SARS-CoV-2 immunogenic compositions and methods

Abstract

The present disclosure relates to compositions and methods for vaccinating a subject against a plurality of SARS-CoV-2 variants that involve preparing and delivering extracellular vesicles that express an engineered spike protein and / or an engineered nucleocapsid protein on the surface to the subject. The present invention also relates to compositions and methods for designing, making, making, formulating and / or using spike display and nucleocapsid display vesicle vaccines designed to elicit potent humoral and cellular immune responses against a variety of SARS-CoV-2 variants.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of the priority dates of U.S. Provisional Application No. 63 / 373,418, filed on August 24, 2022, U.S. Provisional Application No. 63 / 411,090, filed on September 28, 2022, U.S. Provisional Application No. 63 / 413,193, filed on October 4, 2022, U.S. Provisional Application No. 63 / 437,710, filed on January 8, 2023, and U.S. Provisional Application No. 63 / 456,380, filed on March 31, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application includes a sequence listing, which has been electronically submitted in XML file format and is hereby 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.

[0005] Background

[0006] The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created an urgent need for vaccine development strategies that can effectively produce safe, effective, readily available, and accessible vaccines to counter the emergence of evolving SARS-CoV-2 variants. Many vaccines have been developed that attempt to achieve immunity against the virus, mainly by directing the immune response towards the surface spike protein, which binds to the host cell receptor angiotensin-converting enzyme 2 (ACE2) and mediates viral entry and infection of cells. The spike (S) protein is a class I fusion glycoprotein and is the major surface protein on the SARS-CoV-2 virus and the main target for neutralizing antibodies. The spike is also the major site of mutations identified in the SARS-CoV-2 virus, reducing the efficacy of the immune response induced by vaccines. Since the start of the COVID-19 pandemic, five variants of concern (VOCs) and eight variants of interest (VOIs) have been reported, posing challenges to current vaccines and creating a need for more effective vaccines. Vaccines against the SARS-CoV-2 nucleocapsid protein (an internal soluble coronavirus protein) have been largely unsuccessful to date.

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

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

[0009] The present invention discloses a solution to this problem, which solution comprises a safe (LNP-free, adjuvant-free) and effective vaccine that confers long-term humoral and cell-mediated immunity against multiple, emerging, and refractory variants of SARS-CoV-2. The vaccine comprises extracellular vesicles displaying the spike protein of a single variant of SARS-CoV-2, which vaccine confers robust humoral and / or cellular immunity against several SARS-CoV-2 variants of concern or interest, and the vaccine optionally comprises extracellular vesicles displaying the nucleocapsid protein of a single variant of SARS-CoV-2, which vaccine also confers robust humoral and / or cellular immunity against several SARS-CoV-2 variants of concern or interest.

[0010] Overview

[0011] In one embodiment, a synthetic fusion protein is provided that contains the polypeptide sequence of a SARS-CoV-2 protein fused to the polypeptide sequence of an exosomal tetraspanin protein. Such a fusion protein is designed and prepared such that the SARS-CoV-2 protein antigen can be expressed on the surface of the exosome so as to be able to elicit an immune response when administered to a subject.

[0012] In one aspect, the SARS-CoV-2 protein is the spike protein. In another aspect, the SARS-CoV-2 protein is the nucleocapsid protein. In one aspect, the exosomal tetraspanin protein is the CD9 protein.

[0013] In some aspects where the SARS-CoV-2 protein is the spike protein and the exosomal tetraspanin protein is the CD9 protein, the spike protein polypeptide is located at the N-terminus of the CD9 protein polypeptide. In some cases, a linker peptide is located between the spike protein polypeptide and the CD9 protein polypeptide, as Figure 1A depicted in.

[0014] In some aspects where the SARS-CoV-2 protein is the nucleocapsid protein and the exosomal tetraspanin protein is the CD9 protein, the nucleocapsid protein polypeptide is located at the N-terminus of the CD9 protein polypeptide. In some cases, a signal peptide is located at the N-terminus of the nucleocapsid protein polypeptide. In some cases, a hinge peptide, a transmembrane domain peptide, and a linker peptide are located between the spike protein polypeptide and the CD9 protein polypeptide, as Figure 2A depicted in.

[0015] In some aspects where the SARS-CoV-2 protein is the spike protein, the SARS-CoV-2 spike protein polypeptide contains one or more mutations, such as, for example, a furin cleavage site mutation (CSM [682RRAR685 - to - 682GSAG685]) and / or a diproline substitution (2P [986KV987 - to - 986PP987]).

[0016] In one embodiment, there is provided a polynucleotide encoding a synthetic fusion protein, the 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 is designed and prepared such that the SARS-CoV-2 protein antigen can be expressed in cells and sorted to the surface of exosomes so as to be able to elicit an immune response when the exosomes are administered to a subject.

[0017] 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.

[0018] 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 located at the N-terminus of the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some cases, a linker peptide is encoded by a polynucleotide located between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as Figure 1A depicted.

[0019] 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 located at the N-terminus of the encoded CD9 protein polypeptide. In some cases, a signal peptide is located at the N-terminus of the nucleocapsid protein polypeptide. In some cases, a hinge peptide, a transmembrane domain peptide, and a linker peptide are encoded by a polynucleotide located between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as Figure 2A depicted.

[0020] 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, such as, for example, a furin cleavage site mutation (CSM [682RRAR685 - to - 682GSAG685]) and / or a diproline substitution (2P [986KV987 - to - 986PP987]).

[0021] In one embodiment, a cell is provided that contains a polynucleotide encoding a synthetic fusion protein that contains a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosome tetraspanin protein. The encoded fusion protein is designed and prepared such that the SARS-CoV-2 protein antigen can be expressed in the cell and sorted to the surface of the exosome so that an immune response can be elicited when the exosome is administered to a subject.

[0022] 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.

[0023] 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 exosome tetraspanin protein polypeptide that is a CD9 protein polypeptide.

[0024] In some aspects where the polynucleotide in the cell encodes a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide and encodes an exosome tetraspanin protein polypeptide that is a CD9 protein polypeptide, the encoded spike protein polypeptide is located at the N-terminus of the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some cases, the polynucleotide encodes a linker peptide sequence between the spike protein polypeptide and the CD9 protein polypeptide in the encoded synthetic fusion protein, as Figure 1A depicted.

[0025] In some aspects where the polynucleotide in the cell encodes a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein polypeptide and encodes an exosome tetraspanin protein polypeptide that is a CD9 protein polypeptide, the encoded nucleocapsid protein polypeptide is located at the N-terminus of the encoded CD9 protein polypeptide. In some cases, the polynucleotide encodes a signal peptide at the N-terminus of the nucleocapsid protein polypeptide. In some cases, the polynucleotide encodes a hinge peptide, a transmembrane domain peptide, and a linker peptide between the spike protein polypeptide and the CD9 protein polypeptide in the encoded synthetic fusion protein, as Figure 2A depicted.

[0026] In some aspects, the cell contains a polynucleotide encoding a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide, and the encoded SARS-CoV-2 spike protein polypeptide contains one or more mutations, such as, for example, a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and / or a diproline substitution (2P [986KV987-to-986PP987]).

[0027] In one embodiment, a cell containing a synthetic fusion protein is provided, the synthetic fusion protein containing a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosome tetraspanin protein. The synthetic fusion protein is designed and prepared such that the SARS-CoV-2 protein antigen can be expressed in the cell and sorted to the surface of exosomes produced by the cell, so as to be able to elicit an immune response when the exosomes are administered to a subject.

[0028] 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.

[0029] 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 exosome tetraspanin protein polypeptide that is a CD9 protein polypeptide.

[0030] In some aspects where the cell contains a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide and an exosome tetraspanin protein that is a CD9 protein, the spike protein polypeptide is located at the N-terminus of the CD9 protein polypeptide of the contained synthetic fusion protein. In some cases, a linker peptide is located between the spike protein polypeptide and the CD9 protein polypeptide of the contained synthetic fusion protein, as Figure 1A depicted.

[0031] In some aspects where the cell contains a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein polypeptide and an exosome tetraspanin protein polypeptide that is a CD9 protein polypeptide, the nucleocapsid protein polypeptide is located at the N-terminus of the CD9 protein polypeptide. In some cases, a signal peptide is located at the N-terminus of the nucleocapsid protein polypeptide of the contained synthetic fusion protein. In some cases, a hinge peptide, a transmembrane domain peptide, and a linker peptide are located between the spike protein polypeptide and the CD9 protein polypeptide of the contained synthetic fusion protein, asFigure 2A as depicted in

[0032] 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, such as, for example, a furin cleavage site mutation (CSM [682RRAR685 - to - 682GSAG685]) and / or a di-proline substitution (2P [986KV987 - to - 986PP987]).

[0033] In one embodiment, provided are cells produced by transducing cells with a lentivirus containing a synthetic polynucleotide that encodes a synthetic fusion protein, the 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 prepared such that the SARS-CoV-2 protein antigen can be expressed in the cells and sorted to the surface of the exosomes so as to be able to elicit an immune response when the exosomes are administered to a subject.

[0034] 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.

[0035] 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.

[0036] 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 located at the N-terminus of the encoded CD9 protein polypeptide in the encoded synthetic fusion protein. In some cases, the linker peptide is encoded by a polynucleotide located between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as Figure 1A as depicted in

[0037] In some aspects, the encoded SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide and the encoded exosome tetraspanin protein polypeptide is a CD9 protein polypeptide, and the encoded nucleocapsid protein polypeptide is located at the N-terminus of the encoded CD9 protein polypeptide. In some cases, a signal peptide is located at the N-terminus of the nucleocapsid protein polypeptide. In some cases, a hinge peptide, a transmembrane domain peptide, and a linker peptide are encoded by a polynucleotide located between the spike protein polypeptide and the CD9 protein polypeptide of the encoded synthetic fusion protein, as Figure 2A depicted in

[0038] In some aspects, the encoded SARS-CoV-2 protein polypeptide is a spike protein polypeptide, and the encoded SARS-CoV-2 spike protein polypeptide contains one or more mutations, such as, for example, a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and / or a di-proline substitution (2P [986KV987-to-986PP987]).

[0039] In one embodiment, provided are vesicles containing a synthetic fusion protein comprising a polypeptide sequence of a SARS-CoV-2 protein fused to a polypeptide sequence of an exosome tetraspanin protein. Such fusion proteins are designed and prepared such that the SARS-CoV-2 protein antigen can be expressed on the surface of the exosome so as to be able to elicit an immune response when administered to a subject.

[0040] In one aspect, the vesicle contains a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide. In another aspect, the vesicle contains a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein polypeptide. In one aspect, the vesicle contains an exosome tetraspanin protein polypeptide that is a CD9 protein.

[0041] In some aspects where the vesicle contains a SARS-CoV-2 protein polypeptide that is a spike protein and an exosome tetraspanin protein polypeptide that is a CD9 protein, the spike protein polypeptide is located at the N-terminus of the CD9 protein polypeptide. In some cases, a linker peptide is located between the spike protein polypeptide and the CD9 protein polypeptide, as Figure 1A depicted in

[0042] In some aspects where the vesicle contains a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein and an exosome tetraspanin protein polypeptide that is a CD9 protein, the nucleocapsid protein polypeptide is located at the N-terminus of the CD9 protein polypeptide. In some cases, a signal peptide is located at the N-terminus of the nucleocapsid protein polypeptide. In some cases, a hinge peptide, a transmembrane domain peptide, and a linker peptide are located between the spike protein polypeptide and the CD9 protein polypeptide, as Figure 2A depicted in

[0043] In some aspects where the vesicles contain SARS-CoV-2 protein polypeptides that are spike proteins, the SARS-CoV-2 spike protein polypeptides contain one or more mutations, such as, for example, furin cleavage site mutations (CSM [682RRAR685 - to - 682GSAG685]) and / or diproline substitutions (2P [986KV987 - to - 986PP987]).

[0044] In one aspect, the vesicles have a 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 the CD81 protein on their surface.

[0045] In some aspects, the lumen of the vesicles contains cargo molecules. Here, in some cases, the protein or polypeptide displayed on the surface is a targeting ligand for targeting the delivery of the cargo molecules of the vesicles to recipient targets. In some aspects, the cargo molecules are nucleic acids, such as, for example, PMO antisense oligonucleotides, peptides, polypeptides or proteins, hydrophobic small molecule drugs, hydrophilic small molecule drugs, imaging agents, aptamers, trap molecules, nanobodies, antibodies or fragments thereof, receptor tyrosine kinases, and the like. For example, vesicles expressing spike can contain immune effector molecule cargo.

[0046] In one embodiment, a method for preparing vesicles is provided, the vesicles containing a synthetic fusion protein that 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 prepared such that the SARS-CoV-2 protein antigen can be expressed on the surface of the exosomes so as to be able to elicit an immune response when administered to a subject. Here, cells containing a polynucleotide encoding the synthetic fusion protein are cultured in a cell culture medium such that the vesicles are secreted by the cells into the culture medium, then the cell culture medium is collected, and subsequently the vesicles are purified.

[0047] In some aspects, the cells cultured to produce the 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.

[0048] In some aspects, the expression of the polynucleotide that produces the synthetic fusion protein is constitutive. In other aspects, the expression of the polynucleotide in the cell is induced to produce the synthetic fusion protein. Here, in one aspect, the induction is achieved by contacting the cell with tetracycline, doxycycline, or an analogue thereof. Here, in another aspect, the induction is achieved by removing tetracycline, doxycycline, or an analogue thereof from the cell or the cell culture medium.

[0049] In one aspect, the polynucleotide encodes and expresses in the cell a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide. In another aspect, the polynucleotide encodes and expresses in the cell a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein polypeptide. In one aspect, the polynucleotide encodes and expresses in the cell an exosome tetraspanin protein polypeptide that is a CD9 protein polypeptide.

[0050] In some aspects where the polynucleotide in the cell encodes a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide and encodes an exosome tetraspanin protein polypeptide that is a CD9 protein polypeptide, the spike protein polypeptide is located at the N-terminus of the CD9 protein polypeptide in the synthetic fusion protein that is encoded and expressed. In some cases, the polynucleotide encodes a linker peptide sequence between the spike protein polypeptide and the CD9 protein polypeptide in the synthetic fusion protein that is encoded and expressed, such as Figure 1A depicted in.

[0051] In some aspects where the polynucleotide in the cell encodes a SARS-CoV-2 protein polypeptide that is a nucleocapsid protein polypeptide and encodes an exosome tetraspanin protein polypeptide that is a CD9 protein polypeptide, the encoded nucleocapsid protein polypeptide is located at the N-terminus of the encoded CD9 protein polypeptide in the fusion protein that is encoded and expressed. In some cases, the polynucleotide encodes a signal peptide at the N-terminus of the nucleocapsid protein polypeptide. In some cases, the polynucleotide encodes a hinge peptide, a transmembrane domain peptide, and a linker peptide between the spike protein polypeptide and the CD9 protein polypeptide in the synthetic fusion protein that is encoded and expressed, such as Figure 2A depicted in.

[0052] In some aspects where the cell contains a polynucleotide that encodes and expresses a SARS-CoV-2 protein polypeptide that is a spike protein polypeptide as part of the expressed fusion protein, the encoded and expressed SARS-CoV-2 spike protein polypeptide contains one or more mutations, such as, for example, a furin protease cleavage site mutation (CSM [682RRAR685 - to - 682GSAG685]) and / or a di-proline substitution (2P [986KV987 - to - 986PP987]).

[0053] In one embodiment, there is provided an immunogenic composition comprising more than one vesicle containing a synthetic fusion protein. Here, 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 prepared such that the SARS-CoV-2 protein antigen can be expressed on the surface of the exosome so as to be able to elicit an immune response when administered to a subject.

[0054] In some aspects, the immunogenic composition comprises (i) more than one vesicle containing a first synthetic fusion protein and (ii) more than one vesicle containing a second synthetic fusion protein, wherein the first synthetic fusion protein contains a polypeptide sequence of a first SARS-CoV-2 protein fused to a polypeptide sequence of an exosomal tetraspanin protein, and the second synthetic fusion protein contains a polypeptide sequence of a second SARS-CoV-2 protein fused to a 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.

[0055] In one aspect, the immunogenic composition comprises more than one vesicle containing a synthetic fusion protein, wherein the SARS-CoV-2 protein polypeptide is a spike protein polypeptide. In another aspect, the immunogenic composition comprises more than one vesicle containing a synthetic fusion protein, wherein the SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide. In another aspect, the immunogenic composition comprises more than one vesicle containing a combination of vesicles, wherein some vesicles contain a synthetic fusion protein in which the SARS-CoV-2 protein polypeptide is a spike protein polypeptide, and other vesicles contain a synthetic fusion protein in which the SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide. In one aspect, the immunogenic composition comprises more than one vesicle containing a synthetic fusion protein, wherein the exosomal tetraspanin protein polypeptide is a CD9 protein polypeptide.

[0056] In some aspects where the immunogenic composition comprises more than one vesicle containing a synthetic fusion protein, wherein 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 located at the N-terminus of the CD9 protein polypeptide. In some cases, a linker peptide is located between the spike protein polypeptide and the CD9 protein polypeptide, as Figure 1A depicted.

[0057] In some aspects, the immunogenic composition comprises more than one vesicle containing a synthetic fusion protein, wherein the SARS-CoV-2 protein polypeptide is a nucleocapsid protein polypeptide and the exosomal tetraspanin protein polypeptide is a CD9 protein, and the nucleocapsid protein polypeptide is located at the N-terminus of the CD9 protein polypeptide. In some cases, a signal peptide is located at the N-terminus of the nucleocapsid protein polypeptide. In some cases, a hinge peptide, a transmembrane domain peptide, and a linker peptide are located between the spike protein polypeptide and the CD9 protein polypeptide, as Figure 2A depicted in

[0058] In some aspects, the immunogenic composition comprises more than one vesicle containing a synthetic fusion protein wherein the SARS-CoV-2 protein polypeptide is a spike protein polypeptide, and the SARS-CoV-2 spike protein polypeptide contains one or more mutations, such as, for example, a furin cleavage site mutation (CSM [682RRAR685-to-682GSAG685]) and / or a di-proline substitution (2P [986KV987-to-986PP987]).

[0059] In some aspects, the immunogenic composition contains more than one vesicle containing a synthetic fusion protein at a concentration of about 2E9 vesicles / mL - 3E13 vesicles / mL. In those aspects where the immunogenic composition contains two types of vesicles (i.e., one type contains a first synthetic fusion protein and the other type contains a second synthetic fusion protein), each vesicle type can be provided at a concentration of about 1E9 vesicles / mL - 2E13 vesicles / mL or 2E9 vesicles / mL - 3E13 vesicles / mL.

[0060] In some aspects, the immunogenic composition contains more than one vesicle containing a synthetic fusion protein at a concentration of about 0.3 ng / mL - 3 μg / mL of the synthetic fusion protein. In those aspects where the immunogenic composition contains two types of vesicles (i.e., one type contains a first synthetic fusion protein and the other type contains a second synthetic fusion protein), each vesicle type can be provided at a fusion protein concentration of about 0.1 ng / mL - 1.5 μg / mL or about 0.3 ng / mL - 3 μg / mL.

[0061] In some aspects, the immunogenic composition further comprises one or more pharmaceutically acceptable excipients. In one aspect, the immunogenic composition does not contain an adjuvant.

[0062] In some aspects, the vesicles in the more than one vesicle 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.

[0063] In one embodiment, a method of eliciting an immune response in a subject is provided, the method being effected by administering to the subject a dose of the immunogenic composition described in the foregoing embodiments. In some aspects, more than one dose is administered to the subject, such as a second dose administered at a certain time period after the first dose and / or subsequent booster doses. In some embodiments, the time period (e.g., between doses) is 14 days to 1 year.

[0064] In one aspect, the dose contains about 100 μL - 1 mL of the immunogenic composition. In some aspects, the immunogenic composition contains about 0.1 ng / mL - 3 μg / mL of the synthetic fusion protein in a form expressed on the surface of the vesicles. In some aspects, the immunogenic composition contains about 2.81E9 vesicles / mL - 2.81E13 vesicles / mL expressing the synthetic fusion protein on their surface.

[0065] In some aspects, the immune response elicited in the subject is the production of neutralizing antibodies against a virus such as SARS-CoV-2, influenza virus, etc. In some aspects, the immune response elicited in the subject is the production of neutralizing antibodies against two or more SARS-CoV-2 variants (such as, for example, the Delta variant, the Omicron variant, or other variants of interest or concern that are now known or not yet discovered). In some aspects, the immune response elicited in the subject is the production of anti-spike antibodies. In some aspects, the immune response elicited in the subject is the production of anti-nucleocapsid antibodies. In some aspects, the immune response elicited in the subject is a spike-specific T cell response, such as a CD4+ response and / or a CD8+ response. In some aspects, the immune response elicited in the subject is a nucleocapsid-specific T cell response, such as a CD4+ response and / or a CD8+ response.

[0066] Drawings

[0067] Figure 1A Depicts a linear cartoon of the SARS-CoV-2 spike CD9 fusion protein oriented from amino terminus to carboxy terminus from left to right, where the linker sequence is located between the spike protein polypeptide and the CD9 protein polypeptide.

[0068] Figure 1B Depicts Figure 1A a cartoon of the SARS-CoV-2 spike CD9 fusion protein oriented from amino terminus to carboxy terminus from left to right with respect to the vesicle membrane. The spike protein polypeptide spans the membrane once on the outer side of the vesicle with its amino terminus oriented. The linker is on the luminal side of the membrane. The CD9 protein polypeptide spans the membrane 4 times in the lumen of the vesicle with its carboxy terminus oriented.

[0069] Figure 2ADepicts a linear cartoon of the SARS-CoV-2 nucleocapsid CD9 fusion protein oriented from amino-terminus to carboxy-terminus from left to right, where the amino-terminal signal peptide is fused to the nucleocapsid protein polypeptide, the nucleocapsid protein polypeptide is fused to the hinge region peptide, the hinge region peptide is fused to the transmembrane domain peptide, the transmembrane domain peptide is fused to the linker peptide, and the linker peptide is fused to the CD9 protein polypeptide.

[0070] Figure 2B Depicts Figure 2A A cartoon of the SARS-CoV-2 nucleocapsid CD9 fusion protein oriented from amino-terminus to carboxy-terminus from left to right relative to the vesicle membrane. The nucleocapsid protein polypeptide with an amino-terminal signal sequence is located on the outside (cytoplasmic or outside), the hinge sequence connects the nucleocapsid protein polypeptide to the transmembrane domain peptide, the transmembrane domain peptide spans the membrane and is then connected to a linker located in the lumen, and the linker is connected to the CD9 protein polypeptide, which spans the membrane 4 times and has its carboxy-terminus oriented in the lumen of the vesicle.

[0071] Figure 3A Is a flow chart depicting the components and steps for generating cells expressing a spike protein polypeptide fusion protein using a packaging cell (301) and a host cell (311) mediated by a lentiviral vector (304).

[0072] Figure 3B Is a histogram depicting the flow analysis of the relative fluorescence intensity of a host cell expressing the spike protein on its surface.

[0073] Figure 4A Is a graph depicting the variation of the concentration of spike-expressing exosomes per milliliter with the exosome diameter in nanometers.

[0074] Figure 4B Is a western blot stained for the SARS-CoV-2 spike protein. The first column from left to right depicts a lane loaded with size markers, the second column represents a lane loaded with protein from untransduced 293F (host cell), the third column represents a lane loaded with protein from exosomes derived from untransduced 293F cells, the fourth column represents a lane loaded with protein from 293F cells constitutively expressing the spike fusion protein, the fifth column represents a lane loaded with protein from exosomes derived from 293F cells constitutively expressing the spike fusion protein, and the sixth column represents a lane loaded with the spike fusion protein.

[0075] Figure 4C Is a histogram depicting the flow analysis of the relative fluorescence intensity of exosomes with the spike expressed on their surface. The left curve represents exosomes derived from 293F cells not expressing the spike-CD9 fusion protein. The right curve represents exosomes derived from 293F cells expressing the spike-CD9 fusion protein.

[0076] Figure 5A Depicts a transmission electron micrograph of exosomes expressing the SARS-CoV-2 spike protein. The inset is an enlarged view showing exosomes decorated with the spike protein. Arrows point to the SARS-CoV-2 spikes around the exosomes.

[0077] Figure 5B Depicts a transmission electron micrograph at a higher magnification of exosomes expressing the SARS-CoV-2 spike protein. Arrows point to the SARS-CoV-2 spikes around the exosomes.

[0078] Figure 6A Is a graph depicting the concentration (number of exosomes per milliliter) of exosomes expressing the nucleocapsid varying with the diameter of the exosomes in nanometers.

[0079] Figure 6B Is a Western blot stained for the SARS-CoV-2 nucleocapsid protein. The first column from left to right depicts the lane loaded with size markers, the second column represents the lane loaded with the nucleocapsid protein, the third column represents the lane loaded with the protein from exosomes derived from untransduced 293F cells, the fourth column represents the lane loaded with the protein from untransduced 293F cells, and the fifth column represents the lane loaded with the protein from exosomes derived from 293F cells expressing the nucleocapsid fusion protein.

[0080] Figure 6C Is a histogram depicting flow analysis of the relative fluorescence intensity of exosomes with or without nucleocapsid protein expressed on the surface. From left to right, the first curve represents exosomes derived from unmodified 293F cells, the second curve represents exosomes derived from uninduced 293F cells carrying the polynucleotide encoding the nucleocapsid-CD9 fusion protein, the third curve represents exosomes derived from 293F cells expressing the nucleocapsid-CD9 fusion protein, the smaller third curve represents exosomes derived from 293F cells expressing the nucleocapsid-CD9 fusion protein under tet induction, and the larger third curve represents exosomes derived from 293F cells carrying CD9 knockout and expressing the nucleocapsid-CD9 fusion protein.

[0081] Figure 7 is a timeline depicting the process for generating an immune response by administering an immunogenic composition containing exosomes expressing the SARS-CoV-2 fusion protein on their surface.

[0082] Figure 8A - Figure 8D . STX-S exosomes elicited a robust immune response with significantly lower antigen than the recombinant protein vaccine. Data are shown as mean ± SEM. **** p < 0.0005, *** p < 0.001, ** p < 0.01,* p < 0.05, ns = not significant, * One-way ANOVA; ## p < 0.01, # p < 0.05, # - Two-tailed t-test.

[0083] Figure 8A Is a histogram depicting the fold change in serum antibody titers against the spike protein at a 1:100 dilution on day 14 in mice as a function of the dose of spike-expressing exosomes (in nanograms). The leftmost bar represents the PBS control; the second bar represents the 10 ng dose of spike-expressing exosomes (STX-S); the third bar represents the 32 ng dose of STX-S; the rightmost bar represents the serum from mice injected with a 32 ng dose of spike protein in combination with adjuvant.

[0084] Figure 8B Is a histogram depicting the fold change in serum antibody titers against the spike protein at a 1:100 dilution on day 35 in mice as a function of the dose of spike-expressing exosomes (in nanograms). The leftmost bar represents the PBS control; the second bar represents the 10 ng dose of spike-expressing exosomes (STX-S); the third bar represents the 32 ng dose of STX-S; the rightmost bar represents the serum from mice injected with a 32 ng dose of spike protein in combination with adjuvant.

[0085] Figure 8C Is a bar graph of an ELISpot assay depicting the number of spots of IL4 production in response to the spike protein as a function of the antigen dose. On the x-axis from left to right, the first bar represents the spike-free wells from PBS control splenocytes; the second bar represents the spike protein reactivity from PBS control splenocytes; the third bar represents the spike-free wells from splenocytes of subjects administered 10 ng STX-S; the fourth bar represents the spike protein reactivity from splenocytes of subjects administered 10 ng STX-S; the fifth bar represents the spike-free wells from splenocytes of subjects administered 32 ng STX-S; the sixth bar represents the spike protein reactivity from splenocytes of subjects administered 32 ng STX-S; the seventh bar represents the spike-free wells from splenocytes of subjects administered 32 ng of spike protein with adjuvant; the eighth bar represents the spike protein reactivity from splenocytes of subjects administered 32 ng of spike protein with adjuvant.

[0086] Figure 8DIt is a bar graph depicting an ELISpot assay showing the number of IFNγ-producing spots in response to spike protein varying with antigen dose. From left to right on the x-axis, the first bar represents the spike-free wells from PBS control splenocytes; the second bar represents the spike protein reactivity from PBS control splenocytes; the third bar represents the spike-free wells from splenocytes of subjects administered 10 ng STX-S; the fourth bar represents the spike protein reactivity from splenocytes of subjects administered 10 ng STX-S; the fifth bar represents the spike-free wells from splenocytes of subjects administered 32 ng STX-S; the sixth bar represents the spike protein reactivity from splenocytes of subjects administered 32 ng STX-S; the seventh bar represents the spike-free wells from splenocytes of subjects administered 32 ng spike protein with adjuvant; the eighth bar represents the spike protein reactivity from splenocytes of subjects administered 32 ng spike protein with adjuvant.

[0087] Figure 9, Panels A - D. STX-N vaccine elicits a multivalent immune response. Data are shown as mean ± SEM. * p < 0.05, *** p < 0.005, **** p < 0.001, ANOVA, corrected multiple comparisons; ns = not significant. Dose 1 = 0.32 ng N per injection; Dose 2 = 3.2 ng N per injection; Dose 3 = 10 ng N per injection.

[0088] Figure 9A and Figure 9B : A histogram depicting the anti-nucleocapsid antibody titers in serum on day 35 post-immunization varying with dose. The STX-N vaccine induces moderate expression of SARS-CoV-2 nucleocapsid antibodies in two sample bins (N1 and N2). PBS was used as a vehicle control.

[0089] Figure 9C and Figure 9D : A histogram depicting the anti-nucleocapsid IFNγ ELISpot positive wells in splenocytes on day 40 post-immunization varying with dose.

[0090] Figure 10, Panels A - C: A line graph depicting the percentage of SARS-CoV-2 neutralization varying with immune serum as the serum dilution increases. These graphs depict the production of neutralizing antibodies after STX-S injection. Data are shown as mean ± SEM. COV-02-Delta = plasma from patients with breakthrough SARS-CoV-2 Delta spike infection immunized with Moderna's mRNA vaccine. Dose 4 = 9.8 ng S per injection; Dose 2 = 3.2 ng S per injection.

[0091] Figure 10A. The STX-S vaccine produces strong neutralization against the SARS-CoV-2 Delta spike (B.1.617.2).

[0092] Figure 10B . The STX-S vaccine results in the neutralization of the SARS-CoV-2 spike Omicron BA.1.

[0093] Figure 10C . The STX-S vaccine results in the neutralization of the SARS-CoV-2 spike Omicron BA.5.2.1.

[0094] Figure 11 It is a histogram depicting the fold change in anti-spike antibody titers from mice over time and dose. Data are shown as mean ± SEM. * p < 0.05, *** p < 0.005, **** p < 0.001, ANOVA, corrected for multiple comparisons; ns = not significant. From left to right, the first histogram represents serum from mice administered PBS, the second histogram represents serum from mice administered a single dose of 10 ng STX-S (exosomes expressing the CD9-spike fusion) on day 14, the third histogram represents serum from mice administered a first dose of 10 ng STX-S followed by a 7 ng booster on day 21 on day 35, the fourth histogram represents serum from mice administered a first dose of 10 ng STX-S followed by a 7 ng booster on day 21 on day 61, the fifth histogram represents serum from mice administered a first dose of 10 ng STX-S followed by a 7 ng booster on day 21 on day 89, the sixth histogram represents serum from mice administered a first dose of 10 ng STX-S followed by a 7 ng booster on day 21 on day 123, the seventh histogram represents serum from mice administered a first dose of 10 ng STX-S followed by a 7 ng booster on day 21 on day 151, the eighth histogram represents serum from mice administered a first dose of 10 ng STX-S followed by a 7 ng booster on day 21 on day 278, the ninth histogram represents serum from mice administered a first dose of 10 ng STX-S followed by a 7 ng booster on day 21 on day 166, the tenth histogram represents serum from mice administered a first dose of 10 ng STX-S followed by a 7 ng booster on day 21 on day 166 plus 4-month follow-up serum, the eleventh histogram represents serum from mice administered a first dose of 10 ng STX-S followed by a 3 ng booster on day 21 on day 166, and the twelfth histogram represents serum from mice administered a first dose of 10 ng STX-S followed by a 3 ng booster on day 21 on day 166 plus 4-month follow-up serum.

[0095] Figure 12 It is a line graph depicting the percentage of HEK293-hACE2 cell uptake of exosomes varying with exosome concentration expressed as the number of exosomes per mL. Series 1 (blue) represents the HEK293-hACE2 cell uptake of 293F exosomes that do not express spike protein. Series 2 (orange) represents the HEK293-hACE2 cell uptake of exosomes expressing spike.

[0096] Figure 13 It is a histogram depicting the relative fluorescence intensity flow analysis of exosomes with spike expressed on the surface ( Figure 13 A) or exosomes with nucleocapsid expressed on the surface ( Figure 13 B). For each graph, the left curve represents exosomes derived from 293F cells that do not express spike (A) or nucleocapsid (B). For each graph, the right curve represents exosomes derived from 293F cells expressing spike (A) or nucleocapsid (B).

[0097] Figure 14 It is a line graph depicting the size distribution of STX-S and STX-N exosomes varying with exosome concentration by ZetaView nanoparticle tracking analysis (NTA). The blue line represents exosomes expressing spike. The green line represents exosomes expressing nucleocapsid. The grey line represents exosomes from non-engineered 293F.

[0098] Figure 15 Depicts TEM images of purified STX-S (Figure A; scale bar = 200 nm) and STX-N (Figure B; scale bar = 500 nm) exosomes.

[0099] Figure 16 It is a JESS western blot stained for SARS-CoV-2 spike protein. The first column from left to right depicts the lane loaded with size markers, the second column represents the lane loaded with protein from untransduced 293F cells, the third column represents the lane loaded with protein from exosomes derived from untransduced 293F cells, the fourth column represents 293F cells expressing spike fusion protein, and the fifth column represents the lane loaded with protein from exosomes derived from 293F cells expressing nucleocapsid fusion protein. 0.8 μg of protein was loaded per lane as calculated by BCA assay.

[0100] Figure 17A JESS Western blot stained for SARS-CoV-2 nucleocapsid protein. The first column from left to right depicts the lane loaded with size markers, the second column represents the lane loaded with proteins from untransduced 293F cells, the third column represents the lane loaded with proteins from exosomes derived from untransduced 293F cells, the fourth column represents 293F cells expressing the spike fusion protein, and the fifth column represents the lane loaded with proteins from exosomes derived from 293F cells expressing the nucleocapsid fusion protein. 0.8 μg of protein was loaded per lane as calculated by BCA assay.

[0101] Figure 18 A is a histogram depicting the fold change in serum antibody titers against the spike protein in mice on day 14 after 1 i.m. injection as a function of spike dose formulation. The leftmost bar represents the PBS control; the second bar represents the dose of exosomes expressing the spike (STX S); the third bar represents the equivalent dose of spike (S) combined with adjuvant. N = 10 / group. Data are shown as mean ± SEM. **** p < 0.0005, *** p < 0.001, ** p < 0.01, ns = not significant, one-way ANOVA.

[0102] Figure 18 B is a histogram depicting the fold change in serum antibody titers against the spike protein in mice on day 35 after 2 i.m. injections at a 1:100 dilution as a function of spike dose formulation. The leftmost bar represents the PBS control; the second bar represents the dose of exosomes expressing the spike (STX S); the third bar represents the equivalent dose of spike (S) combined with adjuvant. N = 10 / group. Data are shown as mean ± SEM. **** p < 0.0005, *** p < 0.001, ** p < 0.01, ns = not significant, one-way ANOVA.

[0103] Figure 19 A is a histogram depicting the fold change in serum antibody titers against the nucleocapsid protein in mice on day 14 after 1 i.m. injection as a function of nucleocapsid dose formulation. The leftmost bar represents the PBS control; the second bar represents the dose of exosomes expressing the nucleocapsid (STX S); the third bar represents the equivalent dose of nucleocapsid (N) combined with adjuvant. N = 10 / group. Data are shown as mean ± SEM. **** p < 0.0005, *** p < 0.001, ** p < 0.01, ns = not significant, one-way ANOVA.

[0104] Figure 19Panel B is a histogram showing the fold change in serum antibody titers against the spike protein in mice on day 35 after two i.m. injections, varying with the spike dose formulation. The leftmost bar represents the PBS control; the second bar represents the dose of spike-expressing exosomes (STX S); the third bar represents the equivalent dose of spike (S) in combination with an adjuvant. N = 10 per experimental group. Data are shown as mean ± SEM. **** p < 0.0005, *** p < 0.001, ** p < 0.01, ns = not significant, one-way ANOVA.

[0105] Figure 20 Panel is a histogram showing that the STX-S+N combination vaccine elicits strong antibodies in mice. Panel A shows IgG against the spike on day 14. Panel B shows IgG against the spike on day 35. For both Panels A and B, the first column depicts the fold change in IgG due to PBS. The second column depicts the fold change in IgG due to S+N at dose 1 (25 ng S, 2.5 ng N). The third column depicts the fold change in IgG due to S+N at dose 2 (10 ng S, 4 ng N). The fourth column depicts the fold change in IgG due to S+N at dose 3 (3 ng S, 9 ng N). Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, ns = not significant, one-way ANOVA, adjusted for multiple comparisons. N = 10 animals per experimental group.

[0106] Figure 21 Panel is a histogram showing that the STX-S+N combination vaccine elicits strong antibodies in mice. Panel A shows IgG against the nucleocapsid on day 14. Panel B shows IgG against the nucleocapsid on day 35. For both Panels A and B, the first column depicts the fold change in IgG due to PBS. The second column depicts the fold change in IgG due to S+N at dose 1 (25 ng S, 2.5 ng N). The third column depicts the fold change in IgG due to S+N at dose 2 (10 ng S, 4 ng N). The fourth column depicts the fold change in IgG due to S+N at dose 3 (3 ng S, 9 ng N). Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, ns = not significant, one-way ANOVA, adjusted for multiple comparisons. N = 10 animals per experimental group.

[0107] Figure 22 It is a histogram depicting the anti-spike IFNγ ELISpot positive wells (Figure A) and anti-nucleocapsid IFNγ ELISpot positive wells (Figure B) of splenocytes on the 40th day after immunization, varying with 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 shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, ns = not significant, one-way ANOVA, adjusted for multiple comparisons. N = 10 animals per experimental group.

[0108] Figure 23 It is a histogram depicting the change in anti-spike antibody titer (Figure A) or the change in anti-nucleocapsid antibody titer (Figure B) varying with the dose of the combined 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 readout and formulation, the first bar from left to right represents serum from blood collected on the 7th day after the first administration; the second bar represents serum from blood collected on the 14th day after the first administration; the third bar represents serum from blood collected on the 7th day after the second administration, 21 days after the first administration; and the fourth bar represents serum from blood collected on the 14th day after the second administration, 28 days after the first administration. Data are shown as mean ± SEM. N = 8 animals per experimental group.

[0109] Figure 24 , Figures A - C: Line graphs depicting the percentage of SARS-CoV-2 neutralization varying with immune sera as the serum dilution increases. These figures depict the production of neutralizing antibodies after injection of STX-S+N (combined vaccine). Data are shown as mean ± SEM. Data are shown as mean ± SEM. COV-02-Delta = plasma from patients with breakthrough SARS-CoV-2 Delta spike infection immunized with Moderna's mRNA vaccine; CoV-01 = plasma from patients who received two doses of Moderna's covid-19 vaccine and had no history of SARS-CoV-2 infection. N = 8 animals per experimental group.

[0110] Figure 24A. The STX-S vaccine shows strong neutralizing activity against the SARS-CoV-2 Delta spike (B.1.617.2).

[0111] Figure 24 B. The STX-S vaccine induces neutralization of the SARS-CoV-2 Omicron BA.1 spike.

[0112] Figure 24 C. The STX-S vaccine induces neutralization of the SARS-CoV-2 Omicron BA.5.2.1 spike.

[0113] Figure 25 It is a histogram depicting the anti-spike IFNγ ELISpot positive wells (Figure A) and anti-nucleocapsid IFNγ ELISpot positive wells (Figure B) of rabbit splenocytes on day 28 after immunization, varying with the dose of the combined vaccine including exosomes expressing the spike (STX-S) plus exosomes expressing the nucleocapsid (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 shown as mean ± SEM. N = 8 animals per experimental group. Detailed implementation mode

[0114] Definition

[0115] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein, which will become apparent to those skilled in the art after reading this disclosure and the like.

[0116] As used herein, "and / or" means and encompasses any and all possible combinations of one or more of the related listed items, as well as the absence of a combination when interpreted in the alternative (or).

[0117] As used herein, the terms "about" or "approximate", when referring to a measurable value such as an amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, quantity, weight, position, length, etc., mean to encompass variations of ±15%, ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the specified amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, quantity, weight, position, length, etc. In cases where the terms "about" and "approximate" are used in combination with the location or position of a region within a reference polypeptide, these terms encompass variations of ± up to 20 amino acid residues, ± up to 15 amino acid residues, ± up to 10 amino acid residues, ± up to 5 amino acid residues, ± up to 4 amino acid residues, ± up to 3 amino acid residues, ± up to 2 amino acid residues or even ±1 amino acid residue.

[0118] In cases such as "A is derived from B", the term "derived from" means that A is obtained from B in such a way that A is not identical to B.

[0119] The terms "treat", "therapeutic", "prophylactic" and "prevent" are not intended to be absolute terms. Treatment, prevention and prophylaxis can refer to any delay in the onset of disease, improvement in symptoms, improvement in patient survival, increase in survival time or survival rate, etc. Treatment, prevention and prophylaxis can be complete or partial. The term "prophylactic" not only means "prevent", but also minimizes disorders and diseases. For example, a "prophylactic" agent can be administered to a subject (e.g., a human subject) to prevent infection or minimize the extent of disorders and diseases caused by such infection. The effectiveness of treatment can be compared to an individual or group of individuals who have not received treatment, or to the same patient at different times before or during treatment. In some aspects, the severity of the disease is reduced by at least 10% compared to, for example, an individual before administration or a control individual who has not undergone treatment. In some aspects, the severity of the disease is reduced by at least 25%, 50%, 75%, 80% or 90%, or in some cases, is no longer detectable using standard diagnostic techniques.

[0120] As used herein, if, after treatment according to the methods described herein, one or more signs or symptoms of the conditions described herein change in a beneficial manner, other clinically acceptable symptoms improve or even ameliorate, or a desired response is induced, such as at least 2%, 3%, 4%, 5%, 10% or more, then the treatment can be considered "effective". Efficacy, for example, can be evaluated by measuring markers, indicators, symptoms, and / or incidence of the condition being treated according to the methods described herein or any other appropriate measurable parameter. Efficacy can also be measured by the individual not deteriorating, as evaluated by hospitalization, or requiring medical intervention (e.g., the progression of the disease stops). Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or animals) and includes: (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of the disease, e.g., causing the symptoms to resolve. An effective amount for treating a disease means an amount that, when administered to a subject in need thereof, is sufficient to result in an effective treatment of the disease as defined by this term herein. The efficacy of an agent can be determined by evaluating physical indicators of the condition or desired response. One of ordinary skill in the art can monitor the efficacy of administration and / or treatment by measuring any one or combination of such parameters.

[0121] As used herein, the term "effective amount" refers to the amount of a composition or agent required 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. In various instances, an effective amount as used herein can include an amount sufficient to delay the development of disease symptoms, alter the course of disease symptoms (e.g., but not limited to slowing the progression of disease symptoms), or reverse disease symptoms. For example, for a particular parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90% or at least 100% of any one of the therapeutic effects. Therapeutic efficacy can also be expressed as an increase or decrease of "-fold". For example, a therapeutically effective amount can have an effect of at least 1.2-fold, 1.5-fold, 2-fold, 5-fold or more fold compared to a control. A therapeutically effective amount can be administered in one or more doses of a therapeutic agent. A therapeutically effective amount can be administered as a single dose or in multiple doses over a period of time.

[0122] "Administration" as used herein can include any suitable route for administering a therapeutic agent or composition as disclosed herein. Suitable routes of administration include, but are not limited to, oral administration, parenteral administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway (aerosol) administration, pulmonary administration, dermal administration, injection administration, or topical administration. Administration can be local or systemic.

[0123] As used herein, the term "pharmaceutically acceptable" refers to a carrier that is compatible with the other ingredients of the formulation and not harmful to its recipient. This term is used synonymously with "physiologically acceptable" and "pharmacologically acceptable". Depending on the route of administration, the pharmaceutical composition will generally contain agents for buffering and preservation in storage and may contain buffering agents and carriers for proper delivery. The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications and commensurate with a reasonable benefit / risk ratio.

[0124] The terms "dose" and "dosage" are used interchangeably herein. A dose refers to the amount of the active ingredient administered to an individual at each administration. For the present invention, the dose may refer to the concentration of extracellular vesicles or related components, such as the amount of a therapeutic agent or the dose of a radiolabel. The dose will vary depending on a number of factors, including the frequency of administration, the size and tolerance of the individual, the severity of the condition, the risk of side effects, the route of administration, and the imaging modality of the detectable moiety (if any). Those skilled in the art will recognize that the dose can be modified based on the above factors or based on the progress of the treatment. The term "dosage form" refers to the specific form of a drug and depends on the route of administration. For example, the dosage form may be liquid, such as a saline solution for injection.

[0125] The terms "subject", "patient", "individual", and similar terms are used interchangeably and, unless otherwise indicated, refer to mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a specific disease but generally refers to an individual under medical supervision. A patient may be an individual seeking treatment, monitoring, adjustment, or modification of an existing treatment regimen, etc.

[0126] Unless otherwise indicated, the following meanings apply throughout this text. The term "can" is used in a permissive sense (i.e., having the potential), rather than a mandatory sense (i.e., being required). The term "comprising" and others mean including but not limited to. The singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "an element" includes combinations of two or more elements, even though other terms and phrases are used for one or more elements, such as "one or more". Unless otherwise indicated, the term "or" is non-exclusive, i.e., it encompasses both "and" and "or". The term "any one" between a modifier and a sequence means that the modifier modifies each member of the sequence. Thus, for example, the phrase "at least any one of 1, 2, or 3" means "at least 1, at least 2, or at least 3". The phrase "at least one" includes "more than one".

[0127] Definitions of common terms used in cell biology and molecular biology can be found in "The Merck Manual of Diagnosis and Therapy", 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al. eds.

[0128] The term "native form" corresponds to a polypeptide as it is understood to be encoded by the genome of an infectious agent. The term "exosomal form" corresponds to any derivative in which all or part of the protein is fused to an exosome-associated protein. The term "cytoplasmic form" corresponds to any derivative in which all or part of the protein is configured or designed to be expressed within the cytoplasm of a cell rather than enter the classical secretory pathway.

[0129] The expression that a protein is "configured or designed to be expressed in a certain way" means that its nucleotide sequence encodes a certain specific amino acid sequence such that when the protein is expressed in a cell, the protein will be in its native form, exosomal form, or cytoplasmic form by virtue of that specific amino acid sequence. For example, if the spike protein (S) is expressed in its native form, it is configured or designed to elicit a humoral response or a cellular immune response by virtue of the fact that it is a transmembrane protein with an extracellular domain.

[0130] The term "extracellular vesicle" (EV) refers to lipid bilayer-delimited particles that are naturally released from cells. EVs range in diameter from approximately 20 - 30 nanometers to approximately 10 micrometers or larger. EVs can contain proteins, nucleic acids, lipids, and metabolites from the cells that produce them. EVs include exosomes (approximately 50nm to approximately 200nm), microvesicles (approximately 100nm to approximately 300nm), ectosomes (approximately 50nm to approximately 1000nm), apoptotic bodies (approximately 50nm to approximately 5000nm), and lipid-protein aggregates of the same size.

[0131] The term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and chemically modified nucleic acids such as morpholino nucleic acid (PMO), peptide nucleic acid (PNA), 2'-O-methyl, 2'-methoxy-ethyl, phosphoramidate, methylphosphonate, and phosphorothioate. Nucleic acids can be of any size. Nucleic acids include but are not limited to genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules such as aptamers, plasmids, antisense DNA strands, shRNA, ribozymes, conjugated nucleic acids, and oligonucleotides. According to the present invention, nucleic acids can exist as single-stranded or double-stranded and linear or covalently circular closed molecules. Nucleic acids can, for example, be in the form of RNA (which can be prepared by in vitro transcription from a DNA template) for introduction into cells, such as transfection of cells. In addition, RNA can be modified by stabilizing sequences, capping, and polyadenylation prior to application. Generally, nucleic acids can be extracted, isolated, amplified, or analyzed by a variety of techniques such as those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY, pages 2,028 (2012).

[0132] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein and refer to any chain of at least two amino acids joined by covalent chemical bonds. As used herein, a peptide can refer to the complete amino acid sequence encoding an entire protein or a portion thereof. A "protein-coding sequence" or a sequence that "encodes" a particular polypeptide or peptide is a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. Coding sequences can include but are not limited to cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. Transcription termination sequences will generally be located 3' of the coding sequence.

[0133] As used herein, the phrase "protein polypeptide" means a polypeptide sequence that is proteinaceous or derived from a protein. For example, a CD9 protein polypeptide can be any polypeptide of the CD9 protein, such as, for example, the full-length CD9 protein, the transmembrane domain polypeptide of the CD9 protein, the C-terminal extension of the CD9 protein, the extracellular loop region of the CD9 protein, the intracellular (luminal) loop region of the CD9 protein, the C-terminal extension of the CD9 protein, combinations thereof, etc. Here, the protein polypeptide can be at least 10 amino acids in length.

[0134] As used herein, the term "spike protein" includes any SARS-CoV-2 spike glycoprotein, a fragment of a SARS-CoV-2 spike glycoprotein, a monomer of a SARS-CoV-2 spike glycoprotein, a trimer of SARS-CoV-2 spike glycoprotein monomers, a variant of a SARS-CoV-2 spike glycoprotein, a fusion protein or chimeric protein containing a SARS-CoV-2 spike glycoprotein sequence and another non-SARS-CoV-2 spike glycoprotein sequence, a SARS-CoV-2 spike glycoprotein having one or more amino acid deletions, additions or substitutions, and a conservative substitution variant of a SARS-CoV-2 spike glycoprotein having at least 80% amino acid sequence identity in, for example, the stem region such as at least the S2 subunit, the membrane-proximal stem helix region, or the receptor-binding domain or other similar domains.

[0135] Fragments of the SARS-CoV-2 spike glycoprotein include peptides or polypeptides that encompass, include, consist of, or overlap with, for example, antigenic epitopes, specific domains such as the receptor-binding domain (RBD) in an up or down conformational state, the receptor-binding fragment S1, the fusion fragment S2, the N-terminal domain (NTD), the 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), and the like.

[0136] Variants of the SARS-CoV-2 spike glycoprotein include any known or yet to be discovered spike protein sequences, including alpha, beta, gamma, delta, epsilon, eta, iota, kappa, 1.617.3, mu, zeta, Omicron or its subvariants, lineages, and conservative substitutions.

[0137] The SARS-CoV-2 spike glycoprotein can have additions, deletions, substitutions, point mutations. For example, the spike protein can have a deletion of several (2 - 20) amino acids from its C-terminus (see Johnson et al., 2020 and Xiong et al., 2020), or an altered furin cleavage site (see, e.g., Johnson et al., 2020).

[0138] As used herein, the term "nucleocapsid protein" means a soluble coronavirus structural protein that binds to RNA and viral membrane protein (M) to form a complex and is essential for viral genome packaging. The nucleocapsid protein contains (from the amino terminus to the carboxy terminus) an intrinsically disordered region (IDR) and an N-terminal domain (NTD) containing an RNA-binding domain, a serine-arginine-rich linker region (LKR), a C-terminal domain containing RNA-binding and dimerization domains and a nuclear localization signal, followed by a C-terminal IDR. The nucleocapsid protein is typically described in, for example, McBride et al., "The Coronavirus Nucleocapsid Is a Multifunctional Protein," Viruses. 2014 Aug;6(8):2991-3018; Cubuk et al., "The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA," Nature Communications Vol. 12, Article number: 1936 (2021); and the references cited therein.

[0139] A fusion protein or chimeric protein of an SARS-CoV-2 spike glycoprotein or an SARS-CoV-2 nucleocapsid protein comprises a fusion of a spike protein sequence or a nucleocapsid protein sequence with a sequence of another protein to achieve a specific result, such as improved sorting or targeting to endosomes and resulting extracellular vesicles such as exosomes. For example, the spike protein can be a fusion of a spike protein sequence or a nucleocapsid protein sequence with another glycoprotein known to be sorted to exosomes or useful for generating pseudotyped virions (such as VSV glycoprotein, lentiviral glycoprotein, etc.). The spike protein or nucleocapsid protein can be a fusion of a spike protein sequence and other proteins known to be sorted to exosomes (such as various tetraspanins (CD9, CD63, and CD81)). The fusion protein can predominantly contain an SARS-CoV-2 sequence with a short (i.e., dipeptide to 100 amino acid peptide) sequence of another protein.

[0140] As used herein, the term "tetraspanin" or "tetraspanin protein" refers to any member (or chimera thereof) of a family of proteins that have four transmembrane domains and in some cases are present in exosome membranes. Tetraspanin proteins are known to regulate trafficking as well as cellular and membrane compartmentalization. Tetraspanins include, in particular, CD9, CD37, CD63, CD81, CD82, CD151, TSPAN7, TSPAN8, TSPAN12, TSPAN33, peripherin, UP1a / 1b, TSP-15, TSP-12, TSP3A, TSP86D, TSP26D, TSP-2 and their analogs, orthologs and homologs. Useful tetraspanins are contemplated to include chimeras of any one or more canonical tetraspanins, such as, for example, CD9 / CD81 chimeras and the like. Tetraspanins are generally described in: Charrin et al., "Tetraspanins at a glance," J Cell Sci (2014) 127(17):3641-3648; Kummer et al., "Tetraspanins: integrating cell surface receptors to functional microdomains in homeostasis and disease," Med Microbiol Immunol. 2020; 209(4):397-405; and the references cited therein. The CD9 member of the tetraspanin superfamily is generally described in: Umeda et al., "Structural insights into tetraspanin CD9 function," Nature Communications Volume 11, Article number: 1606 (2020) and the references cited therein.

[0141] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percentage identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage identity between two sequences is a function of the number of positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions x 100%). In one embodiment, the two sequences are of the same length. The determination of the percentage identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized, as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Optionally, PSI-BLAST can be used to perform iterative searches to detect distant relationships between molecules (ibid.). When utilizing the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs can be used (e.g., the default parameters of XBLAST and NBLAST) (see, e.g., the NCBI website).

[0142] Another preferred non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0143] The percent identity between two sequences can be determined using techniques that allow gaps or do not allow gaps, similar to those described above. When calculating the percent identity, usually only exact matches are counted.

[0144] For antibodies, the percent sequence identity can be determined when the antibody sequences are maximally aligned by IMGT. After alignment, if the test antibody region (e.g., the entire mature variable region of the heavy or light chain) is compared to the same region of a reference antibody, the percent sequence identity between the test antibody region and the reference antibody region is the number of positions occupied by identical amino acids in both the test antibody region and the reference antibody region divided by the total number of aligned positions in the two regions, multiplied by 100 to convert to a percentage.

[0145] The percent amino acid sequence identity can also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be obtained from the National Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search parameters, all of which are set to default values, including, for example, unmask = yes, strand = all, expect = 10, minimum low complexity length = 15 / 5, multi-pass e-value = 0.01, multi-pass constant = 25, final gap alignment dropoff = 25, and scoring matrix = BLOSUM62.

[0146] In the case of amino acid sequence comparison using NCBI-BLAST2, the percent amino acid sequence identity of a particular amino acid sequence A to, with, or relative to a particular amino acid sequence B (which can also alternatively be stated as a particular amino acid sequence A has or contains a certain percent amino acid sequence identity to, with, or relative to a particular amino acid sequence B) is calculated as follows:

[0147] 100 times the fraction X / Y,

[0148] Where X is the number of amino acid residues in the alignment of A and B that are scored as identical matches by the sequence alignment program NCBI-BLAST2, and where Y is the total number of amino acid residues in B. It is understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not be equal to the percent amino acid sequence identity of B to A. As used herein, the term "nucleic acid sequence" refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars, and inter-sugar (backbone) linkages, and includes cDNA. The term also includes modified or substituted sequences that contain non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA), and can include naturally occurring bases, including adenine, guanine, cytosine, thymine, and uracil. The sequences can also contain modified bases. Examples of such modified bases include azido and deazido adenine, guanine, cytosine, thymine, and uracil; and xanthine and hypoxanthine. It is understood that polynucleotides containing non-transcribable nucleobases can be used, for example, as probes in hybridization assays. The nucleic acid can be double-stranded or single-stranded and represents the sense or antisense strand. In addition, the term "nucleic acid" includes complementary nucleic acid sequences as well as codon-optimized or synonymous codon equivalents.

[0149] As used herein, the term "antibody" refers to an immunoglobulin (Ig) molecule and the immunologically active portions of an immunoglobulin molecule (i.e., molecules that contain antigen-binding sites that specifically bind an antigen). Antibodies are typically heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Light chains from any vertebrate species can be assigned to one of two distinct types based on the amino acid sequence of their constant domains, called kappa (κ) and lambda (λ). Depending on the amino acid sequence of the constant domain of the heavy chain of the immunoglobulin, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. An antibody can have one or more effector functions, which refer to those biological activities that can be attributed to the Fc region of the antibody (native sequence Fc region or amino acid sequence variant Fc region or any other modified Fc region). Non-limiting examples of antibody effector functions include Clq binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor (BCR)); and cross-presentation of antigen by antigen-presenting cells or dendritic cells.

[0150] The term "neutralizing antibody" (Nab) refers to an antibody that protects cells from a pathogen or infectious particle by neutralizing any effect that the pathogen or infectious particle biologically has. Neutralization renders the particle no longer infectious or pathogenic. Neutralizing antibodies are part of the humoral response of the adaptive immune system against viruses, intracellular bacteria, and microbial toxins. By specifically binding to surface antigens on the infectious particle, neutralizing antibodies prevent the particle from interacting with the host cells that it might otherwise infect and destroy.

[0151] Immunity due to neutralizing antibodies is also called sterilizing immunity because the immune system eliminates the infectious particles before any infection occurs.

[0152] The term "antigen" refers to any substance that will elicit an immune response. For example, an antigen relates to any substance that specifically reacts with an antibody or a T lymphocyte (T cell), preferably a peptide or a protein. As used herein, the term "antigen" includes any molecule that contains at least one epitope. For example, an antigen is a molecule that optionally induces an immune response after processing. For example, any suitable antigen that is a candidate for an immune response can be used, where the immune response can be a cellular immune response. For example, an antigen can be presented by a cell, which results in an immune response against the antigen. For example, an antigen is a product that corresponds to or is derived from a naturally occurring antigen. Such antigens include, but are not limited to, the SARS-CoV-2 structural proteins S, N, M, and E and any variants or mutants thereof.

[0153] The term "pharmaceutical composition" refers to a preparation that contains an active ingredient and optionally a pharmaceutically acceptable carrier, diluent, or excipient. The term "active ingredient" can be used interchangeably with "effective ingredient" and refers to any agent that is capable of inducing the desired effect upon administration. "Pharmaceutically acceptable" means that the carrier, diluent, or excipient must be compatible with the other ingredients of the preparation and not harmful to its recipient, nor harmful to the activity of the active ingredient of the preparation. Pharmaceutically acceptable carriers, excipients, or stabilizers are well known in the art, such as Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations employed and can include buffers such as phosphate, citrate, and other organic acid buffers; antioxidants, including ascorbic acid and methionine; preservatives (such as cetyltrimethylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol alcohols, butanol or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG). Examples of carriers include, but are not limited to, liposomes, nanoparticles, ointments, micelles, microspheres, microparticles, creams, emulsions, and gels.

[0154] Examples of excipients include, but are not limited to, anti-adhesives such as magnesium stearate, binders such as sugars and their derivatives (sucrose, lactose, starch, cellulose, sugar alcohols, etc.), proteins such as gelatin and synthetic polymers, lubricants such as talc and silica, and preservatives such as antioxidants, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate, and parabens. Examples of diluents include, but are not limited to, water, alcohols, saline solutions, diols, mineral oil, and dimethyl sulfoxide (DMSO).

[0155] The term "vaccine" or "immunogenic composition" refers to a pharmaceutical product (pharmaceutical composition) or product that induces an immune response that recognizes and attacks pathogens or diseased cells, such as a cellular immune response, upon administration. The term "immune response" refers to the overall body response to an antigen and refers to a cellular immune response and / or a humoral immune response. The immune response can be protective / preventive / prophylactic and / or therapeutic.

[0156] The term "cellular immune response" or "cell-mediated immune response" describes any adaptive immune response in which antigen-specific T cells play a major role. It is operationally defined as all adaptive immunity that cannot be transferred to naive recipients by serum antibodies. In contrast, the term "humoral immune response" describes immunity due to antibodies.

[0157] Cellular responses involve cells called T cells or T lymphocytes that act as "helper" or "killer". Helper T cells (also called CD4+ T cells) play a central role by regulating the immune response, and killer cells (also called cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill diseased cells such as cancer cells to prevent the production of more diseased cells.

[0158] The term "immunoreactive cell", "immune cell", or "immune effector cell" refers to a cell that plays an effector function during an immune response. An "immunoreactive cell" is preferably a cell that is capable of binding an antigen or is characterized by presenting an antigen or an antigenic peptide derived from an antigen and mediating an immune response. For example, such cells secrete cytokines and / or chemokines, secrete antibodies, recognize cancer cells, and optionally eliminate such cells. For example, immunoreactive cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells.

[0159] The term "adjuvant" refers to a pharmacological or immunological agent that modifies the action of other agents. Adjuvants can be added to the vaccine compositions of the present invention to enhance the immune response to produce more antibodies and a more persistent immunity, thereby minimizing the required antigen dose. Adjuvants can also be used to enhance the efficacy of vaccines by assisting in modulating the immune response to specific types of immune system cells: for example, depending on the purpose of the vaccine, enhancing the efficacy of the vaccine 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 they do not provide immunity themselves. Examples of adjuvants include, but are not limited to, analgesic adjuvants; inorganic compounds such as alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide; mineral oils such as paraffin oil; bacterial products, such as killed bacteria (Bordetella pertussis, Mycobacterium bovis, toxoids); non-bacterial organic substances such as squalene; delivery systems, such as detergents (Quil A); plant saponins from soapbark, soybeans, or Polygalasenega; cytokines such as IL-1, IL-2, IL-12; combinations, such as Freund's complete adjuvant, Freund's incomplete adjuvant; food-based oils, such as Adjuvant 65, which is based on peanut oil.

[0160] Detailed Embodiments

[0161] Embodiment 1. In one embodiment, there is provided an immunogenic composition comprising vesicles and a fusion protein, wherein the fusion protein comprises a viral polypeptide and an exosome polypeptide.

[0162] Embodiment 2. There is provided the immunogenic composition of Embodiment 1, further comprising an excipient.

[0163] Embodiment 3. There is provided the immunogenic composition of Embodiment 2, wherein the excipient is a buffer.

[0164] Embodiment 4. There is provided the immunogenic composition of any one of Embodiments 1-3, which does not contain an adjuvant.

[0165] Embodiment 5. There is provided the immunogenic composition of any one of Embodiments 1-4, wherein the fusion protein is present in the membrane of the vesicles.

[0166] Embodiment 6. There is provided the immunogenic composition of any one of Embodiments 1-5, wherein a part or all of the viral polypeptide is present at or on the outer surface of the vesicles.

[0167] Embodiment 7. An immunogenic composition according to any one of Embodiments 1-6 is provided, wherein the fusion protein is present in the composition at a concentration of about 1 ng / 100 μL to about 150 ng / 100 μL.

[0168] Embodiment 8. An immunogenic composition according to any one of Embodiments 1-7 is provided, wherein the exosome polypeptide is a tetraspanin polypeptide.

[0169] Embodiment 9. An immunogenic composition according to any one of Embodiments 1-8 is provided, wherein the exosome polypeptide is a CD9 polypeptide.

[0170] Embodiment 10. An immunogenic composition according to any one of Embodiments 1-9 is provided, wherein the exosome polypeptide comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 10.

[0171] Embodiment 11. An immunogenic composition according to any one of Embodiments 1-10 is provided, wherein the exosome polypeptide comprises the amino acid sequence of SEQ ID NO: 10.

[0172] Embodiment 12. An immunogenic composition according to any one of Embodiments 1-11 is provided, wherein the viral polypeptide is a SARS-CoV-2 polypeptide.

[0173] Embodiment 13. An immunogenic composition according to any one of Embodiments 1-12 is provided, wherein the viral polypeptide is a SARS-CoV-2 spike protein polypeptide.

[0174] Embodiment 14. An immunogenic composition according to any one of Embodiments 1-13 is provided, wherein the viral polypeptide is a SARS-CoV-2 Delta variant spike protein polypeptide.

[0175] Embodiment 15. An immunogenic composition according to any one of Embodiments 1-14 is provided, wherein the viral polypeptide comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 1.

[0176] Embodiment 16. An immunogenic composition according to any one of Embodiments 1-15 is provided, wherein the viral polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

[0177] Embodiment 17. An immunogenic composition according to any one of Embodiments 1-11 is provided, wherein the viral polypeptide is a SARS-CoV-2 nucleocapsid protein polypeptide.

[0178] Embodiment 18. An immunogenic composition according to any one of Embodiments 1-11 and 17 is provided, wherein the viral polypeptide comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 5.

[0179] Embodiment 19. An immunogenic composition according to any one of Embodiments 1-11, 17, and 18 is provided, wherein the viral polypeptide comprises the amino acid sequence of SEQ ID NO:5.

[0180] Embodiment 20. An immunogenic composition according to any one of Embodiments 1-16 is provided, wherein the fusion protein comprises a SARS-CoV-2 spike protein polypeptide and a CD9 protein polypeptide.

[0181] Embodiment 21. An immunogenic composition according to any one of Embodiments 1-16 and 20 is provided, wherein the fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:2.

[0182] Embodiment 22. An immunogenic composition according to any one of Embodiments 1-16, 20, and 21 is provided, wherein the fusion protein comprises an amino acid sequence having the sequence of SEQ ID NO:2.

[0183] Embodiment 23. An immunogenic composition according to any one of Embodiments 1-16 and 20-22 is provided, wherein the fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:3.

[0184] Embodiment 24. An immunogenic composition according to any one of Embodiments 1-16 and 20-23 is provided, wherein the fusion protein comprises an amino acid sequence having the sequence of SEQ ID NO:3.

[0185] Embodiment 25. An immunogenic composition according to any one of Embodiments 1-11 and 17-19 is provided, wherein the fusion protein comprises a SARS-CoV-2 nucleocapsid protein polypeptide and a CD9 protein polypeptide.

[0186] Embodiment 26. An immunogenic composition according to any one of Embodiments 1-11, 17-19, and 25 is provided, wherein the fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:6.

[0187] Embodiment 27. An immunogenic composition according to any one of Embodiments 1-11, 17-19, 25, and 26 is provided, wherein the fusion protein comprises an amino acid sequence having the sequence of SEQ ID NO:6.

[0188] Embodiment 28. An immunogenic composition according to any one of Embodiments 1-11, 17-19, and 25-27 is provided, wherein the fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:7.

[0189] Embodiment 29. An immunogenic composition is provided which is any one of Embodiments 1-11, 17-19, and 25-28, wherein the fusion protein comprises an amino acid sequence having the sequence of SEQ ID NO:7.

[0190] Embodiment 30. The immunogenic composition of any one of Embodiments 1-29, wherein the immunogenic dose of the composition comprises in 0.5 mL (i) from about 1 ng to about 300 ng of the fusion protein or viral polypeptide or (ii) 10 - 200 ng of the total fusion protein.

[0191] Embodiment 31. An immunogenic composition is provided which is any one of Embodiments 1-30, the immunogenic composition comprising a second vesicle and a second fusion protein, wherein the second fusion protein comprises a second viral polypeptide and an exosome polypeptide, and wherein the second fusion protein is present in the membrane of the second vesicle.

[0192] 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.

[0193] 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 wherein the second fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:6.

[0194] Embodiment 34. The immunogenic composition of any one of Embodiments 31-33 is provided, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:2, and wherein the second fusion protein comprises the amino acid sequence of SEQ ID NO:6.

[0195] Embodiment 35. The immunogenic composition of any one of Embodiments 31-34 is provided, wherein the fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:3, and wherein the second fusion protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:7.

[0196] Embodiment 36. The immunogenic composition of any one of Embodiments 31-35 is provided, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:3, and wherein the second fusion protein comprises the amino acid sequence of SEQ ID NO:7.

[0197] Embodiment 37. The immunogenic composition of any one of Embodiments 1-36 is provided, wherein the vesicle is an exosome.

[0198] Embodiment 38. A method of immunizing a subject against viral infection is provided, the method comprising administering to the subject an immunogenically effective dose of an immunogenic composition according to any one of Embodiments 1-37.

[0199] Embodiment 39. The method of Embodiment 38 is provided, wherein the immunogenically effective dose elicits protective immunity against more than one variant of a given virus in the subject.

[0200] Embodiment 40. The method of Embodiment 38 or 39 is provided, wherein the immunogenic composition comprises a SARS-CoV-2 nucleocapsid protein polypeptide.

[0201] Embodiment 41. The method of any one of Embodiments 38-40 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.

[0202] Embodiment 42. The method of any one of Embodiments 38-41 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.

[0203] Embodiment 43. The method of any one of Embodiments 38-42 is provided, wherein the immunogenic composition comprises a fusion protein having the amino acid sequence of SEQ ID NO:6.

[0204] Embodiment 44. The method of any one of Embodiments 38-43 is provided, wherein the immunogenic composition comprises a fusion protein having the amino acid sequence of SEQ ID NO:7.

[0205] 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 the immunogenically effective dose elicits protective immunity against the SARS-CoV-2 Delta variant and the SARS-CoV-2 Omicron variant in the subject.

[0206] 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.

[0207] 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.

[0208] Embodiment 48. There is provided the method of any one of Embodiments 38, 39, and 45 - 47, wherein the immunogenic composition comprises a fusion protein having the amino acid sequence of SEQ ID NO:2.

[0209] Embodiment 49. There is provided the method of any one of Embodiments 38, 39, and 45 - 48, wherein the immunogenic composition comprises a fusion protein having the amino acid sequence of SEQ ID NO:3.

[0210] Embodiment 50. There is provided the method of Embodiment 38 or 39, wherein the immunogenic composition comprises a first fusion protein and a second fusion protein, the first fusion protein comprising a SARS-CoV-2 nucleocapsid protein polypeptide located in the membrane of a first vesicle, and the second fusion protein comprising a SARS-CoV-2 spike protein polypeptide located in the membrane of a second vesicle.

[0211] Embodiment 51. There is provided the method of Embodiment 50, wherein the first fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:6.

[0212] Embodiment 52. There is provided the method of Embodiment 50 or 51, wherein the first fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:7.

[0213] Embodiment 53. There is provided the method of any one of Embodiments 50 - 52, wherein the first fusion protein has the amino acid sequence of SEQ ID NO:6.

[0214] Embodiment 54. There is provided the method of any one of Embodiments 50 - 53, wherein the first fusion protein has the amino acid sequence of SEQ ID NO:7.

[0215] Embodiment 55. There is provided the method of any one of Embodiments 50 - 54, wherein the second fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:2.

[0216] Embodiment 56. There is provided the method of any one of Embodiments 50 - 55, wherein the second fusion protein has an amino acid sequence that is at least 80% identical to SEQ ID NO:3.

[0217] Embodiment 57. There is provided the method of any one of Embodiments 50 - 56, wherein the second fusion protein has the amino acid sequence of SEQ ID NO:2.

[0218] Embodiment 58. There is provided the method of any one of Embodiments 50 - 57, wherein the second fusion protein has the amino acid sequence of SEQ ID NO:3.

[0219] Embodiment 59. The method of any one of embodiments 38 - 58 is provided, wherein the immunogenic effective dose comprises in 0.5 mL (i) about 1 ng to about 300 ng of a fusion protein or viral polypeptide or (ii) 10 - 200 ng of total fusion protein.

[0220] Embodiment 60. The method of any one of embodiments 38 - 59 is provided, further comprising administering to a subject a second effective dose of an immunogenic composition of any one of embodiments 1 - 37.

[0221] Embodiment 61. A synthetic fusion protein is provided, the synthetic fusion protein comprising a viral polypeptide and an exosome polypeptide.

[0222] Embodiment 62. The synthetic fusion protein of embodiment 61 is provided, further comprising a linker polypeptide located between the viral polypeptide and the exosome polypeptide.

[0223] Embodiment 63. The synthetic fusion protein of embodiment 61 or 62 is provided, further comprising a hinge polypeptide located between the viral polypeptide and the exosome polypeptide.

[0224] Embodiment 64. The synthetic fusion protein of any one of embodiments 61 - 63 is provided, further comprising a transmembrane domain polypeptide located between the viral polypeptide and the exosome polypeptide.

[0225] Embodiment 65. The synthetic fusion protein of any one of embodiments 61 - 64 is provided, wherein the exosome polypeptide is a tetraspanin polypeptide.

[0226] Embodiment 66. The synthetic fusion protein of any one of embodiments 61 - 65 is provided, wherein the exosome polypeptide is a CD9 polypeptide.

[0227] Embodiment 67. The synthetic fusion protein of any one of embodiments 61 - 66 is provided, wherein the viral polypeptide is a SARS-CoV-2 structural protein polynucleotide.

[0228] Embodiment 68. The synthetic fusion protein of any one of embodiments 61 - 67 is provided, wherein the viral polypeptide is a SARS-CoV-2 spike protein polypeptide.

[0229] 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 protease cleavage site mutation (CSM[682RRAR685 - to - 682GSAG685]) and a diproline substitution (2P[986KV987 - to - 986PP987]).

[0230] Embodiment 70. A synthetic fusion protein according to any one of embodiments 61-69 is provided, wherein the fusion protein comprises, in order from the amino terminus to the carboxy terminus, a SARS-CoV-2 spike protein polypeptide, a linker polypeptide, and a CD9 polypeptide.

[0231] Embodiment 71. A synthetic fusion protein according to any one of embodiments 61-70 is provided, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:3.

[0232] Embodiment 72. A synthetic fusion protein according to any one of embodiments 61-71 is provided, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:3.

[0233] Embodiment 73. A synthetic fusion protein according to any one of embodiments 61-67 is provided, wherein the viral polypeptide is a SARS-CoV-2 nucleocapsid protein polypeptide.

[0234] Embodiment 74. A synthetic fusion protein according to any one of embodiments 61-67 and 73 is provided, wherein the fusion protein comprises, in order from the amino terminus to the 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.

[0235] Embodiment 75. A synthetic fusion protein according to any one of embodiments 61-67, 73, and 74 is provided, wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:7.

[0236] Embodiment 76. A synthetic fusion protein according to any one of embodiments 61-67 and 73-75 is provided, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:7.

[0237] Embodiment 77. A synthetic polynucleotide is provided, which encodes a synthetic fusion protein according to any one of embodiments 61-76.

[0238] 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.

[0239] Embodiment 79. The synthetic polynucleotide of embodiment 77 or 78 is provided, comprising the nucleic acid sequence set forth in SEQ ID NO:13.

[0240] Embodiment 80. The synthetic polynucleotide of any one of embodiments 77-79 is provided, comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:11.

[0241] Embodiment 81. A synthetic polynucleotide of any one of Embodiments 77 - 80 is provided, comprising the nucleic acid sequence set forth in SEQ ID NO:11.

[0242] Embodiment 82. A synthetic polynucleotide of any one of Embodiments 77 - 81 is provided, comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO:4.

[0243] Embodiment 83. A synthetic polynucleotide of any one of Embodiments 77 - 82 is provided, comprising the nucleic acid sequence set forth in SEQ ID NO:4.

[0244] Embodiment 84. A synthetic polynucleotide of any one of Embodiments 77 - 79 is provided, comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO:12.

[0245] Embodiment 85. A synthetic polynucleotide of any one of Embodiments 77 - 79 and 84 is provided, comprising the nucleic acid sequence set forth in SEQ ID NO:12.

[0246] Embodiment 86. A synthetic polynucleotide of any one of Embodiments 77 - 79, 84 and 85 is provided, comprising a nucleic acid sequence having at least 80% identity with SEQ ID NO:8.

[0247] Embodiment 87. A synthetic polynucleotide of any one of Embodiments 77 - 79 and 84 - 86 is provided, comprising the nucleic acid sequence set forth in SEQ ID NO:8.

[0248] Embodiment 88. A cell is provided, the cell comprising a synthetic polynucleotide of any one of Embodiments 77 - 87.

[0249] Embodiment 89. The cell of Embodiment 88 is provided, wherein the cell is a metazoan cell.

[0250] Embodiment 90. The cell of Embodiment 88 or 89 is provided, wherein the cell is a vertebrate cell.

[0251] Embodiment 91. The cell of any one of Embodiments 88 - 90 is provided, wherein the cell is a mammalian cell.

[0252] Embodiment 92. The cell of any one of Embodiments 88 - 91 is provided, wherein the cell is a primate cell.

[0253] Embodiment 93. The cell of any one of Embodiments 88 - 92 is provided, wherein the cell is a human cell.

[0254] Embodiment 94. There is provided a cell according to any one of Embodiments 88-93, wherein the cell is a primary cell.

[0255] Embodiment 95. There is provided a cell according to any one of Embodiments 88-93, wherein the cell is a human embryonic kidney cell.

[0256] Embodiment 96. There is provided a cell according to Embodiment 95, wherein the cell is a 293 cell.

[0257] Embodiment 97. There is provided a cell according to any one of Embodiments 88-96, wherein the cell is produced by transducing the cell with a lentivirus containing a synthetic polynucleotide.

[0258] Embodiment 98. There is provided a cell according to any one of Embodiments 88-97, wherein the cell comprises a synthetic fusion protein according to any one of Embodiments 61-77.

[0259] Embodiment 99. There is provided a vesicle comprising a synthetic fusion protein according to any one of Embodiments 61-76.

[0260] Embodiment 100. There is provided a vesicle according to Embodiment 99, wherein the vesicle is an exosome.

[0261] Embodiment 101. There is provided a vesicle according to Embodiment 99 or 100, wherein the vesicle has a diameter of about 50-500 nm.

[0262] Embodiment 102. There is provided a vesicle according to any one of Embodiments 99-101, wherein the SARS-CoV-2 spike protein polypeptide is expressed on the outer surface of the vesicle.

[0263] Embodiment 103. There is provided a vesicle according to any one of Embodiments 99-102, wherein the vesicle expresses the SARS-CoV-2 nucleocapsid protein polypeptide on its surface.

[0264] Embodiment 104. There is provided a method for preparing a vesicle according to any one of Embodiments 99-103, the method comprising culturing a cell according to any one of Embodiments 88-98 in a cell culture medium, collecting the cell culture medium, and purifying more than one vesicle comprising the vesicle from the cell culture medium.

[0265] Embodiment 105. There is provided the method of Embodiment 104, further comprising inducing the expression of a synthetic polynucleotide according to any one of Embodiments 77-87 to produce a synthetic fusion protein according to any one of Embodiments 61-76.

[0266] Embodiment 106. The method of Embodiment 105, wherein the induction comprises contacting the cell with tetracycline, doxycycline, or an analogue thereof.

[0267] Embodiment 107. The method of Embodiment 105, wherein said induction comprises removing tetracycline, doxycycline or an analogue thereof from the cells.

[0268] Embodiment 108. A method of eliciting an immune response in a subject is provided, the method comprising administering to the subject a first dose of an immunogenic composition, the immunogenic composition comprising more than one vesicle, the more than one vesicle comprising the vesicle of any one of Embodiments 99 - 103 or the vesicle produced by the method of any one of Embodiments 104 - 107, wherein the synthetic fusion protein of any one of Embodiments 61 - 76 is expressed on the outer surface of the vesicle.

[0269] Embodiment 109. The method of Embodiment 108 is provided, further comprising administering to the subject a second dose of the immunogenic composition at a period of time after administering the first dose.

[0270] Embodiment 110. The method of Embodiment 109 is provided, wherein the period of time is 14 days to 1 year.

[0271] Embodiment 111. The method of any one of Embodiments 108 - 110 is provided, wherein the first dose or the second dose comprises about 100 μL - 1 mL of the immunogenic composition, wherein the immunogenic composition comprises about 0.3 ng / mL - 3 μg / mL of the synthetic fusion protein.

[0272] Embodiment 112. The method of any one of Embodiments 108 - 111 is provided, wherein the first dose or the second dose comprises about 100 μL - 1 mL of the immunogenic composition, the immunogenic composition comprising about 2E9 vesicles / mL - 3E13 vesicles / mL.

[0273] Embodiment 113. The method of any one of Embodiments 108 - 112 is provided, wherein the synthetic fusion protein comprises a SARS-CoV-2 spike protein polypeptide.

[0274] Embodiment 114. The method of any one of Embodiments 108 - 113 is provided, wherein the synthetic fusion protein comprises a SARS-CoV-2 nucleocapsid protein polypeptide.

[0275] Embodiment 115. The method of any one of Embodiments 108 - 114 is provided, wherein the immunogenic composition comprises vesicles expressing a SARS-CoV-2 spike protein polypeptide on the outer surface and other vesicles expressing a SARS-CoV-2 nucleocapsid protein polypeptide on the outer surface.

[0276] Embodiment 116. A method according to any one of embodiments 108 - 115 is provided, wherein the elicited immune response comprises the production of neutralizing antibodies against the antigen present in the synthetic fusion protein.

[0277] Embodiment 117. A method according to any one of embodiments 108 - 116 is provided, wherein the elicited immune response comprises the production of neutralizing antibodies against two or more SARS-CoV-2 variants.

[0278] Embodiment 118. A method according to any one of embodiments 108 - 117 is provided, wherein the elicited immune response comprises the production of anti-spike antibodies.

[0279] Embodiment 119. A method according to any one of embodiments 108 - 118 is provided, wherein the elicited immune response comprises a spike-specific T cell response.

[0280] Embodiment 120. A method according to any one of embodiments 108 - 119 is provided, wherein the elicited immune response comprises the production of anti-nucleocapsid antibodies.

[0281] Embodiment 121. A method according to any one of embodiments 108 - 120 is provided, wherein the elicited immune response comprises a nucleocapsid-specific T cell response.

[0282] Embodiment 122. A method according to any one of embodiments 108 - 121 is provided, wherein the immune response persists in the subject for up to nine months.

[0283] Embodiment 123. A method according to any one of embodiments 108 - 121 is provided, wherein the immune response persists in the subject for at least nine months.

[0284] Sequence

[0285] SEQ ID NO:1 provides the amino acid sequence of a coronavirus spike protein polypeptide.

[0286]

[0287]

[0288] SEQ ID NO:2 provides the amino acid sequence of a spike polypeptide-linker-CD9 proximal region chimera.

[0289]

[0290] SEQ ID NO:3 provides the amino acid sequence of a spike-CD9 fusion protein.

[0291]

[0292] SEQ ID NO:4 provides the nucleic acid sequence encoding the spike-CD9 fusion protein.

[0293]

[0294]

[0295]

[0296] SEQ ID NO:5 provides the amino acid sequence of the coronavirus nucleocapsid protein polypeptide.

[0297]

[0298] SEQ ID NO:6 provides the amino acid sequence of the nucleocapsid polypeptide-linker-transmembrane domain proximal region chimera.

[0299]

[0300] SEQ ID NO:7 provides the amino acid sequence of the nucleocapsid-CD9 fusion protein.

[0301]

[0302]

[0303] SEQ ID NO:8 provides the nucleic acid sequence encoding the nucleocapsid-CD9 fusion protein.

[0304]

[0305]

[0306] SEQ ID NO:9 provides the amino acid sequence of the transmembrane region polypeptide.

[0307]

[0308] SEQ ID NO:10 provides the amino acid sequence of the CD9 protein polypeptide.

[0309]

[0310]

[0311] SEQ ID NO:11 provides the nucleic acid sequence encoding the coronavirus spike protein polypeptide.

[0312]

[0313]

[0314]

[0315] SEQ ID NO:12 provides the nucleic acid sequence encoding the coronavirus nucleocapsid protein polypeptide.

[0316]

[0317]

[0318] SEQ ID NO:13 provides the nucleic acid sequence encoding the CD9 exosome tetraspanin protein polypeptide.

[0319]

[0320] Extracellular Vesicles and Exosomes

[0321] A variety of host cells are known in the art and are suitable for protein expression and extracellular vesicle production. Non-limiting examples of typical cells for transfection include, but are not limited to, bacterial cells, eukaryotic cells, yeast cells, insect cells, or plant cells. For example, human embryonic kidney 293 (HEK293), Escherichia coli (E. coli), Bacillus spp., Streptomyces spp., Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SJ9, CHO, COS (e.g., COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H, or 293F. See, for example, Portolano et al., “Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach,” Journal of Visualized Experiments, October 2014, 92, e51897, pp. 1-8, for a description of recombinant proteins in 293 cells in suspension culture.

[0322] Extracellular vesicles (EVs) are lipid-bound vesicles secreted by cells into the extracellular space. The three main subtypes of EVs are microvesicles (MVs), exosomes, and apoptotic bodies, which are distinguished based on their biogenesis, release pathway, size, content, 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.

[0323] Exosomes include small secretory vesicles with a diameter of about 20 - 200 nm, which are released particularly by mammalian cells and are prepared by budding into endosomes or by budding from the plasma membrane. In some cases, exosomes have a characteristic buoyant density of about 1.1 - 1.2 g / mL and a characteristic lipid composition. Their lipid membranes are usually rich in cholesterol and contain sphingomyelin, ceramides, lipid rafts, and exposed phosphatidylserine. Exosomes express certain marker proteins, such as integrins and cell adhesion molecules, but generally lack markers of lysosomes, mitochondria, or caveolae. In some embodiments, exosomes contain cell-derived components, such as but not limited to proteins, DNA, and RNA (e.g., microRNA [miR] and non-coding RNA). In some embodiments, exosomes can be obtained from cells obtained from allogeneic, autologous, xenogeneic, or syngeneic sources relative to the recipient of the exosomes.

[0324] Certain types of RNA, such as microRNA (miRNA), are known to be carried by exosomes. miRNAs typically act as post-transcriptional regulators by binding to complementary sequences on target messenger RNA transcripts (mRNAs), resulting in translational inhibition, target mRNA degradation, and / or gene silencing.

[0325] Useful exosomes can be obtained from any cell source, including prokaryotes, plants, fungi, metazoans, vertebrates, mammals, primates, humans, autologous cells, and allogeneic cells. See, e.g., Kim et al., “Platform technologies and human cell lines for the production of therapeutic exosomes,” Extracell Vesicles Circ Nucleic Acids 2021;2:3-17. For example, exosomes can be derived from mesenchymal stem cells, embryonic stem cells, iPS cells, immune cells, PBMCs, neural stem cells, HEK293 cells (described, e.g., in Dumont et al., “Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives,” Crit Rev Biotechnol 2016;36:1110-22), HEK293T cells (described, e.g., in Li et al., “Identification and characterization of 293T cell-derived exosomes by profiling the protein, mRNA and MicroRNA components,” PLoS One 2016;11:e0163043), 293F cells (described in Stenkamp et al., “Exosomes represent a novel mechanism of regulatory T cell suppression (P1079),” J Immunol May 1, 2013, 190(1 Suppl) 121.11), amniotic cells, CAR-T cells, cardiospheres, and cardiospherederived cells (CDC) (described, e.g., in WO2014028493, WO2022006178A1, US20210032598A1, US9828603B2, EP2914273A1, US20200316226A1, US20120315252A1, US20170360842A1, and references therein).

[0326] Briefly, methods for preparing exosomes can include the steps of culturing cells in a culture medium, separating the cells from the culture medium, purifying the exosomes by, for example, sequential centrifugation, and optionally, clarifying the exosomes on a density gradient such as a sucrose density gradient. In some cases, the isolated and purified exosomes are substantially free of non-exosome components such as cellular components or whole cells. The exosomes can be resuspended in a buffer such as sterile PBS buffer containing 0.01%-1% human serum albumin. The exosomes can be frozen and stored for future use.

[0327] Exosomes can be collected, concentrated, and / or purified using methods known in the art. For example, differential centrifugation has emerged as a leading technique where secreted exosomes are isolated from the supernatant of cultured cells. This method allows the separation of exosomes from larger extracellular vesicles and most non-particulate contaminants by taking advantage of the size of exosomes. Exosomes can be prepared as described in a variety of papers, including but not limited to Fordjour et al., “A shared pathway of exosome biogenesis operates at plasma and endosome membranes”, bioRxiv, preprint posted February 11, 2019, available 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 are purified using commercial kits such as, but not limited to, the ExoSpin TM Exosome Purification Kit, Total Exosome Purification Kit, Exosome Isolation Kit, and ExoCap TMExosome isolation kit. Methods for isolating exosomes from stem cells are described, for example, in Tan et al., Journal of Extracellular Vesicles, 2:22614 (2013); Ono et al., Sci Signal, 7(332):ra63 (2014); and U.S. Patent Application Publications Nos. 2012 / 0093885 and 2014 / 0004601. Methods for isolating exosomes from cells derived from cardiospheres are described, for example, in Ibrahim et al., “Exosomes as critical agents of cardiac regeneration triggered by cell therapy,” Stem Cell Reports, 2014. Specific methods include ultracentrifugation, density gradient, HPLC, affinity-based substrate adhesion, or size-exclusion-based filtration.

[0328] Size exclusion allows their separation from biochemically similar but biophysically distinct microvesicles with larger diameters up to 1,000 nm. Differences in flotation velocity further allow the separation of exosomes of different sizes. Typically, exosome size will have a diameter in the range of 30 - 200 nm, including sizes of 40 - 100 nm. Further purification can depend on the specific properties of the particular exosomes of interest. This includes, for example, using immunosorption of the protein of interest to select specific vesicles with an ectoplasmic or outward orientation.

[0329] In current methods such as differential centrifugation, discontinuous density gradient, immunoaflinity, ultrafiltration, and high-performance liquid chromatography (HPLC), differential ultracentrifugation is most commonly used for exosome isolation. This technique uses centrifugal forces increasing from 2,000×g to 10,000×g and 100,000×g to separate medium and larger-sized particles and cell debris from the exosome pellet. Centrifugation alone allows significant isolation / collection of exosomes from conditioned media, although it may not be sufficient to remove various protein aggregates, genetic material, particles, and cell debris, which are common contaminants. Enhanced specificity of exosome purification can be achieved by deploying sequential centrifugation in combination with ultrafiltration, or equilibrium density gradient centrifugation in a sucrose density gradient to provide a higher purity exosome preparation (flotation density 1.1 - 1.2 g / mL), or by applying discrete sugar pads in the preparation.

[0330] Ultrafiltration can be used to purify exosomes without compromising their biological activity. Membranes with different pore sizes—such as 100 kDa cut-off molecular weight (MWCO) and gel filtration for eliminating smaller particles—have been used to avoid using non-neutral pH or non-physiological salt concentrations. Current available tangential flow filtration (TFF) systems are scalable (to >10,000 L), allowing not only purification but also concentration of exosome fractions, and such methods are more time-saving than differential centrifugation. HPLC can also be used to purify exosomes into a more uniform-sized particle preparation and maintain their biological activity while the preparation is maintained at physiological pH and salt concentration. Other chemical methods have utilized the different solubility of exosomes for precipitation techniques, in addition to size-excluding polymers (e.g., polyethylene glycol (PEG)), and may incorporate additional rounds of centrifugation or filtration. For example, a precipitation reagent can be added to conditioned cell media to rapidly and promptly precipitate a population of exosomes, although the precipitate prepared via this technique may be difficult to resuspend. Flow field-flow fractionation (FlFFF) is an elution-based technique that has been used to separate and characterize macromolecules (e.g., proteins) and nano- to micron-sized particles (e.g., organelles and cells), and has been successfully applied to fractionate exosomes from media.

[0331] In addition to these techniques that rely on general biochemical and biophysical characteristics, focusing techniques can be applied to isolate specific exosomes of interest. This includes relying on antibody immunaffinity to recognize certain exosome-associated antigens. As described, exosomes also express the extracellular domain of membrane-bound receptors at the membrane surface. This provides an opportunity to isolate and separate exosomes associated with their parental cell origin based on a common antigen profile. Conjugation to magnetic beads (e.g., such as anti-CD81 magnetic beads), chromatographic matrices, plates, or microfluidic devices allows the isolation of specific exosome populations of interest, which may be related to their production from the parental cells of interest or associated cell regulatory states. Other affinity capture methods use lectins that bind to specific sugar residues on the exosome surface.

[0332] For example, exosomes (and other extracellular vesicles) can be produced via 293F cells. 293F cells can be transfected with a polynucleotide encoding a spike protein or a nucleocapsid protein or a chimeric fusion thereof (or transduced with a lentivirus carrying the polynucleotide), as described herein (see Figure 3A ), and express the spike protein or nucleocapsid protein such that the spike protein or nucleocapsid is sorted into or onto the exosomes isolated therefrom and displayed. Exemplary procedures for preparing exosomes from 293F cells can include the following steps: 293F cells (Gibco TM, catalog number 51-0029, ThermoFisher Scientific, Waltham, MA), and found to be free of viral (cytomegalovirus, human immunodeficiency virus I and II, Epstein-Barr virus, hepatitis B virus, and parvovirus B19) and bacterial (mycoplasma) contaminants. The cells can be maintained in FreeStyle TM 293 Expression Medium (Gibco, catalog number 12338-018, ThermoFisher Scientific, Waltham, Ma) and incubated at 37 °C in 8% CO2. For exosome production, 293F cells can be seeded in a shake flask at a density of 1.5E6 cells / ml at a volume of approximately 1 / 4 of the shake flask volume and grown at a shaking speed of approximately 110 rpm. HEK293 cells can be grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum.

[0333] For exosome purification, 293F cells can be cultured continuously in a shake flask for a period of three days. Cells and large cell debris can be removed by centrifugation at 300×g for 5 minutes, followed by centrifugation at 3,000×g for 15 minutes. The resulting supernatant can be passed through a 0.22 μm sterile filtration unit (Thermo Fisher, catalog number 566-0020) to produce a clarified tissue culture supernatant (CTCS). The CTCS can be concentrated by centrifugal filtration (Centricon Plus-70, Ultracel-PL membrane, 100 kDa size exclusion, Millipore Sigma, catalog number UFC710008, St. Louis, Mo), concentrating approximately 120 mL of CTCS to approximately 0.5 mL. The concentrated CTCS can then be purified by size exclusion chromatography (SEC) in 1×PBS (qEV original column / 35 nm: Izon Science, catalog number SP5), where the exosomes present in each 0.5 ml starting sample are eluted in three 0.5 ml fractions. The purified exosomes can be re-concentrated using an Ultra-4 100 kDa cut-off spin column (catalog number UFC810024). This method can produce a population of exosomes / small EVs with the expected ultrastructure and size distribution profile of human exosomes and containing the exosome marker proteins CD9 and CD63, with a concentration effect of approximately 500-fold and a final concentration of 1E10-2E12 exosomes / ml. The concentration and size of the isolated extracellular vesicles can be measured using a NANOSIGHT nanoparticle tracking analysis system (Malvern Panalytical, Malvern, UK).

[0334] SARS-CoV-2 Protein

[0335] Membrane-bound vesicles containing one or more populations of SARS-CoV-2 structural proteins are disclosed. "Containing" means that the proteins contained can be within the lumen of the vesicle, displayed on the surface of the vesicle, or both within the lumen and on the surface. Here, those fusion proteins containing the tetraspanin polypeptide sequence are mostly displayed on the surface of the vesicle. Those proteins displayed on the surface of the vesicle can have a protein portion within the lumen, a protein portion spanning the vesicle membrane (i.e., transmembrane region or domain), and a protein portion extending outside the vesicle. In one embodiment, the SARS-CoV-2 structural protein is the spike glycoprotein (S), nucleocapsid (N) protein, membrane (M) protein, or envelope (E) protein or any combination thereof. See Satarker and Nampoothiri, "Structural Proteins in Severe Acute Respiratory Syndrome Coronavirus-2," Arch Med Res. 2020 Aug;51(6):482-491. See, for example Figure 1A , Figure 1B , Figure 2A and FIG. 2C, which depict the spike / CD9 and nucleocapsid / CD fusion proteins for expressing spike or nucleocapsid antigens on the surface of exosomes.

[0336] In one embodiment, the antigen protein is the SARS-CoV-2 spike glycoprotein (also known as the spike protein or simply "spike"). The spike protein can be any variant of SARS-CoV-2, such as, for example, the Wuhan-1 strain, the Omicron variant (e.g., BA.2 variant), the Delta variant (e.g., B.1.617.2, AY.3, AY.103, AY.44, AY.43 variants, etc.), and the ε variant (e.g., B.1.427 or B.1.429 variants), or any variant now known or yet to be discovered. As used herein, unless otherwise specified, the term spike refers to any SARS-CoV-2 spike glycoprotein, chimera, or fragment thereof.

[0337] In some embodiments, the SARS-CoV-2 spike protein is the SARS-CoV-2 spike protein of the Wuhan-1 strain or the SARS-CoV-2 spike protein of the Delta variant; the furin protease-blocked trimer-stabilized form of the SARS-CoV-2 spike protein of the Wuhan-1 strain; the SARS-CoV-2 spike protein of the Wuhan-1 strain having the D614G amino acid change; the SARS-CoV-2 spike protein of the Wuhan-1 strain having the diproline substitution of 986KV987-to-986PP987 (S-2P); and / or the SARS-CoV-2 spike protein of the Wuhan-1 strain or equivalent (S-CSM) having the cleavage site mutation of 682RRAR685-to-682GSAG685.

[0338] Extracellular Vesicles Exhibiting Spike or Nucleocapsid Proteins

[0339] In one embodiment, the present invention provides extracellular vesicles that express (also referred to as “display”) spike protein or nucleocapsid on their surface, and the extracellular vesicles can be used as vaccines against various variants of SARS-CoV-2. The spike protein can be a Delta variant having any one or more of a trimer stabilization mutation, a prefusion conformation stabilization mutation (e.g., a diproline stabilization mutation), and a furin cleavage site mutation. See, e.g., Walls et al., “Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein,” Cell 180, 281-292, April 16, 2020; Wrapp et al., “Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation,” Science 367, 1260-1263 (2020) 13 March 2020; Kirchdoerfer et al., “Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis,” Sci Rep 8, 15701 (2018), doi.org / 10.1038 / s41598-018-34171-7; Pallesen et al., “Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen,” PNAS, E7348-E7357, published online August 14, 2017 pnas.org / cgi / doi / 10.1073 / pnas.1707304114; Juraszek, et al., “Stabilizing the closed SARS-CoV-2 spike trimer,” Nat Commun 12, 244 (2021), doi.org / 10.1038 / s41467-020-20321-x; Johnson, 2020; and Xiong 20200; and references therein.

[0340] Here, extracellular vesicles are produced that contain a C-terminal tetraspanin protein and the SARS-CoV-2 spike protein ( Figure 1A andFigure 1B ) or a synthetic fusion protein of SARS-CoV-2 nucleocapsid protein ( Figure 2A and Figure 2B ). Importantly, it should be noted that the nucleocapsid protein is a soluble protein that is not expressed at the viral surface. Therefore, an engineered fusion protein containing a four-transmembrane protein and other transmembrane domains can place the nucleocapsid protein on the vesicle surface to provide an accessible antigen for immunization (see Figure 2B ).

[0341] In one embodiment, exosomes expressing the spike protein or nucleocapsid protein on their surface are prepared from 293F cells expressing the spike / nucleocapsid protein. Turning to Figure 3A , in a specific exemplary embodiment, a packaging cell (300) is transfected with a plasmid encoding a spike or nucleocapsid-four transmembrane protein fusion protein (301a) and a plasmid encoding lentiviral structural proteins (302a and 303a). Lentiviral proteins (302b and 303b) are produced and incorporated into the fusion protein RNA to form a lentiviral vector (304) containing the fusion protein RNA. A host cell (311) is transduced with the lentivirus (304) carrying the fusion protein RNA, enabling the production of a SARS-CoV-2 spike or nucleocapsid-four transmembrane protein fusion protein (307a), which is sorted to the plasma membrane (307b) ( Figure 3B ) and the surface of exosomes produced by the transduced host cell ( Figure 4C , Figure 5A , Figure 5B and Figure 6C ).

[0342] Turning to Figure 4A - Figure 4C and Figure 5A and Figure 5B , exosomes are isolated from 293F cells carrying a spike-CD9 construct ( Figure 1A and Figure 1B ). Figure 4A Shows the size distribution of those exosomes from about 50 nm to about 270 nm, with a median of about 100 - 150 nm. Figure 4B Shows the expression of the spike-containing fusion protein in transduced 293F cells (lane 4), and the enriched expression / display 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 on those exosomes as determined by spike flow cytometry.

[0343] Transmission electron microscopy confirmed that the spike protein is expressed on the surface of exosomes derived from spike-CD9 transduced 293F cells ( Figure 5A and Figure 5B arrows point to spikes at the vesicle surface).

[0344] Transfer to Figure 6A - Figure 6C , exosomes were isolated from 293F cells carrying the nucleocapsid-CD9 construct ( Figure 2A and Figure 2B ). Figure 6A The size distribution of those exosomes from approximately 50 nm to approximately 270 nm is shown, with a median of approximately 100 - 150 nm. Figure 6B The expression of the nucleocapsid-containing fusion protein in exosomes derived from transduced 293F cells is shown (lane 5). Figure 6C Significant expression of the spike-containing fusion protein on those exosomes as determined by spike flow cytometry is shown. In one embodiment, the expression of the nucleocapsid-CD9 fusion protein is placed under the control of a tet-inducible promoter. Here, exosomes derived from uninduced but transduced host cells do not express nucleocapsid (see Figure 6C the second curve from the left).

[0345] In one embodiment, exosomes expressing the spike-CD9 fusion protein ( Figure 1A - Figure 1B and Figure 4A - Figure 4C ) are delivered to a subject by intramuscular injection according to a two-dose regimen (see, for example, FIG. 7). Also in another set of experiments, exosomes expressing the nucleocapsid-CD9 fusion protein ( Figure 2A - Figure 2B and Figure 6A - Figure 6C ) are delivered to a subject by intramuscular injection according to the same two-dose regimen (see, for example, FIG. 7).

[0346] Transfer to FIG. 7. On day 1, a 1× dose (i.e., 3E10 vesicles) or 10× dose (i.e., 3E11 vesicles) (710) of exosomes expressing the spike-CD9 fusion protein or the nucleocapsid-CD9 fusion protein is administered to subject mice by intramuscular injection. On day 14, blood is collected from the administered mice and the early humoral immune response is evaluated (720) (see Figure 8A and Figure 9A ). On day 21, the subject mice receive a second dose of exosomes expressing the subject spike or nucleocapsid (730). On day 35, blood is collected from the administered mice and the humoral immune response, including antibody production (see Figure 8B and Figure 9B ) and neutralizing antibody production (740), is evaluated. On day 40, splenocytes are collected from the administered mice and the cellular immune response is evaluated by ELISpot assay (750) (see Figure 8C , Figure 8D , Figure 10A and Figure 10B ).

[0347] Transfer to Figure 8A and Figure 8B , as early as two weeks after the first injection (Figure 8A ) and continuing for at least up to 35 days post - injection Figure 8B ), induces an immune response in the subject against exosomes expressing the COVID - 19 spike antigen (STX - S) at the humoral level. Further, using nanogram amounts of spike protein expressed on the exosomes and without the inclusion of an adjuvant, a robust and long - lasting anti - spike antibody response is elicited. Here, for example, as measured on days 14 and 35, in the absence of an adjuvant, as little as 10 ng of exosomes expressing the spike protein elicits a significant and useful antibody response. Further, compared to 32 ng of purified spike protein (without exosomes) with an adjuvant, 32 ng of exosomes expressing the spike protein without an adjuvant elicits a significantly ≥30 - fold higher antibody response. It is generally known in the art that prior art spike protein vaccines are administered in microgram amounts and with adjuvants, generating antibody responses similar to those observed with the nanogram amounts of exosomes expressing the spike protein antigen of the present invention.

[0348] Turning to Figure 8C and Figure 8D , as shown by ELISpot assays performed on splenocytes obtained on days 35 - 40, an immune response against STX - S is induced in the subject at the cellular level. Here, a dose of as little as 10 ng of exosomes expressing the spike antigen without an adjuvant elicits a significant CD4+ T - cell response against the spike protein in the subject, as shown in the IL4 ELISpot assay of the subject's splenocytes( Figure 8C ). Also here, 10 ng and 32 ng doses of exosomes expressing the spike antigen without an adjuvant elicit a significant CD8+ cytotoxic T - cell response against the spike protein in the subject, as shown in the IFNγ ELISpot assay of the subject's splenocytes( Figure 8D ).

[0349] Exosomes expressing the SARS - CoV - 2 nucleocapsid protein without an adjuvant at similar nanogram doses also elicit useful, robust, and significant humoral and cell - mediated immune responses( Figure 9A - Figure 9D ). The inventors envision that any or most viral antigens expressed on the surface of exosomes according to the present disclosure (such as, for example, influenza hemagglutinin, influenza neuraminidase, respiratory syncytial virus, and / or other viral glycoproteins or other proteins or fragments thereof) will elicit humoral and / or cell - mediated immune responses in recipient subjects.

[0350] Thus, in some embodiments, a low-dose immunogenic composition or vaccine is provided, the low-dose immunogenic composition or vaccine comprising an immunogenic dose of: viral antigen (e.g., SARS-CoV-2 spike or nucleocapsid) between 1 ng and 1 μg (including the endpoints), 1 - 900 ng, 1 - 800 ng, 1 - 700 ng, 1 - 600 ng, 1 - 500 ng, 1 - 400 ng, 1 - 300 ng, 1 - 200 ng, 1 - 100 ng, 1 - 90 ng, 1 - 80 ng, 1 - 70 ng, 1 - 60 ng, 1 - 50 ng, 1 - 40 ng, 5 - 100 ng, 5 - 90 ng, 5 - 80 ng, 5 - 70 ng, 5 - 60 ng, 5 - 50 ng, 5 - 40 ng, ≤100 μg, ≤50 μg, ≤25 μg, ≤20 μg, ≤10 μg, ≤1 μg, ≤900 ng, ≤800 ng, ≤700 ng, ≤600 ng, ≤500 ng, ≤400 ng, ≤300 ng, ≤200 ng, ≤100 ng, ≤90 ng, ≤80 ng, ≤70 ng, ≤60 ng, ≤50 ng, ≤40 ng, about 1 ng, about 1.5 ng, about 2 ng, about 2.5 ng, about 3 ng, about 3.5 ng, about 4 ng, about 4.5 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about 18 ng, about 19 ng, about 20 ng, about 25 ng, about 30 ng, about 35 ng, about 40 ng, about 45 ng, about 50 ng, about 55 ng, about 60 ng, about 65 ng, about 70 ng, about 75 ng, about 80 ng, about 85 ng, about 90 ng, about 95 ng, about 100 ng, about 105 ng, about 110 ng, about 115 ng, about 120 ng, about 125 ng, about 130 ng, about 135 ng, about 140 ng, about 145 ng, 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., SARS-CoV-2 spike or nucleocapsid).

[0351] Go to Figure 9A and Figure 9B , induce an immune response to exosomes expressing the COVID-19 nucleocapsid antigen (STX-N) at the humoral level in a subject, and detected at day 35 post-injection ( Figure 8A andFigure 8B )。In addition, the use of nanogram amounts of spike protein expressed on exosomes and without the inclusion of adjuvants elicits a robust and long-lasting anti-spike antibody response. Here, for example, as measured on day 35, as little as 3.2 ng of exosomes expressing nucleocapsid protein without adjuvant elicits a significant and useful antibody response ( Figure 9A , STX-N dose 2). Thus, in one embodiment, for a dose of approximately 3 ng / injection (dose 2, Figure 9A ), the complete immunization cycle (two i.m. injections) results in a significant 3-fold increase in IgG against the SARS-CoV-2 nucleocapsid protein (N) relative to PBS, up to a 10-fold increase at 10 ng / injection (dose 3, Figure 9B ).

[0352] Moving on to Figure 9C and Figure 9D , to characterize the T cell response to STX-N, the antigen-specific T cell response to the nucleocapsid protein was measured by an ELISpot assay on splenocytes obtained on day 40. Here, vaccination of subjects with STX-N elicits a multifunctional, antigen-specific T cell response against the SARS-CoV-2 nucleocapsid protein on day 40 (after the booster (second) injection). Administration of STX-N results in an effective IFNγ response ( Figure 9C and Figure 9D ). Here, the assessment of IFNγ-secreting cells in response to ex vivo nucleocapsid protein stimulation shows a 6-fold increase in the spleens immunized with STX-N ( Figure 9C and Figure 9D ), indicating a Th1-biased CD8+ T cell response. Since the nucleocapsid protein is not a surface protein and most likely will only be presented to immune cells after the virus is processed within infected cells, this response is of inestimable importance for the development of exosomes expressing immunogenic compositions with the potential to protect against a broad range of SARS-CoV-2 variants.

[0353] As in the humoral antibody response against the nucleocapsid protein ( Figure 9A , Figure 9B ), in this particular embodiment, administration of STX-N in the range of 3 ng to 10 ng (three orders of magnitude lower than existing protein subunit vaccines) without adjuvant achieves a significant CD8+ / IFNγ response against the nucleocapsid protein in subjects.

[0354] Moving on to Figure 10A - Figure 10C, administering picogram amounts of STX-S to subjects elicits effective neutralization of both the Delta and Omicron variants of SARS-CoV-2 induced by STX-S exosome injection. In one embodiment, sera from subjects administered approximately 3.2 ng of STX-S per injection on day 40 (dose 2, Figure 10A ) and sera from subjects administered approximately 9.8 ng of STX-S per injection on days 14 and 40 (dose 4, Figure 10A ) were tested for neutralizing antibodies against the SARS-CoV-2 Delta variant. Here, effective neutralizing activity was elicited by STX-S in each test sample ( Figure 10A ), comparable to SARS-CoV-2 Delta positive vaccine sera. STX-S demonstrated dose-dependent neutralization of the virus, estimated by the ability to protect infected cells from virus-induced cytopathic effects (compare dose 4 with dose 2 in sera on day 40, Figure 10A ).

[0355] Thus, according to one embodiment, STX-S engineered exosome vaccine induces neutralizing antibodies against the Delta spike in one or more subjects by day 14 (after a single i.m. injection). By day 40, i.e., ~4 weeks after STX-S boost, robust neutralization was observed in all subjects regardless of dose ( Figure 10A ). Here, administration of approximately 9 ng of the STX-S spike (dose 4, Figure 10A ) resulted in approximately 65%-75% neutralization of the Delta variant. Additionally, at doses of approximately 3-9 ng of spike delivered by STX-S exosomes, full immunization (i.e., initial injection and at least one booster or two i.m. injections) resulted in approximately 80%-85% neutralization against the Delta variant.

[0356] Delta variant neutralization induced by administration of STX-S in subjects elicited a response comparable to human control plasma (CoV02-Delta, plasma from patients with breakthrough Delta infections immunized with Moderna's mRNA vaccine), with complete neutralization responses at higher dilutions (see e.g., 1:320, day 40, STX-S dose 4) ( Figure 10A ).

[0357] Additionally, turning to Figure 10B and Figure 10C , sera samples from subjects given doses of approximately 9-10 ng of STX-S (here, the spike protein of STX-S is the Delta variant-STX-S δ ) were also tested for neutralizing antibodies against the SARS-CoV-2 Omicron variants (Omicron BA.1 and BA.5.2.1). As Figure 10CAs shown in FIGS. 10D, after full immunization (i.e., at least 2 i.m. injections) with a dose of approximately 9 - 10 ng of Delta spike delivered by STX-S exosomes, strong cross-neutralization was observed for sera obtained from STX-S δ -treated subjects. In this embodiment, approximately 84% neutralization of the Omicron BA1 variant ( Figure 10B ) and 16% to 97% neutralization of the Omicron BA5 variant ( Figure 10C ) were achieved. Thus, in some embodiments, administration of an immunogenic composition containing single variant spike STX-S exosomes provides a certain level of protective immunity against other SARS-CoV-2 variants.

[0358] Turning Figure 11 to, in a murine model, the antibody response to the spike from STX-S persists for at least about 6 months and is expected to persist beyond 6 months. As Figure 11 shown, mice dosed with 10 ng STX-S and boosted with 7 ng STX-S or 3 ng STX-S exhibited strong IgG responses to the spike antigen for at least 166 days after the initial dose.

[0359] In addition to their excellent immune response elicitation, both spike-exosome vaccines and nucleocapsid-exosome vaccines exhibit several other advantages over currently available vaccines. First, the exosome-based vaccines of the present invention deliver antigens through a fully endogenous autologous lipid bilayer that can be easily integrated into the host cell membrane and facilitate engineered antigen presentation to immune cells. Membrane-bound antigens can be easily presented to circulating immune cells to rapidly activate the response, while free antigens contained within the exosomes can additionally be processed by the lysosomal system and activate cytotoxic T lymphocyte responses. Thus, the utilization of a natural delivery system promotes efficient delivery and response compared to synthetic lipid nanoparticle technology.

[0360] Second, spike and nucleocapsid exosome vaccines are protein-based vaccines, such that the antigens are readily available to the subject's immune system without the need for translation in host cells as required for RNA-based vaccines. Incomplete translation and / or incorrect folding of the mRNA encoding the spike protein or nucleocapsid protein limit the amount of antigen available after vaccination, making the immune response highly variable and reducing efficacy.

[0361] Third, the exosome-based spike and nucleocapsid fusion protein vaccine of the present invention does not require an adjuvant or synthetic lipid nanoparticles (LNPs) for delivery and immune response. Traditional protein-based vaccines require adjuvants (such as, for example, aluminum salts and squalene oil-in-water emulsion systems MF59 (Novartis) and AS03 (GlaxoSmithKline)) (see, for example, Wong, S.S. and R.J. Webby, “Traditional and new influenza vaccines,” Clin Microbiol Rev, 2013.26(3): pp. 476-92) to enhance and coordinate the immune response and affect the affinity, specificity, magnitude, and functional profile of B and T cell responses. Current mRNA vaccines do not use adjuvants, which in combination with the need for mRNA translation prior to antigen availability, can also reduce long-term efficacy. Currently approved COVID vaccines use LNPs to deliver the mRNA of the protein or adjuvant, and undesirable side effects have been reported.

[0362] Fourth, the exosome spike fusion protein and nucleocapsid fusion protein immunogenic compositions of the present invention exhibit greater immunogenic efficacy at doses significantly lower (i.e., three orders of magnitude lower) than currently available LNP mRNA and protein vaccines. For example, clinically approved protein candidate vaccines use approximately 5 μg to 25 μg of antigen in combination with an adjuvant to induce immunity (see, e.g., Formica et al., “Different dose regimens of a SARS-CoV-2 recombinant spike protein vaccine (NVX-CoV2373) in younger and older adults: A phase 2 randomized placebo-controlled trial,” PLoS Med, 2021.18(10): p.e1003769; Sun et al., “Development of a Recombinant RBD Subunit Vaccine for SARS-CoV-2,” Viruses, 2021.13(10); Worzner et al., “Adjuvanted SARS-CoV-2 spike protein elicits neutralizing antibodies and CD4 T cell responses after a single immunization in mice. EBioMedicine, 2021.63: p. 103197; and references therein). Here, immunological data from subjects immunized with spike fusion-exosomes or nucleocapsid fusion-exosomes elicit complete immunity, which is associated with high antibody levels, strong virus neutralization, broad variant activity, and both B and T cell memory, with approximately 1 / 1000 (i.e., nanogram amounts versus microgram amounts) of the amount of protein antigen currently administered in currently clinically approved vaccines.

[0363] Fifth, an additional limitation of available vaccines is the need for refrigeration and the short shelf-life of the product at higher temperatures. Due to the high stability of exosomes at physiological pH and temperature, exosomes, including display exosomes, can be stored at 4°C for longer periods. Exosome compositions expressing spike fusions or nucleocapsid fusions can also be lyophilized for long-term storage at convenient temperatures (see, e.g., U.S. Patent Application No. 2017 / 0360842A1).

[0364] Thus, here, when the spike protein or the nucleocapsid protein is delivered via exosomes, a strong CD8+ T cell response as well as a strong B cell response is induced, as shown by IgG production and potentially neutralizing antibodies. Without wishing to be bound by theory, this result can be explained by the role of extracellular vesicles in intercellular communication and antigen presentation. In particular, multiple copies of the spike protein ( Figure 5A and Figure 5B ) or the nucleocapsid protein can be present on the surface of the vesicles, promoting cross-linking with the B cell receptor. In addition, the spike protein in the extracellular vesicle-based vaccine can indirectly activate B cells and CD8(+) T cells via antigen cross-presentation.

[0365] It is also envisioned that the exosomes of the present invention expressing the fusion protein can be engineered to express an antigen of interest to target new COVID variants. The antigen of interest can be easily exchanged and adapted as needed. The antigen can be expressed as a fusion protein (e.g., a chimera), such as an exosome expression or display domain fused to an antigen domain, to enable the display of the antigen on the exosome for delivery to the host subject's immune system.

[0366] It is also envisioned that exosomes can be engineered to selectively target an organ or tissue of interest and allow for the safe and targeted delivery of an antigen to a specific immune subsystem to elicit a specific type of response in a subject.

[0367] Here, turning to Figure 12 , HEK293-hACE2 cells were seeded into 24-well plates and contacted with approximately 1E7 to 1E12 exosomes / mL in serum-reduced 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 better uptake of exosomes expressing spike at concentrations approximately 2.5 to 3-fold lower compared to "wild-type" 293F wt exosomes. See also Table 1.

[0368] Table 1: Uptake of wild-type and spike-expressing exosomes by HEK293-hACE2 cells

[0369]

[0370]

[0371] Thus, exosomes expressing spike can be used to deliver any drug cargo to the lung or other tissues expressing spike receptors such as ACE2. Additionally, the data also demonstrated the fact that the disclosed exosomes expressing recombinant fusion proteins on their surface retain the native structure of the various components of the fusion protein. For example, the spike protein polypeptide has sufficient native structure to engage the native target, i.e., ACE2.

[0372] Examples

[0373] The pandemic emergency has revealed the need for effective, reliable, and broader vaccines to combat evolving viruses and halt their spread. While COVID-19 mRNA vaccines have played an important role in reducing the casualties of SARS-CoV-2 infections in the emergency, their reduced ability to protect against new VOCs, combined with the need for multiple injections to maintain protection, has prompted the scientific community to look for new approaches.

[0374] SARS-CoV-2 has indeed taught us that a successful vaccine will generate a strong antibody response, with neutralizing antibodies and a strong T cell response capable of combating viral infections more broadly and in a more timely manner, with the fewest number of injections. These criteria can be met by exosome-based vaccines and a "multivalent" approach.

[0375] Here, the following examples have shown that exosomes can be used to deliver viral proteins for immunization: the disclosed STX platform has generated two candidate vaccines (STX-S and STX-N), which, by delivering nanogram amounts of protein on the surface of exosomes, independently and in combination (STX-S+N) induced a strong immune response against two SARS-CoV-2 proteins (spike and nucleocapsid) with a single injection in two different animal models. No adjuvant was required, 100-fold less protein was used, and no competition between the proteins was observed.

[0376] "Multivalent" or "combination" vaccines have multiple advantages. First, they require fewer injections, which will positively appeal to the population, thus increasing the percentage of the vaccinated population and having broader epidemiological benefits. Therefore, fewer injections mean lower overall budgets and costs. Of course, there are limitations to consider. First, the selection of antigens: It is crucial that the selected antigens retain strong immunogenicity in the combination, and the antigens hopefully have conserved sequences across species and an ideally low mutagenesis rate. Among them, immunogenic interference is key: Minimal competition or no competition should be observed. According to the exemplary STX-S+N, the multivalent vaccine shows the same efficacy intensity as the single product, with the antibody payload increasing by thousands of times. Additionally, STX-S+N elicits both quantitative and qualitative immune responses: In response to the administration of the disclosed exosome vaccine STX-S+N, a consistent increase in the amount of antibodies produced, the presence of protection described by neutralizing antibodies, and the engagement of T cells are all observed. Importantly, no harmful side effects were recorded: Both mice and rabbits showed no changes in body weight or blood tests, and no changes at the tissue level, indicating an overall safety profile.

[0377] The data provided herein indicate that exosomes are ideal vehicles for vaccination: They can safely deliver the antigen of interest (foreign protein) in a manner that mimics natural viral infection. Exosome-based vaccines constitute an innovative approach to the design of effective virus-free human vaccines. Yoo et al. ("Possibility of exosome-based coronavirus disease," 2019 vaccine (Review). Mol Med Rep, 2022. 25(1)) observed that exosomes or extracellular vesicles can generally support vaccination needs beyond traditional strategies: Compared with viral or vector methods, exosomes themselves are not immunogenic but are carriers that retain the original conformation, three-dimensional structure, and modified proteins, all of which are embedded in the lipid bilayer of their membrane and are themselves ready to be effectively presented to the immune system.

[0378] The strong T cell responses elicited 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 VOCs, T cell populations are able to cross-react with them and confer protection. Here, spike- and Ncap-specific T cell responses were observed: the highly conserved Ncap can further increase and broaden the efficacy of the STX vaccine, eliciting additional immune responses that are not impaired by natural mutations in surface proteins. This suggests that in situations where new variants of concern (VOCs) escape the neutralizing antibody barrier, T cell responses can cross-react and limit infection.

[0379] Other groups have reported a multivalent vaccine for SARS-CoV-2 that uses a more virulent approach of co-expressing the SARS-CoV-2 N and S proteins on a VSV virus (e.g., O'Donnell, K.L. et al., “Protection from COVID-19 with a VSV-based vaccine expressing the spike and nucleocapsid proteins,” Front Immunol, 2022.13: p. 1025500) or via an 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 the multi-protein vaccine broadens the efficacy to distal organs, reducing not only the viral load in the respiratory system but also the viral load in the distal brain (see, e.g., Matchett, W.E. et al., “Cutting Edge: Nucleocapsid Vaccine Elicits Spike-Independent SARS-CoV-2 Protective Immunity,” J Immunol, 2021.207(2): pp. 376 - 379). Nucleocapsid-specific immunity plays a non-essential role during SARS-CoV-2 infection. While antibody responses can block the initial entry of the virus at the proximal site of infection, T cell responses play a key role in controlling the spread of infection, secondary infection, and subsequent spread of the virus to distal sites, providing a synergistic antiviral effect by killing virus-infected cells and further reducing the spread of the virus to peripheral organs.

[0380] A single-dose dual-antigen vaccine effective against multiple SARS-CoV-2 VOCs is disclosed, which has a broader immune capacity and can be used as a booster for existing immunity generated by previously approved vaccines. The disclosed StealthX vaccine technology (STX) uses exosomes to deliver nanogram amounts of viral antigens to elicit a strong and broader immune response without any adjuvant.

[0381] Example 1: Cell Lines

[0382] Human embryonic kidney 293T cells (293T) were purchased from ATCC (CRL-3216). High glucose, Glutamax TM, Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum maintained 293T cells in culture. The 293T cells were incubated at 37 °C / 5% CO2. FreeStyle TM 293F cells (Gibco, 51-0029) were purchased from ThermoFisher, Waltham, MA. 293F cells were used as the parental cell line to generate a stable cell line expressing the spike of SARS-CoV-2 Delta spike: Stealth X-spike cells (STX-S). 293F and STX-S cells were maintained in a MULTITRON incubator (Infors HT, Sulzemoos, DE) at 37 °C, in an 80% humidified atmosphere with 8% CO2, rotating at 110 rpm on an orbital shaker platform.

[0383] Example 2: Lentiviral Vectors

[0384] The lentiviral vectors for expressing the SARS-CoV-2 spike (Delta variant B.1.617.2, NCBI accession number OX014251.1, available at www.ncbi.nlm.nih.gov / nuccore / OX014251.1 on August 15, 2023) and the SARS-CoV-2 nucleocapsid (NCBI accession number OP359729.1, available at www.ncbi.nlm.nih.gov / nuccore / OP359729.1 on August 15, 2023) were designed and synthesized from Genscript together with two packaging plasmids (pMD2.G and psPAX2). According to the manufacturer's instructions, lentiviral particles for transduction were produced by transfecting 293T cells with pMG.2 (Genscript, GenScriptBiotech, Piscataway, NJ), psPAX2 (Genscript), and STX-S_pLenti (Genscript) expressing the spike or STX-N_pLenti (Genscript) expressing the nucleocapsid in a ratio of 5:5:1 using Lipofectamine TM 3000 (ThermoFisher Scientific, Waltham, MA). Spike and nucleocapsid lentiviral particles were collected 72 hours after transfection and used to transduce 293F parental cells to generate STX-S and STX-N, respectively.

[0385] Example 3: Flow Cytometry

[0386] The spike SARS-CoV-2 protein expression on the surface of STX cells was measured using standard flow cytometry methods. Briefly, 250K STX cells were aliquoted, pelleted, and resuspended in 100 uL of eBioscience TM Flow Cytometry Staining Buffer (ThermoFisher). According to the manufacturer's protocol, the cells were incubated for 30 min at room temperature (RT) in the dark in the presence of anti-spike antibody (Abcam, clone 1A9, ab273433, Abcam, Cambridge, UK) or anti-nucleocapsid antibody (Abcam, ab281300) labeled with Alexa -647 (Alexa 647 Conjugation Kit (Fast)-Lightning- )(Abcam, ab269823). After incubation, the STX cells were washed with eBioscience TM Flow Cytometry Staining Buffer (ThermoFisher, catalog number 00-4222-57), resuspended in PBS, and analyzed on a CYTOFLEX S flow cytometer (Beckman Coulter, Brea, California). The data were analyzed by FLOWJO (Becton, Dickinson and Company, Franklin Lakes, NJ).

[0387] Example 4: Cell Sorting

[0388] Cell sorting was performed at the Flow Cytometry Facility at the Scripps Research Institute (San Diego, CA). To enrich the spike-positive population, STX-S cells were flow cytometry stained as described above and then underwent cell sorting (Beckman Coulter MoFlo Astrios EQ) to generate pooled STX-S. Pooled STX-S was used in the examples provided herein unless otherwise indicated.

[0389] Example 5: STX Exosome Production.

[0390] The STX-S and STX-N cells were cultured in FREESTYLE medium (ThermoFisher, 12338018) in a MULTITRON incubator (Infors HT) at 37 °C, in an 80% humidified atmosphere with 8% CO2, on an orbital shaker platform. Subsequently, the cells and cell debris were removed by centrifugation, while microvesicles and other extracellular vesicles larger than ~220 nm were removed by vacuum filtration. Next, exosomes were isolated using filtration and size exclusion. Briefly, the supernatant was subjected to concentration filtration through a Centricon Plus-70 centrifugal filter unit (Millipore, UFC710008, Millipore Sigma, St. Louis, MO), and then to size exclusion chromatography (SEC) using a qEV original SEC column (Izon, SP5, Izon Science, Christchurch, NZ).

[0391] Example 6: Nanoparticle Tracking Analysis

[0392] According to the manufacturer's instructions, exosome size distribution and concentration were determined using ZetaView nanoparticle tracking analysis (Particle Metrix, Inning am Ammersee, DE). The exosome samples were diluted in 0.1 μm filtered 1× PBS (Gibco, 10010072) to fall within the optimal operating range of the instrument.

[0393] Example 7: Protein Expression

[0394] 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), quantified using a BCA assay (ThermoFisher Scientific, 23227), and run for detection. For detection of the spike protein, the separation module 12 - 230 kDa was used following the manufacturer's protocol. Briefly, 0.8 μg of sample and protein standards were run in each capillary, probed with an anti-mouse Ms-RD-SARS-COV-2 antibody (MAB105401, 1:10 dilution; R&D Systems, Minneapolis, MN) or an anti-rabbit nucleocapsid antibody (NBP3-00510, 1:100 dilution; Novus Biologicals, Centennial, CO), and subsequently probed with a secondary antibody provided in the JESS kit (using a pure HRP substrate).

[0395] Example 8: TEM Imaging for Characterizing STX Exosome Morphology

[0396] STX-S and STX-N exosome samples were negatively stained onto copper grids with a carbon film coating and imaged by TEM at the Electron Microscopy Core Facility at UC San Diego (San Diego, CA). Briefly, samples were glow discharged, stained with 2% uranyl acetate, dried, and then imaged. The grids were imaged on a JEM-1400Plus (JEOL Ltd, Japan) at 80 kV and 48 uA. Images were taken at a magnification of 12K - 80K with a resolution of 4k × 4k pixels.

[0397] Example 9: CD81 Bead - Assay

[0398] Mix STX-S or 293F parental exosomes with anti-CD81 labeled magnetic beads (ThermoFisher, 10622D) for 2 hours at room temperature (RT), and wash twice with PBS using a magnetic stand. Next, incubate the bead-exosomes with directly conjugated Alexa Fluor 647 anti-spike antibody (see flow cytometry above), FITC anti-CD81 antibody (BD Biosciences, 551108, Franklin Lakes, NJ), or FITC mouse IgG, κ isotype control (BD Biosciences, 555748) for 1 hour at RT, followed by two PBS washes. 293F exosomes are used as a negative control for spike expression, and the isotype antibody is used as a negative control for CD81 expression. Samples are analyzed on a CytoFlex S (Beckman Coulter) flow cytometer, and the data are analyzed by FLOWJO.

[0399] Example 10: Mouse Studies

[0400] To examine the efficacy of STX exosomes, age-matched BALB / c mice (female, 8 - 10 weeks old) are anesthetized with isoflurane and receive bilateral intramuscular injections of 1) PBS, 2) STX-S, 3) STX-N, or 4) STX-S+N exosomes (50 μL per leg, 100 μL total). Booster injections are performed on day 21. The health changes of the mice are closely monitored, and the body weights are recorded every two weeks. Blood collection is performed on days 14 and 35. Blood (∼50 - 500 μl) is collected from the submandibular vein and processed for plasma separation after centrifugation at 4000 rpm for 5 min at 4°C. For comparative studies, mice are injected with either 1) soluble protein conjugated to an adjuvant (Alhydrogel, 100 μg / dose, vac-alu-250, InvivoGen, San Diego, California), or 2) an equivalent amount of SARS-CoV-2 protein delivered by STX exosomes. Blood is collected 2 weeks after injection and tested for IgG against SARS-CoV-2. The timeline of the mouse study is outlined in Figure 7B Collect mouse tissues (brain, salivary gland, heart, lung, liver, spleen, kidney, gastrointestinal tract (GI), and skeletal muscle (injection site)) and fix in 10% neutral buffered formalin. Sections are stained with hematoxylin and eosin and analyzed for changes.

[0401] Example 11: Rabbit Studies

[0402] To evaluate the potential toxicity and host immune response of the subject STX-S+N vaccine, age-matched rabbits (male / female, New Zealand White, 2.5 - 3.0 kg) received intramuscular (IM) injection of the STX-S+N vaccine at the expected human dose (10 - 200 ng total protein in 0.5 mL). Control animals received phosphate-buffered saline (PBS). A booster injection was given on day 14. The health changes of the rabbits were closely monitored and body weights were recorded. Blood collection was performed once a week on days 0, 7, 14, 21, and 28, and processed for plasma separation after centrifugation at 4000 rpm for 5 min at 4°C. The timeline of the rabbit study is outlined in Figure 7C . Rabbit tissues were collected at the end (day 28), fixed in 10% neutral buffered formalin and processed for pathological changes.

[0403] Example 12: IgG ELISA

[0404] Mouse and rabbit IgG antibodies against SARS-CoV-2 spike or nucleocapsid were measured by enzyme-linked immunosorbent assay (ELISA) at room temperature (RT) using pre-coated 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 in sample buffer (RayBiotech) and added in triplicate to the antigen-coated wells and incubated at RT for 2 h on an orbital shaker (200 rpm). Commercially available antibodies against spike (S1N-S58, Acro Biosystems, Newark, Delaware) or nucleocapsid (NUN-S47, Acro Biosystems) were used as positive controls. The plates were washed 3 times with wash buffer and incubated with HRP-conjugated goat anti-mouse secondary antibody (115-035-003, dilution 1:5000, Jackson ImmunoResearch) or anti-rabbit secondary antibody (111-035-003, dilution 1:5000, Jackson ImmunoResearch, West Grove, PA) diluted in assay buffer (RayBiotech) at RT for 1 h. After washing 3 times, the plates were developed using TMB substrate (RayBiotech). After incubation for 15 min, the reaction was terminated by adding STOP solution and the absorbance at 450 nm was recorded using a BioTeck Gen5 plate reader (Agilent Technologies, Santa Clara, California). The endpoint titer was calculated as the dilution that emitted a light density 4× above the PBS control group.

[0405] Example 13: Neutralizing Antibodies Against Delta SARS-CoV-2

[0406] Vero E6 cells were used to evaluate the neutralizing activity of the test article against replication-competent SARS-CoV-2 Delta variant (B.1.617.2). The samples were pre-incubated with the virus at 37 °C for 1 hour and then added to the cells. After pre-incubation of the plasma / virus samples, the cells were challenged with the mixture. During the course of infection (96 h), the samples were present in the cell culture, at which time 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 the test article against SARS-CoV-2. The test article was evaluated in duplicate using a two-fold serial dilution starting at a 1:40 dilution (total of 8 dilutions). Control wells included "CoV02-Delta" and GS-441524, which were tested as singlet data points on each plate. "CoV02-Delta" is convalescent plasma from an individual who had previously received two doses of the Moderna COVID-19 vaccine (Moderna, Cambridge, MA) prior to infection with the Delta variant. GS-441524 is an antiviral drug from Gilead Sciences (Foster City, California). CoV-01 is plasma from a patient who received two doses of the Moderna COVID-19 vaccine and had no prior history of SARS-CoV-2 infection. The NT50 value of the test article was determined using GraphPad Prism software (GraphPad Software, Boston, MA).

[0407] Example 14: Neutralizing Antibodies Against Omicron (BA.1 and BA.5.2.1) SARS-CoV-2

[0408] The neutralization assay was performed using anti-NP immunostaining (Omicron BA.1 and BA.5.2.1). Briefly, samples were pre-incubated with virus at 37 °C for 1 h and then added to Vero E6 cells. After incubation, the medium was removed and the cells were then challenged with the SARS-CoV-2 / test-item pre-incubation mixture. The amount of the virus inoculum was previously titrated to produce a linear response inhibited by antiviral drugs with known activity against SARS-CoV-2. The cell medium containing the virus inoculum was not removed after virus adsorption, and the test item and virus were maintained in the medium during the assay (48 h). Subsequently, the degree of infection was monitored by incubating the cells with a monoclonal test item against the SARS-CoV-2 nucleocapsid (NP). After incubation with a polyclonal test item against human IgG conjugated to horseradish peroxidase (HRP-goat anti-mouse IgG), the amount of viral antigen in the infected cells was estimated. The reaction was monitored using a colorimetric readout (absorbance at 492 nm). The test item was evaluated in duplicate using a two-fold serial dilution starting at a 1:40 dilution. Control wells included GS-441524 (Gilead Sciences), which was tested as a single data point on each plate.

[0409] Example 15: Splenocyte Isolation

[0410] The spleen was processed to isolate single cells by mechanically disrupting the spleen pouch using a syringe plunger and passing it through a nylon cell strainer with a mesh size of 0.040 mm to remove tissue debris. Red blood cells were lysed using ammonium chloride potassium (ACK) buffer (A1049201, ThermoFisher), and spleen cells were collected by centrifugation at 300 × g for 5 min. The cell pellet was resuspended in complete RPMI 1640 medium (FG1215, Millipore Sigma Aldrich).

[0411] Example 16: ELISPOT

[0412] Spleen cells were isolated by mechanically disrupting the spleen capsule and seeded at a concentration of 5E5 cells / well and incubated for 24 hours in the presence or absence of 10 μg / ml of SARS-CoV-2 spike (S1N-C52H4, AcroBiosystems, Newark, Delaware) or nucleocapsid (NUN-C5227, AcroBiosystems). Commercially available ELISPOT plates were used to evaluate 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). Assays were performed according to the manufacturer's guidelines. Plates were analyzed using an ELISPOT reader S6ENTRY (Immunospot, Cellular Technology Limited).

[0413] Example 17: ELISA for Protein Quantification

[0414] According to the manufacturer's instructions, pre-coated ELISA plates (ELV-COVID19S1, RayBiotech) were used to measure the spike protein levels on exosomes by ELISA at RT. According to the manufacturer's instructions, pre-coated ELISA plates (Legend Max SARS-CoV2 nucleocapsid protein ELISA kit, 448007, BioLegend, San Diego, California) were used to measure the nucleocapsid levels on exosomes by ELISA at RT. Briefly, samples and standards were loaded onto the pre-coated plates and incubated at RT on an orbital shaker (200 rpm) for 2 - 2.5 hours. The plates were washed and incubated with biotin-conjugated detection antibody for 1 hour at RT, followed by incubation in streptavidin solution for 45 minutes. After washing, the plates were developed using TMB substrate. After 30 minutes of incubation, the reaction was terminated by adding STOP solution and the absorbance at 450 nm was recorded using a BIOTECK Gen5 plate reader (Agilent). For nucleocapsid, the absorbance at 450 nm and 570 nm was recorded after 10 min incubation in TMB substrate. For analysis, the absorbance at 570 nm was subtracted from the absorbance at 450 nm and a standard curve was constructed using the optical density (OD).

[0415] Example 18: Pathology

[0416] Mouse tissues (brain, salivary gland, heart, lung, liver, spleen, kidney, gastrointestinal (GI) tract, and skeletal muscle (injection site)) were collected and fixed in 10% neutral buffered formalin. Sections were stained with hematoxylin and eosin and analyzed for changes.

[0417] Example 19: Statistical Analysis.

[0418] Data were analyzed using Excel and GraphPad Prism 9.1 and presented as mean ± sem. One-way ANOVA or two-tailed t-tests with post hoc correction for multiple comparisons were applied as needed.

[0419] Example 20: Expression of SARS-CoV-2 Proteins on the Surface of STX-Producing Cells and Exosomes.

[0420] STX cells were generated by lentiviral transduction and the expression of SARS-CoV-2 proteins on the cell surface was evaluated by flow cytometry ( Figure 13 ). As shown, compared to parental 293F cells, STX cells showed >95% increased expression of spike ( Figure 13 A) and nucleocapsid ( Figure 13 B).

[0421] STX exosomes were purified from engineered 293F cell culture supernatants using the laboratory-scale purification technique described herein (Example 5). The purified STX-S and STX-N exosomes showed expected mean diameters of 144.6 nm and 140.4 nm, respectively ( Figure 4A 、 Figure 6A and Figure 14 ) and expected polydispersity indices (PDIs) of <0.2 (0.152 and 0.129, Figure 14 ).

[0422] STX exosomes were analyzed by TEM imaging. As Figure 15 A- Figure 15 B shows, typical exosome size and morphology were observed with round, smooth nanoparticles with a visualized lipid bilayer detected. Importantly, spike protrusions were visible on the surface of STX-S nanoparticles, indicating the presence of spike protein on the exosomes ( Figure 5A 、 Figure 5B and Figure 15 A). For nucleocapsid, a characteristic lipid bilayer was observed, which resulted in a thicker appearance than the initial exosomes, indicating accumulation of the particles in the exosome membrane ( Figure 15 B).

[0423] As described herein, the expression of spike and nucleocapsid was verified on cell lysates and exosomes using Protein Simple's Jess automated Western blot. Spike protein was detected in both STX-S cells and exosomes, where spike protein was enriched in the exosome sample ( Figure 4B and Figure 16 ). STX-N engineered cells and exosomes expressed abundant levels of nucleocapsid ( Figure 6B and Figure 17 ). In addition, the SARS-CoV-2 protein spike and Ncap were detected on the exosome membrane using a bead-based CD81 assay, with more than >75% expression together with the exosome-specific marker CD81.

[0424] The concentrations of spike antigen in STX-S exosomes and nucleocapsid antigen in STX-N exosomes were further quantified by ELISA. The final STX-S product at 1×10^12 (1E12) exosomes / mL contained an average of 253.77 ng of spike, while the final STX-N product at 1×10^12 (1E12) exosomes / mL contained an average of 40.59 ng of nucleocapsid.

[0425] Example 21: STX-S and STN-N Alone Induce Strong Immunity in the Absence of Adjuvant.

[0426] To verify the ability of STX-S and STX-N exosomes to induce immune responses, spike and nucleocapsid, mice were immunized with 10 ng of the exosome preparations STX-S and STX-N. As a comparison to show the robustness of exosome delivery, 10 ng of spike or Ncap recombinant protein conjugated with an adjuvant (Alhydrogel, InvivoGen) was delivered. PBS was used as a control. Blood collected 2 weeks after the booster injection (second injection) showed that both STX-S and STX-N vaccines increased antibody production against spike and nucleocapsid, respectively, in all animals. There was no statistical difference between the spike protein or Ncap protein combined with the adjuvant and the PBS control (negative control), and no antibody production was observed ( Figure 18 and Figure 19 ).

[0427] Example 22: The STX-S+N vaccine induces strong immunity against SARS-CoV2 proteins in mice.

[0428] The immune responses of the multivalent vaccine obtained by the combination of STX-S and STX-N exosomes were evaluated. STX-S+N was administered to mice by two i.m. injections at three different doses (Table 2). The second i.m. injection, i.e., the booster injection, was delivered after a 3-week interval. PBS was used as the negative control in the study.

[0429] Table 2. Concentrations of spike and Ncap used in the study.

[0430] Spike ng / injection Ncap ng / injection Dose 1 25 2.5 Dose 2 10 4 Dose 3 3 9

[0431] Immunization with the STX-S+N vaccine was evaluated by quantifying antibodies against Ncap and spike Ncap ( Figure 20 A- Figure 20 B and Figure 21 A- Figure 21 B), respectively). An increase in 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 up to 30-fold increases in IgG against the spike, with no significant differences overall between the three doses. After full immunization, dose 1 (25 ng / spike) and dose 2 (10 ng / spike) resulted in 1500-fold increases in antibodies against the spike, while dose 3 (3 ng / spike, significantly lower spike amount) resulted in a 280-fold increase. On the other hand, a dose response for Ncap was observed. A 1.5-fold increase was observed for the low dose 1 (2.5 ng / Ncap), a ~4-fold increase for dose 2 (4 ng / Ncap), and up to a 7-fold increase for dose 3 (9 ng / Ncap). After completion of the immunization cycle (2 i.m. injections), no significant differences were observed between the doses, with a 24-43-fold increase in IgG against Ncap observed in STX-S+N-treated mice.

[0432] To characterize the T cell response to STX-S+N, antigen-specific T cell responses were measured by ELISpot ( Figure 22 ). Vaccination with STX-S+N elicited a multifunctional, antigen-specific T cell response. Spleen cells were isolated from animals on day 35 (2 weeks after the booster (second) injection) and evaluated using ELISpot plates pre-coated with IFNγ. As described above, PBS was used as a control in the study. Baseline expression was compared to stimulation with 10 μg / ml of spike or Ncap protein (AcroBiosystem). Although baseline IFNγ responses were comparable between the groups, ex vivo evaluation of cells secreting IFNγ in response to spike ( Figure 22 A) or Ncap ( Figure 22 B) stimulation showed a strong increase in spleens immunized with the STX-S+N vaccine, indicating a Th1-biased CD8+ T cell response. After spike stimulation, a mean 7-fold increase in IFNγ response was observed despite the STX-S+N dose administered ( Figure 22 A). 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 a ~3-fold increase in mice receiving dose 2 (4 ng Ncap) or dose 3 (9 ng Ncap) ( Figure 22 B).

[0433] Example 23: The STX-S+N vaccine induces strong immunity against SARS-CoV2 proteins in rabbits.

[0434] The immune response to the STX-S+N vaccine using clinically relevant doses was evaluated in rabbits. The STX-S+N combination vaccine was administered to rabbits by two i.m. injections at two different doses (Table 3). The second i.m. injection, i.e., the booster injection, was delivered after a 2-week interval. PBS was used as a control in the study.

[0435] Table 3. Concentrations of spike and Ncap used in the study.

[0436] Spike ng / injection Ncap ng / injection Dose 1 125 10 Dose 2 50 20

[0437] An increase in antibody production was detected as early as one week after the first injection and continued to increase after the booster injection ( Figure 7C ). After completion of the immunization cycle (two i.m. injections), an up to 3600-fold increase in IgG against spike and an up to 170-fold increase in IgG against Ncap were observed in STX-S+N-treated rabbits ( Figure 23 A) and ( Figure 23 B), respectively. No significant differences were observed between the doses in terms of antibody production against either antigen.

[0438] The immunity to the STX-S+N vaccine was further evaluated by assessing neutralizing antibodies against SARS-CoV-2 variants ( Figure 24 A- Figure 24 C). Neutralizing antibodies against the SARS-CoV-2 Delta variant were tested in the plasma of rabbits from dose 2 (50 ng S and 20 ng N, 8 animals) and 2 PBS controls. In all analyzed animals, STX-S+N elicited effective neutralizing activity. Importantly, STX-S+N in rabbits induced a response comparable to that of human control plasma (COV-02-Delta, plasma from patients with breakthrough Delta infections who were fully immunized with Moderna's mRNA vaccine), with complete neutralizing responses at higher dilutions (i.e., 1:320, range 116.74%-881.47%; mean: 96.22% ± 8.9%) ( Figure 24 A). In addition, STX-S+N performed better in rabbits than the CoV-01 control (plasma from patients who received two doses of the Moderna covid-19 vaccine and had no prior history of SARS-CoV-2 infection).

[0439] Additionally, neutralizing antibodies against the SARS-CoV-2 Omicron variants (Omicron BA.1 and BA.5.2.1) were tested in the same samples. As shown in Figure 24 B and Figure 24 As shown in C, strong cross-neutralization was observed for rabbits treated with STX-S+N, with an average neutralization of 75% (range 40.7% - 100%) achieved for Omicron BA.1( Figure 24 B), and a range of 10% to 91% achieved for Omicron BA5( Figure 24 C). These data indicate that protein-based vaccines delivered by exosomes, specifically STX-S+N, can generate a broader protective effect against SARS-CoV-2 variants. In all assays, rabbits receiving PBS showed no neutralization.

[0440] The T cell responses to STX-S+N immunization were measured by ELISpot( Figure 25 A and Figure 25 B). Vaccination with STX-S+N elicited multifunctional, antigen-specific T cell responses. Spleen cells were isolated from the animals at day 28 (2 weeks after the booster (second) injection) and evaluated using ELISpot plates pre-coated with IFNγ. As described above, PBS was used as a control in the study. Baseline expression was compared to stimulation with 10 μg / ml spike or Ncap protein (AcroBiosystem). Although the baseline IFNγ responses were comparable between groups, stimulation with spike( Figure 25 A) or Ncap( Figure 25 B) led to a strong increase in IFNγ production in the spleens of rabbits immunized with the STX-S+N vaccine, indicating a Th1-biased CD8+ T cell response. For spike, a greater apparent dose response was observed in rabbits receiving dose 1 (125 ng S, +28-fold) compared to dose 2 (50 ng S, +10-fold), although not statistically significant( Figure 25 A). For Ncap, dose 1 (10 ng N, +10-fold) produced a better immune response than dose 2 (20 ng N, +2-fold)( Figure 25 B).

[0441] All patents and other publications cited throughout this application, including literature references, issued patents, published patent applications, and co-pending patent applications, such as methods described in such publications that can be used in combination with the techniques described herein, are hereby expressly incorporated by reference for the purpose of description and disclosure. These publications are provided only for their disclosure prior to the filing date of this application. In this regard, nothing should be construed as an admission by the present inventors that they are not entitled to rely on prior inventions or for any other reason prior to such disclosure. Regarding dates or statements, all representations as to the content of these documents are based on information available to the applicant and do not constitute any admission as to the correctness of the dates or content of these documents.

[0442] The description of embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed. While specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications within the scope of the present disclosure are possible, as will be recognized by those skilled in the relevant art. For example, while method steps or functions are presented in a particular order, alternative embodiments may perform the functions in a different order or may perform the functions substantially concurrently. The teachings of the present disclosure provided herein may be applied to other programs or methods as appropriate. The various embodiments described herein may be combined to provide additional embodiments. Aspects of the present disclosure may be modified, as necessary, to employ the combinations, functions, and concepts of the above references and applications to provide yet additional embodiments of the present disclosure. In addition, due to considerations of biological functional equivalence, some changes may be made to the protein structure without affecting the biological or chemical action in terms of type or amount. These and other changes may be made to the present disclosure in accordance with the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0443] Specific elements of any of the above embodiments may be combined or replaced with elements in other embodiments. In addition, while advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments need to exhibit such advantages to fall within the scope of the present disclosure.

Claims

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

2. The immunogenic composition according to claim 1, the immunogenic composition not comprising an adjuvant.

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

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

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

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

3.

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

7.

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

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

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

7.

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

14. The method according to claim 13, wherein the immunogenically effective dose elicits protective immunity against more than one variant of a given virus in the subject.

15. The method according to claim 14, wherein 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; and b. more than one exosome; wherein the nucleocapsid protein polypeptide and / or the spike protein polypeptide is present on the outer surface of the exosomes in the more than one exosome.

16. The method according to claim 15, wherein 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.

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

18. The method according to claim 17, wherein 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.

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

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

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

22. The synthetic fusion protein according to claim 21, wherein the synthetic fusion protein further comprises a transmembrane domain polypeptide located between the viral polypeptide and the exosome polypeptide.

23. The synthetic fusion protein according to claim 21 or claim 22, wherein the exosome polypeptide is a CD9 polypeptide.

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

25. The synthetic fusion protein according to claim 24, wherein the fusion protein comprises, in order from the amino terminus to the carboxyl terminus, a SARS-CoV-2 spike protein polypeptide, a linker polypeptide, and a CD9 polypeptide.

26. The synthetic fusion protein according to claim 25, wherein the fusion protein comprises: (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.

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

28. The synthetic polynucleotide according to claim 27, wherein the synthetic polynucleotide comprises: (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.

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

30. The cell according to claim 29, wherein the cell is a human cell.

31. The cell according to claim 29, wherein the cell is a human embryonic kidney cell.

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

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

34. The vesicle according to claim 33, wherein the vesicle is an exosome.

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

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

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