mean that the contained proteins are within the lumen of the vesicle, displayed on the surface of the vesicle, or both within the lumen and on the surface. Here, fusion proteins containing tetraspanin protein polypeptide sequences are primarily displayed on the surface of the vesicle. These proteins displayed on the surface of the vesicle can have a portion of the protein within the lumen, a portion of the protein spanning the membrane of the vesicle (i.e., a transmembrane region or domain), and a portion of the protein extending outside the vesicle. In one embodiment, the SARS-CoV-2 structural protein is the spike glycoprotein (S), nucleocapsid (N), membrane (M), or envelope (E), protein, or any combination thereof. See Satarker and Nampoothiri, "Structural Proteins in Severe Acute Respiratory Syndrome Coronavirus-2," Arch Med Res. 2020 Aug;51(6):482-491. See, for example, Figures 1A, 1B, 2A, and 2C, which show spike / CD9 and nucleocapsid / CD9 fusion proteins for expression of spike or nucleocapsid antigens on the surface of exosomes.

[0257] In one embodiment, the antigenic protein is the SARS-CoV-2 spike glycoprotein (also known as spike protein or simply "spike"). The spike protein can be of any variant of SARS-CoV-2, such as the Wuhan-1 strain, an omicron variant (e.g., a BA.2 variant), a delta variant (e.g., a B.1.617.2, AY.3, AY.103, AY.44, AY.43 variant, etc.), and an epsilon variant (e.g., a B.1.427 or B.1.429 variant), or any currently known or undiscovered variant. As used herein, the term spike refers to any SARS-CoV-2 spike glycoprotein, chimera, or fragment thereof, unless otherwise specified.

[0258] In some embodiments, the SARS-CoV-2 spike protein is a Wuhan-1 strain SARS-CoV-2 spike protein or a delta-variant SARS-CoV-2 spike protein; a furin-blocked, trimer-stabilized form of the Wuhan-1 strain SARS-CoV-2 spike protein; a Wuhan-1 strain SARS-CoV-2 spike protein comprising an amino acid change of D614G; a Wuhan-1 strain SARS-CoV-2 spike protein comprising a diproline substitution of 986KV987-to-986PP987 (S-2P); and / or a Wuhan-1 strain SARS-CoV-2 spike protein comprising a cleavage site mutation of 682RRAR685-to-682GSAG685, or equivalent (S-CSM).

[0259] Extracellular vesicles displaying spike or nucleocapsid proteins In one embodiment, the present invention provides extracellular vesicles that express (also known as "displaying") spike proteins or nucleocapsids on their surface, which are useful as vaccines against multiple variants of SARS-CoV-2. The spike proteins can be delta variants that have any one or more of trimer-stabilizing mutations, prefusion conformation-stabilizing mutations (e.g., diproline-stabilizing mutations), and furin cleavage site mutations.For example, Walls et al.,"Structure,Function,and Antigenicity of the SARS-CoV-2 Spike Glycoprotein,"Cell 180,281-292,April 16,2020;Wrapp et al.,"Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation,"Science367,1260-1263(2020)13 March 2020;Kirchdoerfer et al.,"Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis,"Sci Rep 8,15701(2018),doi.org / 10.1038 / s41598-018-34171-7;Pallesen et al.,"Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen," PNAS, E7348-E7357, published online August 14, 2017 pnas.org / cgi / doi / 10.1073 / pnas.1707304114; Juraszek, et al., "Stabilizing the closed SARS-CoV-2 spike trimer," Nat Commun 12, 244(2021), doi.org / 10.1038 / s41467-020-20321-x; Johnson, 2020; and Xiong 2020; and references therein.

[0260] Here, we generated synthetic fusion proteins containing a C-terminal tetraspanin protein and either the SARS-CoV-2 spike protein (Figures 1A and 1B) or the SARS-CoV-2 nucleocapsid protein (Figures 2A and 2B). It is important to note that the nucleocapsid protein is a soluble protein that is not expressed on the viral surface; therefore, engineered fusion proteins containing tetraspanins and other transmembrane domains allow for the localization of the nucleocapsid protein to the vesicle surface, providing a readily accessible antigen for immunization (see Figure 2B).

[0261] In one embodiment, exosomes expressing spike or nucleocapsid proteins on their surface were generated from 293F cells expressing spike / nucleocapsid proteins. Referring to Figure 3A, in a specific exemplary embodiment, packaging cells (300) were transfected with a plasmid (301a) encoding a spike or nucleocapsid-tetraspanin fusion protein and plasmids (302a and 303a) encoding lentiviral structural proteins. Lentiviral proteins were produced (302b and 303b), which incorporated the fusion protein RNA, forming a lentiviral vector (304) containing the fusion protein RNA. Host cells (311) were transduced with lentivirus (304) carrying the fusion protein RNA, which allowed the SARS-CoV-2 spike or nucleocapsid-tetraspanin fusion protein (307a) to be produced and sorted to the plasma membrane (307b) (Figure 3B) and the surface of exosomes produced by the transduced host cells (Figures 4C, 5A, 5B, and 6C).

[0262] Referring to Figures 4A-C and 5A and 5B, exosomes were isolated from 293F cells harboring the spike-CD9 construct (Figures 1A and 1B). Figure 4A shows the size distribution of these exosomes, ranging from approximately 50 nm to approximately 270 nm, with a median of approximately 100-150 nm. Figure 4B shows the expression of the spike-containing fusion protein in transduced 293F cells (lane 4) and the enriched expression / presentation of the spike-containing fusion protein in exosomes derived from transduced 293F cells (lane 5). Figure 4C shows significant expression of the spike-containing fusion protein in these exosomes, as determined by flow cytometry.

[0263] Transmission electron microscopy confirmed that spike protein was expressed on the surface of exosomes derived from spike-CD9-transduced 293F cells (arrows in Figures 5A and 5B point to spikes on the surface of the vesicles).

[0264] Referring to Figures 6A-C, exosomes were isolated from 293F cells harboring the nucleocapsid-CD9 construct (Figures 2A and 2B). Figure 6A shows the size distribution of these exosomes, ranging from approximately 50 nm to approximately 270 nm, with a median of approximately 100-150 nm. Figure 6B shows the expression of the nucleocapsid-containing fusion protein in exosomes derived from transduced 293F cells (lane 5). Figure 6C shows significant expression of the spike-containing fusion protein in these exosomes, as determined by spike flow cytometry. In one embodiment, expression of the nucleocapsid-CD9 fusion protein was placed under the control of a tet-inducible promoter. Here, exosomes derived from transduced but uninduced host cells did not express nucleocapsid (see the second curve from the left in Figure 6C).

[0265] In one embodiment, exosomes expressing a spike-CD9 fusion protein (FIGS. 1A-B and 4A-C) were delivered to a subject by intramuscular injection according to a two-dose schedule (see, e.g., FIG. 7). Similarly, in another set of experiments, exosomes expressing a nucleocapsid-CD9 fusion protein (FIGS. 2A-B and 6A-C) were delivered to a subject by intramuscular injection according to the same two-dose schedule (see, e.g., FIG. 7).

[0266] Referring to Figure 7, on day 1, subject mice were administered a 1x dose (i.e., 3E10 vesicles) or a 10x dose (i.e., 3E11 vesicles) expressing either a spike-CD9 fusion protein or a nucleocapsid-CD9 fusion protein by intramuscular injection (710). On day 14, blood was collected from the administered mice and assessed for initial humoral immune responses (720) (see Figures 8A and 9A). On day 21, subject mice received a second dose of exosomes expressing spike or nucleocapsid of the invention (730). On day 35, blood was collected from the administered mice and assessed for humoral immune responses, including antibody production (see Figures 8B and 9B) and neutralizing antibody production (740). On day 40, splenocytes were collected from the administered mice and assessed for cellular immune responses via ELISpot assay (750) (see Figures 8C, 8D, 10A, and 10B).

[0267] Referring to Figures 8A and 8B, humoral immune responses to exosomes (STX-S) expressing COVID-19 spike antigens were induced in subjects as early as two weeks after the first injection (Figure 8A) and persisted until at least day 35 post-injection (Figure 8B). Furthermore, robust and sustained anti-spike antibody responses were elicited using nanogram amounts of spike protein expressed on exosomes, without the inclusion of an adjuvant. Here, for example, as little as 10 ng of exosome-expressed spike protein induced significant and useful antibody responses measured on days 14 and 35 without an adjuvant. Furthermore, 32 ng of exosome-expressed spike protein induced a more than 30-fold greater antibody response than 32 ng of purified spike protein with or without an adjuvant (exosome-less). It is generally known in the art that prior art spike protein vaccines are administered with adjuvants in microgram amounts to generate antibody responses similar to those observed with nanogram amounts of the exosome-expressed spike protein antigen of the present invention.

[0268] Referring to Figures 8C and 8D, immune responses to STX-S at the cellular level were induced in subjects, as shown in ELISpot assays performed on splenocytes obtained on days 35 to 40. Here, a dose of as little as 10 ng of adjuvant-free exosome-expressed spike antigen induced a significant CD4+ T cell response against the spike protein in subjects, as evidenced by an IL4 ELISpot assay of splenocytes from the subjects (Figure 8C). Here, doses of 10 ng and 32 ng of adjuvant-free exosome-expressed spike antigen also induced a significant CD8+ cytotoxic T cell response against the spike protein in subjects, as evidenced by an IFNγ ELISpot assay of splenocytes from the subjects (Figure 8D).

[0269] Similar nanogram doses of adjuvant-less exosomes expressing SARS-CoV-2 nucleocapsid protein also elicit useful, robust, and significant humoral and cell-mediated immune responses (Figures 9A-D). The inventors anticipate that any or most viral antigens (e.g., influenza hemagglutinin, influenza neuraminidase, respiratory syncytial virus, and / or other viral glycoproteins or other proteins or fragments thereof) expressed on the surface of exosomes according to the present disclosure will elicit humoral and / or cell-mediated immune responses in recipient subjects.

[0270] Thus, in some embodiments, 1 ng to 1 μg (inclusive) of viral antigen (e.g., spike or nucleocapsid of SARS-CoV-2), 1 to 900 ng, 1 to 800 ng, 1 to 700 ng, 1 to 600 ng, 1 to 500 ng, 1 to 400 ng, 1 to 300 ng, 1 to 200 ng, 1 to 100 ng, 1 to 90 ng, 1 to 80 ng, 1 to 70 ng, 1 to 60 ng, 1 to 50 ng, 1 to 40 ng, 5 to 100 ng, 5 to 90 ng, 5 to 80 ng, 5 to 70 ng, 5 to 60 ng, 5 to 50 ng, 5~40ng, 100μg or less, 50μg or less, 25μg or less, 20μg or less, 10μg or less, 1μg or less, 900ng or less, 800ng or less, 700ng or less, 600ng or less, 500ng or less, 400ng or less, 300ng or less, 200ng or less, 10 0ng or less, 90ng or less, 80ng or less, 70ng or less, 60ng or less, 50ng or less, 40ng or less, about 1ng, about 1.5ng, about 2ng, about 2.5ng, about 3ng, about 3.5ng, about 4ng, about 4.5ng, about 5ng, about 6ng, about 7ng, about 8ng , about 9ng, about 10ng, about 11ng, about 12ng, about 13ng, about 14ng, about 15ng, about 16ng, about 17ng, about 18ng, about 19ng, about 20ng, about 25ng, about 30ng, about 35ng, ​​about 40ng, about 45ng, about 50ng, about 55ng, about 60ng, about 65ng, about 70ng, about 75ng, about 80ng, about 85ng, about 90ng, about 95ng, about 100ng, about 105ng, about 110ng, about 115ng, about 120ng, about 125ng, about 130ng, about 135ng, ​​about 140ng, about 145 Low dose immunogenic compositions or vaccines are provided that contain an immunogenic dose of about 150 ng, about 155 ng, about 160 ng, about 165 ng, about 170 ng, about 175 ng, about 180 ng, about 185 ng, about 190 ng, about 195 ng, about 200 ng, about 205 ng, about 210 ng, about 215 ng, about 220 ng, about 225 ng, about 230 ng, about 235 ng, about 240 ng, about 245 ng, or about 250 ng of viral antigen (e.g., spike or nucleocapsid of SARS-CoV-2).

[0271] Referring to Figures 9A and 9B, humoral immune responses against COVID-19 nucleocapsid antigen-expressing exosomes (STX-N) were induced in subjects and detected 35 days after injection (Figures 8A and 8B). Furthermore, robust and sustained anti-spike antibody responses were elicited using nanogram amounts of spike protein expressed on exosomes, without the inclusion of an adjuvant. Here, for example, only 3.2 ng of exosome-expressed nucleocapsid protein induced a significant beneficial antibody response measured at day 35 without an adjuvant (Figure 9A, STX-N Dose 2). Thus, in one embodiment, a full immunization cycle (two im injections) induced a significant increase in IgG against SARS-CoV-2 nucleocapsid protein (N) compared to PBS, a 3-fold increase for a dose of approximately 3 ng / injection (Dose 2, Figure 9A) and a 10-fold increase for a dose of 10 ng / injection (Dose 3, Figure 9B).

[0272] Referring to Figures 9C and 9D, to characterize T cell responses to STX-N, antigen-specific T cell responses to nucleocapsid protein were measured by ELISpot assay performed on splenocytes obtained on day 40. Here, vaccination of subjects with STX-N induced polyfunctional antigen-specific T cell responses to SARS-CoV-2 nucleocapsid protein on day 40 (after the booster (second) injection). STX-N administration resulted in a strong IFNγ response (Figures 9C and 9D). Here, evaluation of cells secreting IFNγ in response to ex vivo nucleocapsid protein stimulation showed a 6-fold increase in STX-N-immunized spleens (Figures 9C and 9D), thereby suggesting a Th1-biased CD8+ T cell response. Because the nucleocapsid protein is not a surface protein and is most likely presented to immune cells only after the infecting virus has been processed within the infected cell, this response is crucial for the development of immunogenic compositions expressing exosomes that have the potential to protect against a wide range of SARS-CoV-2 variants.

[0273] Similar to the humoral antibody responses against the nucleocapsid protein (Figures 9A and 9B), in this specific embodiment, administration of doses of STX-N in the range of 3 to 10 ng, approximately three orders of magnitude lower than prior art protein subunit vaccines, resulted in significant CD8+ / IFNγ responses against the nucleocapsid protein in subjects without an adjuvant.

[0274] Referring to Figures 10A-C, administration of nanogram amounts of STX-S to subjects induced potent neutralization of both the delta and omicron variants of SARS-CoV-2 induced by STX-S exosome injection. In one embodiment, day 40 serum from a subject receiving approximately 3.2 ng of STX-S per injection (Dose 2, Figure 10A) and day 14 and day 40 serum from a subject receiving approximately 9.8 ng of STX-S per injection (Dose 4, Figure 10A) were tested for neutralizing antibodies against the SARS-CoV-2 delta variant. Here, STX-S induced potent neutralizing activity in each sample tested, comparable to SARS-CoV-2 delta-positive vaccine sera (Figure 10A). STX-S showed dose-dependent neutralization of the virus, as estimated by its ability to protect infected cells from virus-induced cytopathic effects (compare dose 4 with dose 2 in serum on day 40, Fig. 10A ).

[0275] Thus, according to one embodiment, the engineered STX-S exosome vaccine induced neutralizing antibodies against the delta spike in subjects by day 14 (after a single im injection). By day 40, approximately four weeks after the STX-S boost, robust neutralization was observed in all subjects, regardless of dose (Figure 10A). Here, a single injection of approximately 9 ng of STX-S spike (dose 4, Figure 10A) resulted in approximately 65-75% neutralization against the delta variant. Furthermore, full immunization (i.e., a primary injection and at least one booster, or two im injections) resulted in approximately 80-85% neutralization against the delta variant with a dose of approximately 3-9 ng of spike delivered by STX-S exosomes.

[0276] Delta variant neutralization induced in subjects by administration of STX-S induced responses comparable to human control plasma (CoV02-delta, plasma from a patient immunized with Moderna's mRNA vaccine who developed a breakthrough Delta infection), with full neutralization responses induced at higher dilutions (e.g., 1:320, see day 40 of STX-S dose 4) (Figure 10A).

[0277] Further, referring to Figures 10B and 10C, a dose of approximately 9-10 ng of STX-S (where the spike protein of STX-S is the delta variant, STX-S δ Day 40 serum samples from subjects receiving STX-S were also tested for neutralizing antibodies against SARS-CoV-2 Omicron variants (Omicron BA.1 and BA.5.2.1). As shown in Figures 10C and 10D, STX-S δStrong cross-neutralization was observed for sera obtained from subjects treated with STX-S exosomes, in which approximately 84% neutralization of the Omicron BA1 variant (Figure 10B) and a range of 16% to 97% neutralization of the Omicron BA5 variant (Figure 10C) were achieved after full immunization (i.e., at least two im injections) with an approximately 9-10 ng dose of delta spike delivered by STX-S exosomes. Thus, in some embodiments, administration of immunogenic compositions containing single-variant spike STX-S exosomes provides some level of protective immunity against other SARS-CoV-2 variants.

[0278] Referring to Figure 11, the antibody response to the spike from STX-S in the mouse model persists for at least about 6 months and is expected to persist for longer than 6 months. As shown in Figure 11, mice administered 10 ng STX-S and boosted with 7 ng STX-S or 3 ng STX-S show strong IgG responses to the spike antigen at least 166 days after the first dose.

[0279] In addition to eliciting superior immune responses, both spike-exosome vaccines and nucleocapsid-exosome vaccines offer several other advantages over currently available vaccines. First, the exosome-based vaccines of the present invention deliver antigens through a fully internalized, self-assembled lipid bilayer, which can easily integrate into host cell membranes and facilitate presentation of engineered antigens to immune cells. Membrane-bound antigens are readily presented to circulating immune cells and can rapidly activate responses, while free antigens contained within exosomes can be further processed by the lysosomal system and activate cytotoxic T lymphocyte responses. Therefore, the use of natural delivery systems promotes efficient delivery and responses compared to synthetic lipid nanoparticle technology.

[0280] Second, spike exosome vaccines and nucleocapsid exosome vaccines are protein-based vaccines, and therefore antigens are readily available to the subject's immune system without the translation required in host cells for RNA-based vaccines. Incomplete translation and / or incorrect folding of the mRNA encoding the spike protein or nucleocapsid protein limits the amount of antigen available after vaccination, resulting in highly variable immune responses and reduced efficacy.

[0281] Third, the exosome-based spike fusion protein vaccine and nucleocapsid fusion protein vaccine of the present invention do not require adjuvants or synthetic lipid nanoparticles (LNPs) for delivery and immune response. Traditional protein-based vaccines require adjuvants (e.g., aluminum salts and squalene oil-in-water emulsion systems MF59 (Novartis) and AS03 (GlaxoSmithKline)) to enhance and coordinate immune responses and influence the affinity, specificity, magnitude, and functional profile of B and T cell responses (see, e.g., Wong, S.S., and R.J. Webby, "Traditional and New Influenza Vaccines," Clin Microbiol Rev, 2013.26(3):476-92). Current mRNA vaccines do not use adjuvants, which, combined with the requirement for mRNA translation before antigens become available, may reduce long-term efficacy. Currently approved COVID vaccines use LNPs to deliver mRNA or adjuvants for proteins, and undesirable side effects have been reported.

[0282] Fourth, the immunogenic exosome compositions of spike fusion proteins and nucleocapsid fusion proteins of the present invention exhibit greater immunogenic efficacy at doses significantly lower, i.e., three orders of magnitude lower, than currently available LNP mRNA and protein vaccines. For example, clinically approved protein candidate vaccines use approximately 5µg to 25µg of antigen along with an adjuvant to induce immunization (e.g., Formica, et al., "Different dose regimens of a SARS-CoV-2 recombinant spike protein vaccine (NVX-CoV2373) in younger and older adults: A phase 2 randomized placebo-controlled trial," PLoS Med, 2021.18(10):p.e1003769; Sun et al., "Development of a Recombinant RBD Subunit Vaccine for SARS-CoV-2," Viruses, 2021.13(10); Worzner et al., "Adjuvanted SARS-CoV-2 spike protein elicits neutralizing antibody and CD4 T cell responses after a single immunization in (See mice. EBioMedicine, 2021. 63:103197; and references therein.) Here, immunological data from subjects immunized with either spike-fused exosomes or nucleocapsid-fused exosomes demonstrate that approximately 1 / 1000th the amount of protein antigen currently administered in clinically approved vaccines (i.e., nanogram amounts versus microgram amounts) elicits complete immunization correlated with high antibody levels, potent virus neutralization, broad multivariate activity, and both B cell and T cell memory.

[0283] Fifth, a further limitation of available vaccines is the need for refrigeration and the short shelf life of the product at elevated temperatures. Due to their high stability at physiological pH and temperature, exosomes containing display exosomes can be stored at 4°C for extended periods of time. Exosome compositions expressing spike fusions or nucleocapsid fusions may be lyophilized for long-term storage at convenient temperatures (see, e.g., U.S. Patent Application No. 2017 / 0360842 A1).

[0284] Thus, here, when spike or nucleocapsid proteins were delivered via exosomes, they induced not only strong CD8+ T cell responses but also strong B cell responses, as evidenced by IgG production and potential neutralizing antibodies. Without wishing to be bound by theory, this result may be explained by the role of extracellular vesicles in cell-to-cell communication and antigen presentation. Specifically, multiple copies of spike protein (Figure 5A and B) or nucleocapsid protein may be present on the vesicle surface, facilitating cross-linking to B cell receptors. Furthermore, the spike protein of extracellular vesicle-based vaccines may indirectly activate B cells and CD8(+) T cells through antigen cross-presentation.

[0285] It is further contemplated that the fusion protein-expressing exosomes of the present invention can be engineered to express an antigen of interest to target new COVID variants. The antigen of interest can be easily exchanged and adapted to meet the needs. To enable presentation of the antigen in exosomes for delivery to the host subject's immune system, the antigen can be expressed as a fusion protein (e.g., chimera) comprising, for example, an exosomal expression or presentation domain fused to an antigen domain.

[0286] It is further envisioned that exosomes can be genetically engineered to selectively target organs or tissues of interest and to allow for safe and targeted delivery of antigens to specific immune subsystems to elicit specific types of responses in a subject.

[0287] Referring now to Figure 12, HEK293-hACE2 cells were seeded in 24-well plates and contacted with approximately 1E7 to 1E12 exosomes per mL in low-serum medium at approximately 37°C for approximately 3.5 hours. In one series (series 2), the exosomes expressed spike-CD9. In another series (series 1), the exosomes did not express spike-CD9 or any other form of spike. Here, HEK293-hACE2 cells showed greater uptake of spike-expressing exosomes compared to "wild-type" 293F wt exosomes at approximately 2.5- to 3-fold lower concentrations. See also Table 1.

[0288] Table 1. Uptake of wild-type and spike-expressing exosomes by HEK293-hACE2 cells TIFF2025529904000020.tif87152

[0289] Thus, spike-expressing exosomes can be used to deliver any drug cargo to the lung or other tissues that express spike receptors, such as ACE2. Furthermore, this data further demonstrates the fact that exosomes of the present disclosure that express recombinant fusion proteins on their surface maintain the native structure of the various components of the fusion protein, e.g., the spike protein polypeptide has sufficient native structure to associate with its natural target, i.e., ACE2. [Example]

[0290] The pandemic emergency has revealed the need for potent, reliable, and more widespread vaccines to counter the ever-changing virus and stop viral transmission. While COVID-19 mRNA vaccines played a crucial role in the emergency in reducing the toll of SARS-CoV-2 infection, their diminishing ability to protect against emerging VOCs, along with the need for multiple injections to sustain protection, has forced the scientific community to search for new approaches.

[0291] SARS-CoV-2 has taught us that a successful vaccine must generate a strong antibody response with neutralizing antibodies and a strong T cell response that can counter viral infection more broadly and in a timely manner with a minimum number of injections. These criteria can be met by an exosome-based vaccine, along with a "multivalent" approach.

[0292] The following examples demonstrate that exosomes can be used to deliver viral proteins for immunization. The disclosed STX platform generated two vaccine candidates (STX-S and STX-N) that, both individually and in combination (STX-S+N), induced potent immune responses against two SARS-CoV-2 proteins (spike and nucleocapsid) with a single shot in two different animal models by delivering a few nanograms of protein on the surface of exosomes. No adjuvant was required, 100-fold less protein was used, and no inter-protein competition was observed.

[0293] "Multivalent" or "combination" vaccines have several advantages. First, they require fewer injections, which increases population acceptance and, consequently, increases the percentage of the population vaccinated, providing broader epidemiological benefits. Consequently, fewer injections mean lower overall budgets and costs. Naturally, there are limitations to consider. First, antigen selection: the selected antigens must retain strong immunogenicity after combination, preferably have sequences that are conserved across species, and ideally have a low mutagenesis rate. Among these, immunogenic interference is crucial; minimal or no competition should be observed. As in the case of the exemplified STX-S+N, multivalent vaccines exhibit the same potency as single products, increasing antibody levels by several thousand times. Furthermore, STX-S+N induced both quantitative and qualitative immune responses, and consistent increases in the amount of antibodies produced, protection demonstrated by neutralizing antibodies, and T cell engagement were all observed in response to administration of the disclosed exosome vaccine STX-S+N. Importantly, no adverse side effects were recorded, and both mice and rabbits showed no changes in body weight or blood tests, nor any alterations at the tissue level, suggesting an overall safety profile.

[0294] The data presented herein suggest that exosomes are ideal vehicles for vaccination, capable of safely delivering antigens of interest (foreign proteins) in a manner that mimics natural viral infection. Exosome-based vaccines constitute an innovative approach for designing efficient virus-free human-derived vaccines. Yoo et al. ("Possibility of exosome-based coronavirus disease vaccines," 2019 Vaccine (Review), Mol Med Rep, 2022, 25(1)) observed that exosomes, or extracellular vesicles, may generally support vaccination needs beyond traditional strategies. Compared to viral or vector approaches, exosomes are not immunogenic per se; rather, they are carriers of proteins that retain their original conformation, three-dimensional structure, and modifications, all embedded in the lipid biolayer of their membrane, ready for efficient presentation to the immune system.

[0295] The strong T cell responses initiated by STX-S+N administration are clinically relevant. Keeton et al. ("T cell responses to SARS-CoV-2 spike cross-recognize Omicron," Nature, 2022, 603(7901):pp.488-492) observed that while neutralizing antibodies may not recognize new variants of concern (VOCs), T cell populations can cross-react with them and confer protection. Here, spike- and Ncap-specific T cell responses were observed. The highly conserved Ncap could further increase the efficacy of STX vaccines, ensuring an additional immune response that is not compromised by naturally mutating surface proteins. This suggests that if new variants of concern (VOCs) escape the barrier of neutralizing antibodies, T cell responses may cross-react and limit infection.

[0296] Other groups have reported multivalent vaccines for SARS-CoV-2 using additional viral approaches, co-expressing the N and S proteins of SARS-CoV-2 in VSV (e.g., O'Donnell, KL, et al., "Protection from COVID-19 with a VSV-based vaccine expressing the spike and nucleocapsid proteins," Front Immunol, 2022.13:p.1025500) or by adenovirus (e.g., Dangi, T., et al., "Combining spike- and nucleocapsid-based vaccines improves distal control of SARS-CoV-2," Cell Rep, 2021.36(10):p.109664). Interestingly, the use of multiprotein vaccines extends efficacy to distal organs, lowering viral load not only in the respiratory system but also in the distant brain (see, for example, Matchett, W.E., et al., "Cutting Edge: Nucleocapsid Vaccine Elicits Spike-Independent SARS-CoV-2 Protective Immunity," J Immunol, 2021, 207(2):376-379). Nucleocapsid-specific immunity plays an essential role in SARS-CoV-2 infection. While antibody responses can block initial viral entry at proximal sites of infection, it is T cell responses that regulate transmission, secondary infection, and subsequent viral spread to distal sites, providing a synergistic antiviral effect by killing virally infected cells and further suppressing viral spread to peripheral organs.

[0297] A single-dose dual-antigen vaccine with efficacy against multiple SARS-CoV-2 VOCs is disclosed that has broader immunopotency and can be used as a boost to existing immunity generated by a previously approved vaccine. The disclosed StealthX Vaccine Technology (STX) uses exosomes to deliver nanogram quantities of viral antigens to elicit a strong, broader immune response without adjuvants.

[0298] Example 1: Cell lines Human embryonic kidney 293 T cells (293T) were purchased from ATCC (CRL-3216). 293T cells were maintained in culture using Dulbecco's Modified Eagle Medium (DMEM), high glucose, and Glutamax™ containing 10% fetal bovine serum. 293T cells were incubated at 37°C / 5% CO2. FreeStyle™ 293F cells (Gibco, 51-0029) were purchased from ThermoFisher (Waltham, MA). 293F cells were used as the parent cell line to generate Stealth X-Spike cells (STX-S), a stable cell line expressing the SARS-CoV-2 delta spike. 293F and STX-S cells were maintained in a MULTITRON incubator (Infors HT, Sulzemoos, DE) at 37°C in an 80% humidified atmosphere containing 8% CO on an orbital shaker platform rotating at 110 rpm.

[0299] Example 2: Lentiviral Vectors Lentiviral vectors for expression of SARS-CoV-2 spike (delta variant B.1.617.2, NCBI accession number OX014251.1, available August 15, 2023, at www.ncbi.nlm.nih.gov / nuccore / OX014251.1) and SARS-CoV-2 nucleocapsid (NCBI accession number OP359729.1, available August 15, 2023, at www.ncbi.nlm.nih.gov / nuccore / OP359729.1), along with two packaging plasmids (pMD2.G and psPAX2), were designed and synthesized from Genscript. Lentiviral particles for transduction were generated by transfecting 293T cells with pMG.2 (Genscript, GenScript Biotech, Piscataway, NJ), psPAX2 (Genscript), and spike-expressing STX-S_pLenti (Genscript) or nucleocapsid-expressing STX-N_pLenti (Genscript) at a 5:5:1 ratio using Lipofectamine™ 3000 (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's instructions. Spike and nucleocapsid lentiviral particles were harvested 72 hours posttransfection and used to transduce 293F parental cells to generate STX-S and STX-N, respectively.

[0300] Example 3: Flow cytometry Standard flow cytometry methods were applied to measure spike SARS-CoV-2 protein expression on the surface of STX cells. Briefly, 250K STX cells were aliquoted, pelleted, and resuspended in 100uL of eBioscience™ Flow Cytometry Staining Buffer (ThermoFisher). Cells were incubated in the presence of anti-spike antibody (Abcam, clone 1A9, ab273433, Abcam, Cambridge, UK) or anti-nucleocapsid (Abcam, ab281300) labeled with Alexa Fluor®-647 (Alexa Fluor® 647 Conjugation Kit (Fast)-Lightning-Link® (Abcam, ab269823) according to the manufacturer's protocol, protected from light, for 30 minutes at room temperature (RT). After incubation, STX cells were washed with eBioscience™ Flow Cytometry Staining Buffer (ThermoFisher, catalog no. 00-4222-57), resuspended in PBS, and analyzed on a CYTOFLEX S flow cytometer (Beckman Coulter, Brea, California). Data were analyzed by FLOWJO (Becton, Dickinson and Company, Franklin Lakes, NJ).

[0301] Example 4: Cell sorting Cell sorting was performed at the flow cytometry facility at Scripps Research Institute (San Diego, CA). To enrich for spike-positive populations, STX-S cells were stained as described above for flow cytometry and then subjected to cell sorting (Beckman Coulter MOFLO ASTRIOS EQ) to generate pooled STX-S. Unless otherwise noted, pooled STX-S was used in the examples presented herein.

[0302] Example 5: STX exosome production STX-S and STX-N cells were cultured in FREESTYLE medium (ThermoFisher, 12338018) in a MULTITRON incubator (Infors HT) at 37°C with an 80% humidified atmosphere containing 8% CO2 on an orbital shaker platform. Cells and cell debris were then removed by centrifugation, and microvesicles larger than approximately 220 nm and other extracellular vesicles were removed by vacuum filtration. Exosomes were then isolated using filtration and size exclusion. Briefly, the supernatant was subjected to concentration filtration on a Centricon Plus-70 centrifugal filter unit (Millipore, UFC710008, MilliporeSigma, St. Louis, MO) and then subjected to size exclusion chromatography (SEC) using a qEV Original SEC column (Izon, SP5, Izon Science, Christchurch, NZ).

[0303] Example 6: Nanoparticle tracking analysis Exosome size distribution and concentration were determined using ZetaView nanoparticle tracking analysis (Particle Metrix, Inning am Ammersee, DE) according to the manufacturer's instructions. Exosome samples were diluted with 0.1 μm-filtered 1× PBS (Gibco, 10010072) to be within the optimal operating range of the instrument.

[0304] Example 7: Protein Expression Detection of SARS-CoV-2 spike and nucleocapsid proteins in cell lysates and exosomes was performed using the JESS Capillary Protein Detection System (ProteinSimple, San Jose, CA). Samples were lysed in RIPA buffer (ThermoFisher Scientific, 8990) supplemented with protease / phosphatase inhibitors (ThermoFisher Scientific, A32961) and quantified using the BCA assay (ThermoFisher Scientific, 23227). For spike protein detection, a 12-230 kDa separation module was used according to the manufacturer's protocol. Briefly, 0.8 μg of sample and protein standards were run in each capillary and probed with anti-mouse Ms-RD-SARS-COV-2 (MAB105401, 1:10 dilution; R&D Systems, Minneapolis, MN) or anti-rabbit nucleocapsid (NBP3-00510, 1:100 dilution; Novus Biologicals, Centennial, CO), followed by the secondary antibody provided in the JESS kit (HRP substrate used directly).

[0305] Example 8: TEM imaging for characterization of STX exosome morphology STX-S and STX-N exosome samples were negatively stained onto carbon film-coated copper grids and imaged by TEM at the UC San Diego Electron Microscopy Core Facility (San Diego, CA). Briefly, samples were processed by glow discharge, stained with 2% uranyl acetate, dried, and then imaged. Grids were imaged on a JEM-1400 Plus (JEOL Ltd, Japan) at 80 kV and 48 uA. Images were taken at a resolution of 4k × 4k pixels at magnifications ranging from 12k to 80k.

[0306] Example 9: CD81 bead assay STX-S exosomes or 293F parent exosomes were mixed with anti-CD81-labeled magnetic beads (ThermoFisher, 10622D) for 2 hours at room temperature (RT) and washed twice with PBS using a magnetic stand. The bead-exosomes were then incubated with either directly conjugated AlexaFluor 647 anti-spike (see Flow Cytometry above), FITC anti-CD81 antibody (BD Biosciences, 551108, Franklin Lakes, NJ), or FITC mouse IgG, kappa isotype control (BD Biosciences, 555748) for 1 hour at RT, followed by two washes with PBS. 293F exosomes were used as a negative control for spike expression, and the isotype antibody was used as a negative control for CD81 expression. Samples were analyzed on a CytoFlex S (Beckman Coulter) flow cytometer, and data were analyzed by FLOWJO.

[0307] Example 10: Mouse studies To investigate the efficacy of STX-exosomes, age-matched BALB / c mice (female, 8-10 weeks old) were anesthetized using isoflurane and given bilateral intramuscular injections (50 μl per leg, totaling 100 μl) of either (1) PBS, (2) STX-S exosomes, (3) STX-N exosomes, or (4) STX-S+N exosomes. A booster injection was administered on day 21. Mice were closely monitored for changes in health status, and body weights were recorded biweekly. Blood collection was performed on days 14 and 35. Blood (approximately 50-500 μl) was collected from the submandibular vein and centrifuged at 4000 rpm for 5 minutes at 4°C before being processed for plasma isolation. For comparative studies, mice were injected with equal amounts of SARS-CoV-2 protein delivered either (1) as soluble protein with adjuvant (Alhydrogel, 100 μg / dose, vac-alu-250, InvivoGen, San Diego, California) or (2) as STX exosomes. Blood was collected two weeks after injection and tested for IgG against SARS-CoV-2. The timeline of the mouse study is outlined in Figure 7B. Mouse tissues (brain, salivary gland, feces, lung, liver, spleen, kidney, gastrointestinal tract (GI), and skeletal muscle (injection site)) were collected and fixed in 10% neutralized formalin. Sections were stained with hematoxylin and eosin and analyzed for alterations.

[0308] Example 11: Rabbit Study To evaluate the potential toxicity and host immune response of the STX-S+N vaccine of the present invention, age-matched rabbits (male / female, New Zealand White, 2.5-3.0 kg) were given intramuscular (IM) injections of the intended human dose of STX-S+N vaccine (10-200 ng total protein in 0.5 mL). Control animals received phosphate-buffered saline (PBS). A booster injection was administered on day 14. Rabbits were closely monitored for changes in health status, and body weights were recorded. Blood collections were performed weekly on days 0, 7, 14, 21, and 28. After centrifugation at 4000 rpm for 5 minutes at 4°C, the rabbits were processed for plasma isolation. The timeline of the rabbit study is outlined in Figure 7C. Rabbit tissues were collected at the end (day 28), fixed in 10% neutralized formalin, and processed for pathological changes.

[0309] Example 12: IgG ELISA Mouse and rabbit IgG antibodies against the SARS-CoV-2 spike or nucleocapsid were measured by enzyme-linked immunosorbent assay (ELISA) at room temperature (RT) using precoated ELISA plates (IEQ-CoV-S-RBD-IgG and IEQ-CoV-N-IgG, RayBiotech, Peachtree Corners, GA) according to the manufacturer's instructions. Briefly, mouse plasma samples were diluted with sample buffer (RayBiotech) and added in triplicate to antigen-coated wells and incubated for 2 hours at RT on a shaker (200 rpm). Commercially available antibodies against the spike (S1N-S58, Acro Biosystems, Newark, Delaware) or nucleocapsid (NUN-S47, Acro Biosystems) were used as positive controls. Plates were washed three times with wash buffer and incubated for 1 hour at room temperature with HRP-conjugated goat anti-mouse secondary antibody (115-035-003, dilution 1:5000, Jackson ImmunoResearch) or anti-rabbit (111-035-003, dilution 1:5000, Jackson ImmunoResearch, West Grove, PA) diluted in assay buffer (RayBiotech). After three washes, plates were developed using TMB substrate (RayBiotech). After a 15-minute incubation, the reaction was stopped by adding stop solution, and absorbance at 450 nm was recorded using a BIOTECK Gen5 plate reader (Agilent Technologies, Santa Clara, California). Endpoint titers were calculated as the dilution that emitted an optical density greater than four times that of the PBS control.

[0310] Example 13: Neutralizing antibodies against delta SARS-CoV-2 Vero E6 cells were used to evaluate the neutralizing activity of test articles against the replication-competent SARS-CoV-2 delta variant (B.1.617.2). Samples were preincubated with virus at 37°C for 1 hour before being added to the cells. After preincubation of the plasma / virus sample, cells were challenged with the mixture. Samples were allowed to remain in cell culture for the 96-hour infection period, at which point a Neutral Red uptake assay was performed to determine the extent of virus-induced cytopathic effect (CPE). Prevention of virus-induced CPE was used as a surrogate marker to determine the neutralizing activity of test articles against SARS-CoV-2. Test articles were evaluated in duplicate using two-fold serial dilutions (8 total dilutions), starting with a 1:40 dilution. Control wells contained "CoV02-delta" and GS-441524, which were tested as single data points on each plate. "CoV02-Delta" is convalescent plasma from an individual infected with the Delta variant after previously receiving two doses of the Moderna COVID-19 vaccine (Moderna, Cambridge, MA). GS-441524 is an antiviral drug manufactured by Gilead Sciences (Foster City, California). CoV-01 is plasma from a patient who received two doses of the Moderna COVID-19 vaccine and has no prior SARS-CoV-2 infection. NT50 values ​​of test articles were determined using GraphPad Prism software (GraphPad Software, Boston, MA).

[0311] Example 14: Omicron (BA.1 and BA.5.2.1) Neutralizing Antibodies Against SARS-CoV-2 Neutralization assays were performed using anti-NP immunostaining (Omicron BA.1 and BA.5.2.1). Briefly, samples were preincubated with virus at 37°C for 1 hour before being added to Vero E6 cells. After incubation, the medium was removed, and the cells were then challenged with the preincubated SARS-CoV-2 / test substance mixture. The amount of virus inoculum was previously titrated to produce a linear response that was inhibited by antiviral drugs known to be active against SARS-CoV-2. The cell culture medium containing the virus inoculum was not removed after virus adsorption; the test substance and virus were maintained in the medium for the duration of the assay (48 hours). The extent of infection was then monitored by incubating the cells with a monoclonal test substance against the SARS-CoV-2 nucleocapsid (NP). The amount of viral antigen in infected cells was estimated after incubation with a polyclonal test substance conjugated with horseradish peroxidase against human IgG (HRP-goat anti-mouse IgG). The reaction was monitored using a colorimetric readout (absorbance at 492 nm). Test articles were evaluated in duplicate using two-fold serial dilutions starting at a 1:40 dilution. Control wells contained GS-441524 (Gilead Sciences), which was tested at a single data point on each plate.

[0312] Example 15: Splenocyte isolation Spleens were processed for single-cell isolation by mechanically disrupting the spleen pouch using a syringe stopper and passing it through a 0.040 mm mesh nylon cell strainer to remove tissue debris. Red blood cells were lysed using ammonium chloride potassium (ACK) buffer (A1049201, ThermoFisher), and splenocytes were collected by centrifugation at 300 × g for 5 minutes. The cell pellet was resuspended in complete RPMI 1640 medium (FG1215, Millipore Sigma-Aldrich).

[0313] Example 16: ELISPOT Splenocytes were isolated by mechanical disruption of the spleen pouch, seeded at a concentration of 5E5 cells / well, and incubated for 24 hours in the presence or absence of 10 μg / ml SARS-CoV-2 spike (S1N-C52H4, AcroBiosystems, Newark, Delaware) or nucleocapsid (NUN-C5227, AcroBiosystems). Commercially available ELISPOT plates for the evaluation of IL-4 (MuIL4, Immunospot, Cellular Technology Limited, Shaker Heights, OH) and IFNg (MuIFNg, Immunospot, Cellular Technology Limited; catalog number 3110-4APW-10, rabbit, MabTech, Cincinnati, OH) were used. Assays were performed according to the manufacturer's guidelines. Plates were analyzed using the ELISPOT reader S6ENTRY (Immunospot, Cellular Technology Limited).

[0314] Example 17: ELISA for protein quantification Spike protein levels on exosomes were measured by ELISA at RT using precoated ELISA plates (ELV-COVID19S1, RayBiotech) according to the manufacturer's instructions. Nucleocapsid levels on exosomes were measured by ELISA at RT using precoated ELISA plates (Legend Max SARS-CoV2 Nucleocapsid Protein ELISA Kit, 448007, BioLegend, San Diego, California) according to the manufacturer's instructions. Briefly, samples and standards were loaded onto the precoated plates and incubated at RT for 2–2.5 h on a shaker (200 rpm). The plates were washed and incubated with a biotin-conjugated detection antibody at RT for 1 h, followed by incubation in streptavidin solution for 45 min. After washing, the plates were developed using TMB substrate. After 30 minutes of incubation, the reaction was stopped by adding stop solution, and the absorbance at 450 nm was recorded using a BIOTECK Gen5 plate reader (Agilent). For nucleocapsids, the absorbance at 450 nm and 570 nm was recorded after 10 minutes of incubation in TMB substrate. For analysis, the absorbance at 570 nm was subtracted from the absorbance at 450 nm, and the optical density (OD) can be used to construct a standard curve.

[0315] Example 18: Pathology Mouse tissues (brain, salivary glands, feces, lungs, liver, spleen, kidneys, gastrointestinal (GI) tract, and skeletal muscle (injection site)) were collected and fixed in 10% neutralized formalin. Sections were stained with hematoxylin and eosin and analyzed for alterations.

[0316] Example 19: Statistical Analysis Data were analyzed using Excel and GraphPad Prism 9.1 and presented as mean ± s.e.m. One-way ANOVA or two-tailed t-tests with post-hoc correction for multiple comparisons were applied, where appropriate.

[0317] Example 20: SARS-CoV-2 protein expression on the surface of STX-producing cells and exosomes STX cells were generated by lentiviral transduction, and the expression of SARS-CoV-2 proteins on the cell surface was assessed by flow cytometry (Figure 13). As shown, STX cells exhibited over 95% increased expression of spike (Figure 13A) and nucleocapsid (Figure 13B) compared to parental 293F cells.

[0318] STX exosomes were purified from genetically engineered 293F cell culture supernatants using the lab-scale purification technique described herein (Example 5). Purified STX-S and STX-N exosomes exhibited predicted mean diameters of 144.6 nm and 140.4 nm, respectively (Figures 4A, 6A, and 14), and predicted polydispersity indices (PDIs) of less than 0.2 (0.152 and 0.129, Figure 14).

[0319] STX-S exosomes were analyzed by TEM imaging. As shown in Figures 15A-B, typical exosome size and morphology were observed, with round, smooth nanoparticles with a visible lipid bilayer. Importantly, spikes were visible on the surface of STX-S nanoparticles, indicating the presence of spike proteins on the exosomes (Figures 5A, 5B, and 15A). Nucleocapsids exhibited a characteristic lipid bilayer that was thicker than that of naive exosomes, suggesting particle accumulation in the exosome membrane (Figure 15B).

[0320] Spike and nucleocapsid expression was verified in cell lysates and exosomes using Protein Simple's Jess automated Western blot, as described herein. Spike protein was detected in both STX-S cells and exosomes, with spike protein enriched in exosome samples (Figures 4B and 16). Engineered STX-N cells and exosomes expressed high levels of nucleocapsid (Figures 6B and 17). Furthermore, SARS-CoV-2 proteins, spike and Ncap, were also detected in exosome membranes using a bead-based CD81 assay, with over 75% expressing the exosome-specific marker CD81.

[0321] The concentrations of spike antigen in STX-S exosomes and nucleocapsid antigen in STX-N exosomes were further quantified by ELISA. Final STX-S preparations at 1 x 10^12 (1E12) exosomes / mL contained an average of 253.77 ng of spike, and final STX-N preparations at 1 x 10^12 (1E12) exosomes / mL contained an average of 40.59 ng of nucleocapsid.

[0322] Example 21: STX-S and STN-N individually induced potent immunization in the absence of adjuvant To verify the ability of STX-S and STX-N exosomes to induce immune responses against spike and nucleocapsid, mice were immunized with 10 ng of exosome formulations STX-S and STX-N. To demonstrate the robustness of exosome delivery, mice were immunized with 10 ng of either spike or Ncap recombinant protein delivered with an adjuvant (Alhydrogel, InvivoGen). PBS was used as a control. Blood collected 2 weeks after the boost injection (second injection) showed that both the STX-S and STX-N vaccines increased antibody production against spike and nucleocapsid, respectively, in all animals. Neither spike nor Ncap protein combined with adjuvant was statistically different from the PBS control (negative control), and no antibody production was observed (Figures 18 and 19, respectively).

[0323] Example 22: STX-S+N vaccine induces potent immunization against SARS-CoV2 proteins in mice The immune response of a multivalent vaccine obtained by combining STX-S and STX-N exosomes was assessed. STX-S+N was administered to mice by two im injections at three different doses (Table 2). A second im injection, a boost injection, was delivered after a 3-week interval. PBS was used as a negative control in this study.

[0324] Table 2. Spike and Ncap concentrations used in the study TIFF2025529904000021.tif31128

[0325] Immunization with the STX-S+N vaccine was evaluated by quantification of antibodies against Ncap and spiked Ncap (Figures 20A-B and 21A-B, respectively). Increased antibody production was detected after the first injection and continued to increase after the booster injection (second injection). A single injection of STX-S+N induced a maximum 30-fold increase in IgG against spike, with no significant differences overall among the three doses. After full immunization, dose 1 (25 ng / spike) and dose 2 (10 ng / spike) resulted in a 1500-fold increase in antibody against spike, while dose 3 (3 ng / spike, a significantly lower spike dose) resulted in a 280-fold increase. Meanwhile, a dose-response was observed for Ncap: a 1.5-fold increase was observed with low dose 1 (2.5 ng / Ncap), a nearly 4-fold increase with dose 2 (4 ng / Ncap), and a maximum 7-fold increase with dose 3 (9 ng / Ncap). After a full immunization cycle (two im injections), no significant differences were observed between doses, and a 24- to 43-fold increase in IgG against Ncap was observed in mice treated with STX-S+N.

[0326] To characterize T cell responses to STX-S+N, antigen-specific T cell responses were measured by ELISpot (Figure 22). Vaccination with STX-S+N induced polyfunctional antigen-specific T cell responses. Splenocytes were isolated from animals on day 35 (2 weeks after the boost (second) injection) and evaluated using IFNγ-precoated ELISpot plates. PBS was used as a control in the study. Baseline expression was compared to stimulation with 10 μg / ml of either spike or Ncap protein (AcroBiosystem). While baseline IFNγ responses were comparable between groups, evaluation of IFNγ-secreting cells in response to ex vivo stimulation with either spike (Figure 22A) or Ncap (Figure 22B) showed a strong increase in spleens immunized with the STX-S+N vaccine, suggesting a Th1-biased CD8+ T cell response. After spike stimulation, an average 7-fold increase in IFNγ responses was observed regardless of the dose of STX-S+N administered (Figure 22A). After Ncap stimulation, a dose-response effect was observed, with a 7-fold increase in mice receiving the lowest Ncap dose (dose 1, 2.5 ng) and an approximately 3-fold increase in mice receiving either dose 2 (4 ng Ncap) or 3 (9 ng Ncap) (Figure 22B).

[0327] Example 23: STX-S+N vaccine induces potent immunization against SARS-CoV2 proteins in rabbits The immune response to the STX-S+N vaccine was evaluated in rabbits using clinically relevant doses. The STX-S+N combination vaccine was administered to rabbits by two im injections at two different doses (Table 3). The second im injection, a boost injection, was delivered after a two-week interval. PBS was used as a control in this study.

[0328] Table 3. Spike and Ncap concentrations used in the study TIFF2025529904000022.tif28128

[0329] Increased antibody production was detected as early as one week after the first injection and continued to increase after booster injections (Figure 7C). After a full immunization cycle (two im injections), up to a 3600-fold increase in IgG against spike (Figure 23A) and up to a 170-fold increase in IgG against Ncap (Figure 23B) were observed in rabbits treated with STX-S+N. No significant differences were observed between doses in antibody production against either antigen.

[0330] Immunization with the STX-S+N vaccine was further evaluated by assessing neutralizing antibodies against SARS-CoV-2 variants (Figure 24A-C). Plasma from eight rabbits receiving dose 2 (50 ng S and 20 ng N) and two PBS controls was tested for neutralizing antibodies against the SARS-CoV-2 delta variant. Potent neutralizing activity was elicited by STX-S+N in all animals analyzed. Importantly, STX-S+N induced responses in rabbits comparable to human control plasma (COV-02-delta, plasma from a patient who had been fully immunized with Moderna's mRNA vaccine and developed a breakthrough delta infection), and induced complete neutralization responses at higher dilutions (i.e., 1:320, range: 116.74–881.47%; mean: 96.22 ± 8.9%) (Figure 24A). Furthermore, STX-S+N was superior to the CoV-01 control (plasma derived from patients who received two doses of the Moderna covid-19 vaccine and had no prior SARS-CoV-2 infection) in rabbits.

[0331] Additionally, the same samples were also tested for neutralizing antibodies against SARS-CoV-2 omicron variants (omicron BA.1 and BA.5.2.1). As shown in Figures 24B and 24C, strong cross-neutralization was observed in rabbits treated with STX-S+N, achieving an average neutralization of 75% (range 40.7-100%) for omicron BA.1 (Figure 24B) and 10%-91% for omicron BA5 (Figure 24C). These data suggest that exosomes, specifically protein-based vaccines delivered by STX-S+N, may confer broader protection against SARS-CoV-2 variants. In all assays, rabbits receiving PBS showed no neutralization.

[0332] T cell responses to immunization with STX-S+N were measured by ELISpot (Figures 25A and 25B). Vaccination with STX-S+N induced polyfunctional, antigen-specific T cell responses. Splenocytes were isolated from animals on day 28 (2 weeks after the boost (second) injection) and evaluated using IFNγ-precoated ELISpot plates. PBS was used as a control in the study. Baseline expression was compared with stimulation with 10 μg / ml of either spike or Ncap protein (AcroBiosystem). While baseline IFNγ responses were comparable between groups, stimulation with either spike (Figure 25A) or Ncap (Figure 25B) resulted in a strong increase in IFNγ production in spleens immunized with the STX-S+N vaccine, suggesting a Th1-biased CD8+ T cell response. For spike, a clear dose response was observed, with a greater response in rabbits receiving dose 1 (125 ng S, +28-fold) than dose 2 (50 ng S, +10-fold), although not statistically significant (Figure 25A). For Ncap, dose 1 (10 ng N, +10-fold) resulted in a greater immune response than dose 2 (20 ng N, +2-fold) (Figure 25B).

[0333] All patents and other publications cited throughout this application, including, for example, references, issued patents, published patent applications, and co-pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing the methodologies described in such publications, which may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0334] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes; however, as those skilled in the relevant art will recognize, various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or may perform the functions substantially simultaneously. The teachings of the present disclosure provided herein may be applied to other approaches or methods, as appropriate. The various embodiments described herein may be combined to provide additional embodiments. Aspects of the present disclosure may be modified, as appropriate, to utilize the compositions, functions, and concepts of the above-mentioned references and applications to provide additional embodiments of the present disclosure. Furthermore, considerations of biological functional equivalence allow some changes to be made to protein structure without affecting the type or amount of biological or chemical action. These and other changes may be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0335] Specific elements of any of the above aspects may be combined with or substituted for elements in other aspects. Additionally, although advantages associated with certain aspects of the present disclosure have been described with respect to those aspects, other aspects may also exhibit such advantages, and not all aspects need exhibit such advantages to fall within the scope of the present disclosure.

Claims

1. An immunogenic composition comprising an extracellular vesicle and a fusion protein, wherein the fusion protein comprises a viral polypeptide and an exosome polypeptide.

2. 10. The immunogenic composition of claim 1, which does not contain an adjuvant.

3. 3. The immunogenic composition of claim 1, wherein the fusion protein is present in the composition at a concentration of about 1 ng / 100 μL to about 300 ng / 100 μL.

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

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

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

7. The immunogenic composition of claim 5, wherein the fusion protein comprises (i) an amino acid sequence having at least 80% identity to SEQ ID NO:3 or (ii) the amino acid sequence of SEQ ID NO:

3.

8. The immunogenic composition of claim 6, wherein the fusion protein comprises (i) an amino acid sequence having at least 80% identity to SEQ ID NO:7 or (ii) the amino acid sequence of SEQ ID NO:

7.

9. 7. The immunogenic composition of claim 5 or claim 6, wherein an immunogenic dose of the composition comprises from about 1 ng to about 300 ng of the fusion protein or viral polypeptide.

10. 3. The immunogenic composition of claim 2, comprising a second vesicle and a second fusion protein, wherein the second fusion protein comprises a second viral polypeptide and an exosomal polypeptide, and the second fusion protein is present in the membrane of the second vesicle.

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

12. the fusion protein comprises (i) an amino acid sequence having at least 80% identity to SEQ ID NO:3, or (ii) the amino acid sequence of SEQ ID NO:3; and The second fusion protein comprises (III) an amino acid sequence having at least 80% identity to SEQ ID NO:7 or (iv) the amino acid sequence of SEQ ID NO:7; The immunogenic composition of claim 11.

13. 12. A method of immunizing a subject against a viral infection, comprising administering to the subject an immunogenically effective dose of the immunogenic composition of claim 2 or claim 11.

14. 14. The method of claim 13, wherein the immunogenic effective dose induces protective immunity against multiple variants of a given virus in the subject.

15. The immunogenic composition comprises: (a) a first fusion protein comprising a SARS-CoV-2 spike protein polypeptide or a second fusion protein comprising a SARS-CoV-2 nucleocapsid protein polypeptide; (b) Multiple exosomes and Including, the nucleocapsid protein polypeptide and / or the spike protein polypeptide are present on the outer surface of an exosome among the plurality of exosomes; 15. The method of claim 14.

16. the first fusion protein comprises (i) an amino acid sequence that is at least 80% identical to SEQ ID NO:3 or (ii) the amino acid sequence set forth in SEQ ID NO:3; and the second fusion protein comprises (i) an amino acid sequence that is at least 80% identical to SEQ ID NO:7, or (ii) the amino acid sequence set forth in SEQ ID NO:7; 16. The method of claim 15.

17. 16. The method of claim 15, wherein the immunogenic composition comprises a first fusion protein comprising a SARS-CoV-2 spike protein polypeptide located in the membrane of a first exosome and a second fusion protein comprising a SARS-CoV-2 nucleocapsid protein polypeptide located in the membrane of a second exosome.

18. the first fusion protein comprises (i) an amino acid sequence that is at least 80% identical to SEQ ID NO:3 or (ii) the amino acid sequence set forth in SEQ ID NO:3; and the second fusion protein comprises (i) an amino acid sequence that is at least 80% identical to SEQ ID NO:7, or (ii) the amino acid sequence set forth in SEQ ID NO:7; 18. The method of claim 17.

19. 18. The method of claim 15 or claim 17, wherein the immunogenic effective dose comprises from about 1 ng to about 300 ng of each of the fusion proteins.

20. 15. The method of claim 14, further comprising administering to the subject a second effective dose of the immunogenic composition.

21. A synthetic fusion protein comprising a viral polypeptide, an exosomal polypeptide, and a linker polypeptide.

22. 22. The synthetic fusion protein of claim 21, further comprising a transmembrane domain polypeptide positioned between said viral polypeptide and said exosomal polypeptide.

23. 23. The synthetic fusion protein of claim 21 or claim 22, wherein the exosomal polypeptide is a CD9 polypeptide.

24. 22. The synthetic fusion protein of claim 21, wherein the viral polypeptide is a SARS-CoV-2 spike protein polypeptide or a SARS-CoV-2 nucleocapsid protein polypeptide.

25. 25. The synthetic fusion protein of claim 24, comprising, in order from amino terminus to carboxy terminus, a SARS-CoV-2 spike protein polypeptide, a linker polypeptide, and a CD9 polypeptide.

26. (i) an amino acid sequence that is at least 80% identical to SEQ ID NO:3; (ii) the amino acid sequence of SEQ ID NO:3; (iii) an amino acid sequence that is at least 80% identical to SEQ ID NO:7; or (iv) the amino acid sequence of SEQ ID NO:7 26. The synthetic fusion protein of claim 25, comprising:

27. 27. A synthetic polynucleotide encoding the synthetic fusion protein of claim 26.

28. (i) a nucleic acid sequence that is at least 80% identical to SEQ ID NO:4; (ii) the nucleic acid sequence of SEQ ID NO:4; (iii) a nucleic acid sequence that is at least 80% identical to SEQ ID NO:8; or (iv) the nucleic acid sequence of SEQ ID NO:8 28. The synthetic polynucleotide of claim 27, comprising:

29. 29. A cell comprising the synthetic polynucleotide of claim 27 or claim 28.

30. 30. The cell of claim 29, which is a human cell.

31. 30. The cell of claim 29, which is a human embryonic kidney cell.

32. 30. The cell of claim 29, wherein the cell is produced by transducing the cell with a lentivirus comprising the synthetic polynucleotide.

33. 22. A vesicle comprising the synthetic fusion protein of claim 21.

34. 34. The vesicle of claim 33, which is an exosome.

35. The vesicle of claim 34, wherein the SARS-CoV-2 spike protein polypeptide is expressed on the outer surface of the exosome.

36. The vesicle of claim 34, wherein the SARS-CoV-2 nucleocapsid protein polypeptide is expressed on the outer surface of the exosome.

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