WO2022011014A1

WO2022011014A1 - Bacteria-derived vesicles and use thereof for generating immune response to sars-cov-2

Info

Publication number: WO2022011014A1
Application number: PCT/US2021/040688
Authority: WO
Inventors: Kyong-Su Park; Jan LÖTVALL
Original assignee: Exocure Biosciences, Inc.
Priority date: 2020-07-09
Filing date: 2021-07-07
Publication date: 2022-01-13

Abstract

A composition comprising artificial outer membrane vesicles (aOMVs) generated from a gram-negative bacterium is disclosed. A composition comprising extruded outer membrane vesicles (exOMVs) generated from a gram-negative bacterium is also disclosed. The gram-negative bacterium may be genetically modified for expression of a SARS-CoV-2 protein in the outer membrane. In certain embodiments, the gram-negative bacterium is not genetically modified for expression of a SARS-CoV-2 protein in the outer membrane but the composition comprises recombinant SARS-CoV-2 protein. In certain embodiments, the gram-negative bacterium is genetically modified for expression of a SARS-CoV-2 protein in the outer membrane and the composition comprises recombinant SARS-CoV-2 protein. Any immunogenic SARS-CoV-2 protein may be used. In certain embodiments, the SARS-CoV-2 protein may be a surface exposed protein. In certain embodiments, the SARS-CoV-2 protein may be a spike protein, e.g., S protein, S1 protein, S2 protein, or a fragment thereof. In certain aspects, the SARS-CoV-2 protein is a fragment of S1 protein, e.g., receptor binding domain (RBD). Methods for making the compositions and using the compositions as a vaccine for inducing an immune response against the virus are also disclosed.

Description

BACTERIA-DERIVED VESICLES AND USE THEREOF FOR GENERATING IMMUNE RESPONSE TO

SARS-CoV-2

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/049,967, filed July 9, 2020, which application is incorporated herein by reference in its entirety.

INTRODUCTION

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally. Epidemiological data so far suggest that COVID-19 has case fatality rate of about 2.3%, several times greater than that of seasonal influenza. The elderly and individuals with underlying medical comorbidities such as cardiovascular disease, diabetes mellitus, chronic lung disease, chronic kidney disease, obesity, hypertension or cancer have a much higher mortality rate than healthy young adults.

[0003] Naturally released bacterial vesicles, including outer membrane vesicles (OMVs), have been developed as vaccines. One example is the meningococcus vaccine, which is available clinically. However, such vaccines are known to activate innate immunity resulting in severe side effects at the time of immunization, including fever and flu-like symptoms.

[0004] There is a need to produce compositions that when used as a vaccine do not evoke serious side effects caused by activation of innate immunity but can induce protective immunity against viruses, like, SARS-CoV-2. The present disclosure addresses the above issues as well as other issues and provides related advantages.

SUMMARY

[0005] A composition comprising artificial outer membrane vesicles (aOMVs) generated from a gram-negative bacterium is disclosed. A composition comprising extruded outer membrane vesicles (exOMVs) generated from a gram-negative bacterium is also disclosed. The gram-negative bacterium may be genetically modified for expression of a SARS-CoV-2 protein in the outer membrane. In certain embodiments, the gram-negative bacterium is not genetically modified for expression of a SARS-CoV-2 protein in the outer membrane but the composition comprises recombinant SARS-CoV-2 protein. In certain embodiments, the gram-negative bacterium is genetically modified for expression of a SARS- CoV-2 protein in the outer membrane and the composition comprises recombinant SARS-CoV-2 protein.

[0006] Any immunogenic SARS-CoV-2 protein may be used. In certain embodiments, the SARS-CoV-2 protein may be a surface exposed protein. In certain embodiments, the SARS-CoV-2 protein may be a spike protein. In certain embodiments, the SARS-CoV-2 protein may be a spike protein, e.g., S protein, SI protein, S2 protein, or a fragment thereof. In certain aspects, the SARS-CoV-2 protein is a fragment of SI protein, e.g., receptor binding domain (RBD).

[0007] Methods for making the compositions and using the compositions as a vaccine for inducing an immune response against the virus are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 depicts steps for generation of artificial outer membrane vesicles (aOMVs) from a gram -negative bacterium according to an embodiment of the present disclosure.

[0009] Figure 2 depicts E. coli OMV- or aOMV-induced pro-inflammatory cytokines in the supernatants of RAW 264.7 cells. Indicated numbers of OMVs or aOMVs were added to the cells for 15 h, and then TNF-a (left) and IL-6 (right) were measured by ELISA. *, P<0.05; **, P<0.01; ***,

P<0.001; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3.

[0010] Figure 3 depicts the body weight (left) and body temperature (right) at 6 h of mice injected intraperitoneally with E. coli OMVs (5 x 10⁹), aOMVs (5 x 10⁹), or 4-fold excess amount of aOMVs (2 x 10¹⁰). ***, P<0.001; ns, not significant; versus PBS group; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3.

[0011] Figure 4 depicts inflammatory cytokines TNF-a (left) and IL-6 (right) in the peritoneum at 6 h of mice injected intraperitoneally with E. coli OMVs (5 x 10⁹), aOMVs (5 x 10⁹), or 4-fold excess amount of aOMVs (2 x 10¹⁰). *, P<0.05; ns, not significant; versus PBS group; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3.

[0012] Figure 5 depicts the number of total leukocytes (left) and platelets (right) in the blood at 6 h of mice injected intraperitoneally with E. coli OMVs (5 x 10⁹), aOMVs (5 x 10⁹), or 4-fold excess amount of aOMVs (2 x 10¹⁰). *, P<0.05; **, P<0.01; ns, not significant; versus PBS group; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3.

[0013] Figure 6, Panel A is a graph of TNF-a level (pg/ml) in the supernatants of bone marrow-derived dendritic cells treated with E. coli OMVs or aOMVs for 24 h. ***, P<0.001; versus control; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3.

Figure 6, Panel B is a graph of IL-6 level (pg/ml) in the supernatants of bone marrow-derived dendritic cells treated with E. coli OMVs or aOMVs for 24 h. ***, P<0.001; versus control; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3. Figure 6, Panel C is a graph of IL-12p70 level (pg/ml) in the supernatants of bone marrow -derived dendritic cells treated with E. coli OMVs or aOMVs for 24 h. **, P<0.01; ***, P<0.001; versus control; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3. Figure 6, Panel D is a graph of IL-4 level (pg/ml) in the supernatants of bone marrow-derived dendritic cells treated with E. coli OMVs or aOMVs for 24 h. Versus control; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3.

[0014] Figure 7, Panel A depicts the level of TNF-a secreted from mouse splenic CD4+ T cells upon ex vivo treatment with E. coli OMVs after the mice were immunized with 5 x 10⁹ of . coli OMVs or aOMVs. ***, P<0.001; versus sham group; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3. Figure 7, Panel B depicts the level of IFN-g secreted from mouse splenic CD4+ T cells upon ex vivo treatment with E. coli OMVs after the mice were immunized with 5 x 10⁹ of E. coli OMVs or aOMVs. **, P<0.01; ***, P<0.001; versus sham group; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3. Figure 7, Panel C depicts the level of IL-4 secreted from mouse splenic CD4+ T cells upon ex vivo treatment with E. coli OMVs after the mice were immunized with 5 x 10⁹ of E. coli OMVs or aOMVs. Versus sham group; one-way ANOVA with Tukey’s multiple comparison test Error bars indicate SEM. N=3.

[0015] Figure 8 depicts P. aeruginosa OMV- or aOMV-induced pro-inflammatory cytokines in the supernatants of MH-S cells. Various particle numbers of OMVs or aOMVs were added to the cells for 18 h, and then TNF-a (left) and IL-6 (right) were measured by ELISA. ***, PO.OOl; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3.

[0016] Figure 9 is a graph showing the levels of P. aeruginosa protein-specific antibodies measured in the course of three intraperitoneal injection of 5 x 10⁹ of P. aeruginosa OMVs or aOMVs at regular intervals of one week. *, P<0.05; ***, P<0.001; ns, not significant; versus sham group; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=4.

[0017] Figure 10, Panel A depicts the level of TNF-a secreted from mouse splenic CD4+ T cells upon ex vivo treatment with P. aeruginosa proteins ( 1 pg/mL) after the mice were immunized with 5 x 10⁹ of . aeruginosa OMVs or aOMVs. ***, P<0.001; ns, not significant; versus sham group; one way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3. Figure 10, Panel B depicts the level of IFN-g secreted from mouse splenic CD4+ T cells upon ex vivo treatment with P. aeruginosa proteins (1 pg/mL) after the mice were immunized with 5 x 10⁹ of P. aeruginosa OMVs or aOMVs. ***, P<0.001; versus sham group; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3. Figure 10, Panel C depicts the level of IL-4 secreted from mouse splenic CD4+ T cells upon ex vivo treatment with P. aeruginosa proteins (1 pg/mL) after the mice were immunized with 5 x 10⁹ of P. aeruginosa OMVs or aOMVs. Versus sham group; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3. Figure 10, Panel D depicts the level of IL-17 secreted from mouse splenic CD4+ T cells upon ex vivo treatment with P. aeruginosa proteins (1 pg/mL) after the mice were immunized with 5 x 10⁹ of P. aeruginosa OMVs or aOMVs. ***, P<0.001; versus sham group; one-way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3.

[0018] Figure 11 shows schematic presentation of structure of the chimeric Lpp-OmpA construct with SARS-CoV-2 spike protein SI in the bacterial outer membrane.

[0019] Figure 12 shows transmission electron microscopy images of engineered aOMVs (aOMV^sl).

[0020] Figure 13 shows Western blot analysis of bacterial lysates and aOMV^sl with anti -His tag antibody.

[0021] Figure 14A is a graph showing the levels of SI protein-specific antibodies measured in the course of three subcutaneous immunization of aOMV^sl at regular intervals of one week. *, P<0.05; versus sham group; one way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=6.

[0022] Figure 14B is a graph showing the levels of aOMV protein-specific antibodies measured in the course of three subcutaneous immunization of aOMV^sl at regular intervals of one week. ***, P<0.001; versus sham group; one way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=6.

[0023] Figure 15A shows schematic presentation of structure of the chimeric Lpp-OmpA construct with SARS-CoV-2 RBD protein in the bacterial outer membrane.

[0024] Figure 15B shows Western blot analysis of aOMV¹*™ with anti-RBD antibody.

[0025] Figure 16 is a graph showing the levels of RBD protein-specific antibodies measured in the course of three subcutaneous injection of wild-type aOMVs plus recombinant RBD protein at regular intervals of one week. *, P<0.05; versus sham group; one way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=5.

[0026] Figure 17 shows optimization diagram for isolation of extruded OMVs overexpressing SARS-CoV-2 spike protein SI.

[0027] Figure 18 shows transmission electron microscopy images of extruded OMV^sl isolated in various conditions.

[0028] Figure 19 shows Western blot analysis of extruded OMV^sl (100 ng) isolated in various conditions with anti-His tag antibody.

[0029] Figure 20 shows optimization diagram for isolation of aOMVs overexpressing SARS- CoV-2 spike protein SI.

[0030] Figure 21 shows Western blot analysis of aOMV^sl (100 ng) isolated in various conditions with anti -SI antibody. [0031] Figure 22 shows the level of IFN-gamma secreted from mouse splenic CD4+ T cells upon ex vivo treatment with S 1 after the mice were immunized with 5 x 10⁹ of aOMV^sl . * * * , P<0.001; versus sham group; one way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3.

[0032] Figure 23 shows the percentage of various adaptive immune cell types in spleens, after mice were subcutaneously immunized with SyBVSl three times at regular intervals of one week. SyBVSl is an alternate term for aOMV^sl.

[0033] Figure 24 is a graph of IFN-beta in the supernatants of mouse BMDCs treated with lyophilized SyBV^sl (5 x 10⁹) for 24 h. *, P<0.05; **, P<0.01; one way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3.

[0034] Figure 25A is a graph showing the levels of RBD protein-specific antibodies measured in the course of three subcutaneous immunization of SyBV^RliD (5 x 10⁹, at weekly intervals). *, P<0.05; versus sham group; one way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=5. SyBV^RliD is an alternate term for aOMV^RliD.

[0035] Figure 25B shows the level of IFN-gamma secreted from mouse splenic CD4⁺ T cells upon ex vivo treatment with RBD after the mice were immunized with 5 x 10⁹ of SyBV^RliD. ***, P<0.001; versus sham group; one way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3.

[0036] Figure 26 shows the level of IFN-gamma secreted from mouse splenic CD4⁺ T cells upon ex vivo treatment with RBD after the mice were immunized with SyBV (5 x 10⁹) and RBD (1, 10 pg). **, P<0.01; ***, P<0.001; versus sham group; one way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3.

DEFINITIONS

[0037] The term “pathogenic” as used herein refers to organisms that cause disease, particularly in animals and especially in humans. Pathogenic organisms include viruses, bacteria, fungi, and parasites.

[0038] The term “non-pathogenic” refers to organisms that do not cause disease in animals, in particular in humans. The term includes commensal organisms. Commensal organisms are those that can colonize a host organism without signs of disease.

[0039] A “gram-negative bacterium” refers to bacterium that has been classified as such based on Gram stain test. A gram-negative bacterium includes a cell wall composed of an outer membrane, periplasmic space that includes proteins such as lipoproteins and a peptidoglycan layer. The outer membrane (OM) is made of an asymmetric lipid bilayer, the inner leaflet of the lipid layer is composed of phospholipids and the outer leaflet of the lipid layer is composed of lipopoly saccharides (LPS). The OM houses outer membranes proteins, such as, outer membrane protein A (OmpA), OmpE, OmpC,

LptD, BamA, etc.

[0040] A “gram-positive bacterium” refers to bacterium that has been classified as such based on Gram stain test. A gram-positive bacterium includes a cell wall composed of peptidoglycan, teichoic acid, and lipoteichoic acid. A gram-positive bacterium lacks an outer membrane. The cell wall in both gram-positive bacterium and gram-negative bacterium enclose an inner membrane made of a phospholipid bilayer, which includes several proteins. The inner membrane is also referred to as a cell membrane, plasma membrane, or a cytoplasmic membrane. The plasma membrane in prokaryotic cells surrounds a cytoplasm that includes nucleic acids, cytoplasmic proteins, and ribosomes.

[0041] The term “outer membrane vesicle(s)” or “OMV(s)” as used herein refers to vesicles that include an outer membrane enclosing periplasmic contents, cytoplasmic contents and inner membrane components. The term OMVs includes blebs produced by budding of the outer membrane of organisms, such as, gram -negative bacteria. Such OMVs can also be referred to as native OMVs. OMVs can also be produced by disrupting (e.g., by extrusion, sonication, detergents, or osmotic shock) a gram-negative bacterium in a hydrophilic solution thereby forcing the cell to form vesicles. Extruded OMVs are similar to natural OMVs since they are produced by fragmentation of the bacteria.

[0042] The term “spheroplast,” as used herein, refers a spherical structure produced from a cell by removing the peptidoglycan layer of the cell. A spheroplast includes an outer membrane and an inner membrane. A spheroplast may be generated from a bacterial, archaeal, fungal, or plant cell. A spheroplast may be generated from a gram -negative bacterium.

[0043] The term “protoplast,” as used herein, refers to a bacterial, archaeal, fungal, or plant cell in which the cell wall is partially or completely removed, exposing the cytoplasmic membrane. A protoplast may be produced from a gram-negative or a gram-positive bacterium.

[0044] The term “vesicle” as used herein refers to a spherical structure which contains an interior volume that is separated from the outside environment by a lipid bilayer membrane. A vesicle can be secreted from cells or can be artificially synthesized from a cell, a spheroplast, or a protoplast. A vesicle is generally smaller than the cell, a spheroplast, or a protoplast from which it is derived. A vesicle may be an outer membrane vesicle or a cell membrane vesicle (e.g. a protoplast derived vesicle).

[0045] The term “deficient” as used in the context of a component present in the non-naturally occurring outer membrane vesicles (artificial OMVs, aOMVs) derived from a bacterium as disclosed herein means having at least 50% less of the component, for example, 60%, 70%, 80%, 90%, or 99%, as compared to amount of the component present in naturally occurring OMVs produced from the bacterium. Examples of vesicles that are similar to naturally occurring OMVs but are produced in the laboratory are extruded OMVs generated from a bacterium by serially extruding the bacterial cell sequentially through fdters of reduced pore size.

[0046] The term “enriched” as used in the context of a protein (e.g., an outer membrane protein, OMP) present in the aOMVs derived from a bacterium as disclosed herein means that the component makes up a bigger fraction of the total amount of protein in the aOMVs as compared to the fraction of the same protein in naturally occurring OMVs produced by the bacterium or extruded OMVs made from the bacterium. For example, the enriched protein may represent at least 25% or more of the total proteins in the aOMVs while the same protein may represent at most 20% of the total proteins in the naturally occurring OMVs or extruded OMVs made from the bacterium. An enriched component may be present in the aOMVs at a higher concentration by total weight, e.g., at least a three-fold greater concentration by total weight, e.g., at least 5-fold greater concentration, at least 10-fold greater concentration, at least 30- fold greater concentration, at least 50-fold greater concentration, or at least 100-fold greater concentration than the concentration of that component by total weight in OMVs isolated from the same bacterium from which the aOMVs were derived. Naturally occurring OMVs refer to OMVs that bud off from the bacterium. OMVs similar to the naturally occurring OMVs can be prepared by using serial extrusion in which the bacterial cells are forced through a first filter comprising a first pore size and the filtrate then forced through a second filter comprising a second pore size that is smaller than the first pore size. Such OMVs that are similar in composition to natural OMVs are referred to as extruded OMVs.

[0047] The term “protective immunity” means that a vaccine or immunization schedule that is administered to a mammal induces an immune response that prevents, retards the development of, or reduces the severity of a disease that is caused by a pathogen, or diminishes or altogether eliminates the symptoms of the disease.

[0048] The term “inflammatory response” as used herein refers to secretion of proinflammatory cytokines, activation of toll-like receptors (TLR) and/or systemic inflammation. Examples of proinflammatory cytokines include IL-1, TNF-a and IL-6.

[0049] The term “reduced” in the context of inflammatory response means production of a lower level of a proinflammatory cytokine upon administration of aOMVs as compared to that produced by administering naturally occurring OMVs produced by the bacterium or exOMVs produced by the bacterium. In some embodiments, production of cytokines is lowered by at least 5%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or more as compared to that produced by administering naturally occurring OMVs or exOMVs produced by the bacterium. Examples of proinflammatory cytokines include IL-2, IL-4, IL-6, IL-12, IL-12p70, IL-17, tumor necrosis factor alpha (TNF-a) and interferon gamma (IFN-g). [0050] The term “toxic response” as used herein refers to any harmful reaction that occurs in the affected cells and adjacent tissues in a subject in response to administration of a composition to the subject. Examples of toxic responses include, but are not limited to, reduction in the number of leukocytes, reduction in the number of platelets, and/or body weight.

[0051] The phrase “specifically binds to an antibody” or “specifically immunoreactive with”, when referring to an antigen such as a polysaccharide, phospholipid, protein or peptide, refers to a binding reaction which is based on and/or is probative of the presence of the antigen in a sample which may also include a heterogeneous population of other molecules. Thus, under designated immunoassay conditions, the specified antibody or antibodies bind(s) to a particular antigen or antigens in a sample and do not bind in a significant amount to other molecules present in the sample. Specific binding to an antibody under such conditions may require an antibody or antiserum that is selected for its specificity for a particular antigen or antigens.

[0052] The phrase “in a sufficient amount to elicit an immune response to epitopes present in said preparation” means that there is a detectable difference between an immune response indicator measured before and after administration of a particular antigen preparation. Immune response indicators include but are not limited to: antibody titer or specificity, as detected by an assay such as enzyme-linked immunoassay (ELISA), bactericidal assay, flow cytometry, immunoprecipitation, Ouchter-Lowny immunodiffusion, spot blot, Western blot, antigen arrays, etc.

[0053] A “surface antigen” is an antigen that is present in a surface structure of a cell.

[0054] The term “endogenous” refers to a naturally-occurring biological component of a cell, i.e., as found in nature.

[0055] The term “heterologous” refers to two biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to a coding sequence. “Heterologous” as used herein in the context of genes or proteins includes bacterial genes or proteins that are naturally expressed in two different bacterial strains. Genes and proteins are also said to be “heterologous” where they expressed in the same strain, but originate from different strains. For example, a strain that expresses an endogenous outer membrane polypeptide and also expresses a recombinant outer membrane polypeptide that differs in amino acid sequence from the endogenous outer membrane polypeptide (e.g., is of a different variant group or variant subtype) is said to contain a heterologous outer membrane polypeptide.

[0056] “Recombinant” as used herein refers to nucleic acid encoding a gene product, or a gene product (e.g., polypeptide) encoded by such a nucleic acid, that has been manipulated by the hand of man, and thus is provided in a context or form in which it is not found in nature. “Recombinant” thus encompasses, for example, a nucleic acid encoding a gene product operably linked to a heterologous promoter (such that the construct that provides for expression of the gene product from an operably linked promoter with which the nucleic acid is not found in nature). For example, a “recombinant outer membrane polypeptide” encompasses an outer membrane polypeptide encoded by a construct that provides for expression from a promoter heterologous to the outer membrane polypeptide coding sequence, outer membrane polypeptides that are modified relative to a naturally-occurring outer membrane (e.g., as in a fusion protein), and the like. It should be noted that a recombinant outer membrane polypeptide can be endogenous to or heterologous to a cell in which such a recombinant nucleic acid is present.

[0057] A “knock-out” or “knockout” of a target gene refers to an alteration in the sequence of the gene that results in a decrease of function of the target gene, e.g., such that target gene expression is undetectable or insignificant, and/or the gene product is not functional or not significantly functional. For example, a “knockout” of a gene involved in LPS synthesis indicates means that function of the gene has been substantially decreased so that the expression of the gene is not detectable or only present at insignificant levels and/or a biological activity of the gene product (e.g., an enzymatic activity) is significantly reduced relative to prior to the modification or is not detectable. “Knock-outs” encompass conditional knock-outs, where alteration of the target gene can occur upon, for example, exposure to a predefined set of conditions (e.g., temperature, osmolarity, exposure to substance that promotes target gene alteration, and the like.

[0058] “Isolated” refers to an entity of interest that is in an environment different from that in which it may naturally occur. “Isolated” is meant to include entities that are within samples that are substantially enriched for the entity of interest and/or in which the entity of interest is partially or substantially purified.

[0059] The terms “subject” and “patient” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, bovine, equine, canine, ovine, or feline.

[0060] The terms "treatment," "treat," or "treating," as used herein cover any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition, i.e., arresting its development; (c) relieving and or ameliorating the disease or condition, i.e., causing regression of the disease or condition; or (d) curing the disease or condition, i.e., stopping its development or progression. The population of subjects treated by the methods of the invention includes subjects suffering from the undesirable condition or disease, as well as subjects at risk for development of the condition or disease.

[0061] The term “therapeutic effect” refers to some extent of relief of one or more of the symptoms of a disorder (e.g., infection) or its associated pathology. “Therapeutically effective amount” as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying, and the like beyond that expected in the absence of such treatment. “Therapeutically effective amount” is intended to qualify the amount required to achieve a therapeutic effect. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the “therapeutically effective amount” (e.g., ED50) of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the vesicles of the present disclosure employed in a pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0062] Any cells, agents, vesicles, compositions or methods provided herein can be combined with one or more of any of the other cells, agents, vesicles, compositions and methods provided herein, regardless of whether they are disclosed in separate sections of the application or within the same section of the application.

[0063] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0064] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0066] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a vesicle” includes a plurality of such vesicles and reference to “the vesicle” includes reference to one or more vesicles and equivalents thereof known to those skilled in the art, reference to “a bacterium” includes a plurality of bacteria of the same type, reference to “a spheroplast” includes reference to a plurality of such spheroplasts, reference to “a heterologous protein” includes reference to one or more heterologous protein that may be different, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0067] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub combination was individually and explicitly disclosed herein.

[0068] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

[0069] A composition comprising artificial outer membrane vesicles (aOMVs) generated from a gram-negative bacterium is disclosed. A composition comprising extruded outer membrane vesicles (exOMVs) generated from a gram-negative bacterium is also disclosed. The gram-negative bacterium may be genetically modified for expression of a SARS-CoV-2 protein in the outer membrane. In certain embodiments, the gram-negative bacterium is not genetically modified for expression of a SARS-CoV-2 protein in the outer membrane but the composition comprises recombinant SARS-CoV-2 protein. In certain embodiments, the gram-negative bacterium is genetically modified for expression of a SARS- CoV-2 protein in the outer membrane and the composition comprises recombinant SARS-CoV-2 protein.

[0070] Any immunogenic SARS-CoV-2 protein may be used. In certain embodiments, the SARS-CoV-2 protein may be a surface exposed protein. In certain embodiments, the SARS-CoV-2 protein may be a spike protein. In certain embodiments, the SARS-CoV-2 protein may be a spike protein, e.g., S protein, SI protein, S2 protein, or a fragment thereof. In certain aspects, the SARS-CoV-2 protein is a fragment of SI protein, e.g., receptor binding domain (RBD).

[0071] Methods for making the compositions and using the compositions as a vaccine for inducing an immune response against the virus are also disclosed.

COMPOSITIONS

[0072] The compositions of the present disclosure comprise non-naturally occurring aOMVs or exOMVs derived from a gram -negative bacterium. In certain embodiments, the gram -negative bacterium may be genetically modified for expression of a SARS-CoV-2 protein in the outer membrane. In certain embodiments, the composition may include aOMVs or exOMVs and a recombinant SARS-CoV-2 protein. In certain embodiments, the composition may include aOMVs or exOMVs, wherein the aOMVs or exOMVs are derived from a gram -negative bacterium genetically modified for expression of at least one SARS-CoV-2 protein in the outer membrane, and a recombinant SARS-CoV-2 protein.

[0073] Any immunogenic SARS-CoV-2 protein may be used. In certain embodiments, the SARS-CoV-2 protein may be a surface exposed protein. In certain embodiments, the SARS-CoV-2 protein may be soluble protein, i.e., not including a membrane spanning region. In certain embodiments, the SARS-CoV-2 protein may be a spike protein. In certain embodiments, the SARS-CoV-2 protein, e.g., spike protein presented on the surface of the gram-negative bacterium may be conjugated to an outer membrane protein. The outer membrane protein may be OmpA, OmpE, OmpC, LptD, BamA, or a fragment thereof comprising a number of membrane-spanning domains sufficient to localize the SARS- CoV-2 protein in the outer membrane of the bacterium. In certain embodiments, OmpA protein is conjugated to a lipoprotein signal peptide. In certain embodiments, the OmpA protein is conjugated to a lipoprotein signal peptide via an N-terminal fragment of E. coli lipoprotein (Lpp). In certain embodiments, the N-terminal fragment may be about 5-30 amino acids in length, e.g., 5-25 amino acids, 5-20 amino acids, or 5-15 amino acids in length. In certain embodiments, the SARS-CoV-2 spike protein comprises S 1 protein or a fragment thereof. In certain embodiments, the fragment of S 1 protein comprises receptor binding domain (RBD). As used herein, OmpA protein refers to OmpA protein from any gram -negative bacterium. The OmpA protein may be full length or a membrane spanning fragment thereof. The fragment of OmpA protein may include at least one, at least two, at least three, at least four, or at least five membrane spanning domains. The OmpA may be from Haemophilus influenzae. In certain embodiments, the OmpA protein includes transmembrane region B3-B7 (amino acids 46-159 of UniProtKB-P45996) of the H. influenzae OmpA. In certain embodiments, the SARS-CoV-2 spike protein is expressed as a fusion protein that comprises from N-terminus to C-terminus, a signal peptide, an N-terminal fragment of E. coli Lpp, OmpA protein, and SARS-CoV-2 spike protein. In certain embodiments, instead of the OmpA protein, a different outer membrane protein may be used to localize the SARS-CoV-2 spike protein to the surface of the gram -negative bacterium.

[0074] In certain embodiments, the recombinant SARS-CoV-2 spike protein present in the compositions comprising aOMVs may be an isolated SARS-CoV-2 spike protein purified from a recombinant host cell genetically modified to express the SARS-CoV-2 spike protein. The SARS-CoV-2 spike protein may be S 1 protein or a fragment thereof. In certain embodiments, the fragment of S 1 protein comprises receptor binding domain (RBD).

[0075] SARS-CoV-2 spike protein SI as used herein refers to a polypeptide comprising an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence:

[0076] SDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVT GFHTINHTF GNP VIPFKDGIYFA ATEKSNVVRGWVF GSTMNNKS Q S VIIINNSTNVVIRACNFELC DNPFFAV SKPMGTQTHTMIFDNAFNCTFEYISDAFSLDV SEKSGNFKHLREFVFKNKDGFLYVY KGYQPIDVVRDLPSGFNTLKPIFKLPLGINITNFRAILTAFSPAQDIWGTSAAAYFVGYLKPTTFM LKYDENGTITD A VD CS QNPLAELKC S VKSFEIDKGIY QTSNFRVVP SGD VVRFPNITNLCPF GEVF NATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGD DVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDI SNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLI KNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITP GTNASSEVAVLY QDVNCTDV STAIHADQLTPAWRIY STGN VFQTQAGCLIGAEHVDTSYECDI PIGAGICASYHTV SLLR (SEQ ID NO: 1).

[0077] A fragment of the SI protein may be a polypeptide that is up to 600 amino acids long, e.g., about 200-600 amino acids long, about 200-550 amino acids long, about 200-500 amino acids long, about 200-450 amino acids long, about 200-400 amino acids long, about 200-350 amino acids long, about 200-300 amino acids long, or about 200-250 amino acids long, and comprising an amino acid sequence having at least 80% or higher sequence identity to a contiguous region of the amino acid sequence set forth in SEQ ID NO: 1. For example, the contiguous region may be an N-terminus region of the amino acid sequence set forth in SEQ ID NO: 1.

[0078] In certain embodiments, a fragment of SI protein comprising an RBD may comprise an amino acid sequence having at least 80% identity or higher to the amino acid sequence: [0079] RWPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNST FF STFKCY GV S ATKLNDLCF SNVY AD SFV VKGDD VRQIAPGQTGVIADYNYKLPDDFMGCVLA WNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTT TGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNF (SEQ ID NO:2).

[0080] The fragment may be up to 270 amino acids long, e.g., 150-270 amino acids long, 160- 260 amino acids long, 170-250 amino acids long, 180-240 amino acids long, 190-230 amino acids long, or 200-230 amino acids long.

[0081] In certain embodiments, the amino acid differences that account for the difference in amino acid sequence identity may be conservative amino acid substitutions. “Conservative amino acid substitution” refers to a substitution of one amino acid residue for another sharing chemical and physical properties of the amino acid side chain (e.g., charge, size, hydrophobicity/hydrophilicity). “Conservative substitutions” are intended to include substitution within the following groups of amino acid residues: gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr. Guidance for such substitutions can be drawn from alignments of amino acid sequences of polypeptides presenting the epitope of interest.

[0082] As used herein, an amino acid sequence identity higher than 80% refers to at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity.

[0083] The aOMVs derived from a gram -negative bacterium are distinguishable from the naturally occurring outer membrane vesicles (OMVs) produced by the gram-negative bacterium or extruded OMVs made from the bacterium on the basis of one or more of the following features: the aOMVs are enriched in outer membrane proteins and are deficient in one or more of the following components present in the gram-negative bacterium: inner membrane proteins, nucleic acids, cytoplasmic proteins, and ribosomes.

[0084] In certain aspects, in the aOMVs enriched in outer membrane proteins, the outer membrane proteins represent a higher fraction of the total protein content of the aOMVs as compared to naturally occurring OMVs or extruded OMVs made from the bacterium. In certain aspects, the aOMVs have a higher amount of an outer membrane protein (OMP) per total amount of proteins, e.g., at least a three-fold greater concentration by total concentration, e.g., at least 5-fold greater concentration, at least 10-fold greater concentration, at least 30-fold greater concentration, at least 50-fold greater concentration, or at least 100-fold greater concentration than the concentration of that OMP by total protein concentration in OMVs produced from the same bacterium from which the aOMVs are derived.

[0085] In certain aspects, in the aOMVs enriched in outer membrane proteins, the outer membrane protein representing a higher fraction of the total protein content of the aOMVs as compared to naturally occurring OMVs secreted by the same bacterium from which the aOMVs are derived, may be a porin or BamA. In certain aspects, the aOMVs described herein may have a higher amount of a porin and/or Bam A per total amount of proteins in the aOMVs as compared to OMVs and to OMVs prepared by a process different from the process of making the aOMVs as disclosed herein.

[0086] In certain aspects, the aOMVs provided herein may be derived from a gram -negative bacterium by disrupting a spheroplast prepared from the gram-negative bacterium to generate vesicles comprising outer membrane and vesicles comprising inner membrane; exposing the vesicles to an ionic surfactant to disrupt the vesicles comprising inner membrane and to an alkaline pH to open the vesicles comprising outer membrane thereby generating outer membrane sheets; purifying the outer membrane sheets; and applying energy or force to the purified outer membrane sheets sufficient to convert the outer membrane sheets into OMVs, thereby generating the non-naturally occurring OMVs. The step of removing vesicles that include inner membrane by treatment with an ionic surfactant reduces contamination from inner membrane components. The step of opening the vesicles releases any inner membrane, periplasmic, cytoplasmic components present within the vesicles and revesiculation of the open outer membrane sheets provides OMVs that are enriched in OMPs and are deficient in one or more of inner membrane, periplasmic, and cytoplasmic components. The aOMVs may have the same topology of the lipid bilayer as the cell from which they are derived. In certain aspects, the aOMVs of the present disclosure are prepared by removing inner membrane contamination by exposing vesicles generated from spheroplasts to an ionic surfactant and to an alkaline pH to open the vesicles to release any inner membrane, periplasmic, or cytoplasmic components present within the vesicles prior to reforming the vesicles.

[0087] In certain aspects, the aOMVs provided herein are enriched in outer membrane proteins such that the outer membrane proteins are at least 25% of the total protein content of the aOMVs. In contrast, outer membrane proteins generally form at most only 15% of the total protein content of OMVs produced by the same gram-negative bacterium from which the aOMVs are derived. In certain aspects, the aOMVs provided herein are enriched in outer membrane proteins such that the outer membrane proteins form at least 30%, at least 35%, at least 40%, at least 50%, or more (e.g., 25%-80%, 25%-75%, 25%-70%, 25%-50%, 25%-40%, 30%-80%, 30%-75%, 30%-70%, 30%-50%, 30%-40%) of the total protein content of the aOMVs.

[0088] In certain aspects, the aOMVs provided herein are deficient in one, two, or more of the following non-outer membrane components present in the bacterium from which the aOMVs are derived: peptidoglycans, periplasmic proteins, inner membrane proteins, nucleic acids, cytoplasmic proteins, and ribosomes. In certain aspects, the aOMVs are deficient in one, two, or more of the non outer membrane components such that the amount of a non-outer membrane component present in the aOMVs is reduced by at least 50% (e.g., at least 55%, 60%, 65%, 70%, or more, e.g., up to 90%, 95%, 99%, or 100%) as compared to the amount of the same non-outer membrane component present in OMVs produced by the gram -negative bacterium.

[0089] In certain aspects, the aOMVs provided herein are deficient in cytosolic proteins such that the amount of cytosolic proteins present in the aOMVs is less than 25%, e.g., less than 20%, 15%, or 10% of the total protein content of the aOMVs. In contrast, the OMVs produced by the same gram negative bacterium may include an amount of cytoplasmic proteins that is at least 50% (e.g., at least 60%, 65%, 70%, or more) of the total protein content of the OMVs.

[0090] In certain aspects, the aOMVs provided herein are deficient in inner membrane proteins such that the amount of inner membrane proteins present in the aOMVs is less than 20%, e.g., less than 15%, or 10% of the total protein content of the aOMVs. In contrast, the OMVs produced by the same gram-negative bacterium may include an amount of inner membrane proteins that is at least 25% (e.g., at least 30%, 40%, or up to 50%) of the total protein content of the OMVs.

[0091] In certain aspects, the aOMVs provided herein are enriched in outer membrane proteins such that at least 25% (e.g., at least 30%-40%) of the total protein content of the aOMVs are outer membrane proteins and these aOMVs are deficient in cytosolic proteins such that less than 25% (e.g. l%-20% or less) of the total protein content of the aOMVs are cytosolic proteins.

[0092] The aOMVs of the present disclosure may be roughly spherical in shape. The aOMVs may range in diameter or greatest dimension from 20 nm - 200 nm, e.g., 40 nm - 200 nm, 50 nm - 200 nm, 30 nm - 175 nm, 30 nm - 150 nm, 40 nm - 175 nm, or 50 nm - 150 nm. The aOMVs of the present disclosure may also be referred to as aOMV particles, reOMVs, or revesiculated aOMVs.

[0093] The exOMVs may be produced from a gram negative bacterium by extrusion. Extrusion may include forcing the bacterium through pores smaller than the size of the bacterium. In the context of extrusion, bacterium may be forced to sequentially pass through a series of filters having decreasing pore sizes. For example, bacterium are sequentially passed through three filters with respective pore sizes of

0.45 mhi®0.22 pm to form exOMVs. In certain embodiments, the filtrate comprising the exOMVs may be concentrated by, e.g., centrifugation or precipitation. Centrifugation may be ultracentrifugation, e.g., at 150,000 x g. Precipitation may be PEG precipitation.

[0094] In certain embodiments, the exOMVs may be further processed. In certain embodiments, the exOMVs are exposed to a high pH solution to open the exOMVs. the exOMVs exposed to the high pH solution are purified by density gradient centrifugation or precipitation. The purified exOMVs may be closed by applying energy to the exOMVs exposed to the reclose the opened exOMVs. applying energy comprises applying shear force or acoustic energy (e.g., sonication) to the opened exOMVs. [0095] In certain aspects, the aOMVs or exOMVs may be derived from a gram -negative bacterium that is genetically modified for decreased production of lipopolysaccharides (LPS). The genetic modification may include a mutation resulting in decreased activity in one or more proteins required for LPS synthesis. The genetic modification may include a mutation resulting in reduced Lipid A.

[0096] In certain aspects, the gram-negative bacterium is genetically modified for increased expression of an endogenous outer membrane protein, such as, an OMP that is a virulence factor and increases immunogenicity of the aOMVs as compared to immunogenicity of aOMVs derived from a gram-negative bacterium not genetically modified for decreased production of the OMP.

[0097] In certain aspects, the gram-negative bacterium is genetically modified for expression of a heterologous protein in the outer membrane. The heterologous protein may be a viral antigen. Any viral antigen may be expressed in the gram -negative bacterium. In certain aspects, the viral antigen may be localized to the outer membrane. In certain aspects, the heterologous protein is an outer membrane protein from a different gram -negative bacterium. The heterologous protein may be an OMP from a gram-negative bacterium that is a different strain, a different species, or different genus as compared to the genetically modified gram -negative bacterium.

[0098] In certain aspects, the compositions may include the aOMVs or exOMVs (e.g., derived from a gram -negative bacterium expressing a SARS Co-V-2 protein as described herein or from a gram negative bacterium that is not genetically modified), optionally a recombinant viral protein (e.g., a viral protein described herein), and a carrier, diluent, vehicle, excipient, and the like. In certain aspects, the carrier, diluent, vehicle, excipient, and the like may be a pharmaceutically acceptable carrier, diluent, vehicle, excipient, and the like. In certain aspects, the compositions may further include an additional prophylactic or therapeutic agent. As used herein, a carrier, diluent, vehicle, excipient, and the like includes salt, buffer, antioxidant (e.g., ascorbic acid and sodium bisulfate), preservative (e.g., benzyl alcohol, methyl parabens, ethyl or n-propyl, p-hydroxybenzoate), emulsifying agent, suspending agent, dispersing agent, solvent, filler, bulking agent, detergent, and/or adjuvant. For example, a suitable vehicle may be physiological saline solution or buffered saline, possibly supplemented with other materials common in pharmaceutical compositions for, e.g., parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Those skilled in the art will readily recognize a variety of buffers that could be used in the compositions. Typical buffers include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. As an example, the buffer components can be water soluble materials such as phosphoric acid, tartaric acids, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof. Acceptable buffering agents include, for example, a Tris buffer, N-(2-Hydroxyethyl)piperazine- N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N- Morpholino)ethanesulfonic acid sodium salt (MES), 3 -(N-Morpholino)propane sulfonic acid (MOPS), and N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS). In certain aspects, an adjuvant included in the disclosed compositions may be poly-ICLC, 1018 IS S, aluminum salts, Amplivax, AS 15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP- EC, ONTAK, PEPTEL, vector system, PLGA microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, acrylic or methacrylic polymers, or copolymers of maleic anhydride and Aquila's QS21 stimulon.

[0099] In certain aspects, a composition may include a first population of aOMVs generated from a first gram-negative bacterium and a second population of aOMVs generated from a second gram negative bacterium, where the first and second gram-negative bacterium are different from each other, e.g., are different strains, different species, or different genus. In certain aspects, a composition may include a first, a second, a third, a fourth, or more populations of aOMVs derived from different types of gram-negative bacteria. The first, second, third, fourth, or more gram negative bacterium may or may not be genetically modified.

[00100] The aOMVs described herein may be synthesized from any gram-negative bacterium, e.g., a non-pathogenic gram-negative bacterium, such as a laboratory strain, a pathogenic gram-negative bacterium, such as, any human and/or animal pathogen. In certain aspects, the bacterium may be from genus Escherichia, Pseudomonas, Moraxella, Shigella, Treponema, Porphyromonas, Helicobacter, Neisseria, Kingella, Acinetobacter, Brucella, Bordetella, Haemophilus, Chlamydia, Legionella, Proteus, or Yersinia. In certain aspects, the gram-negative bacterium may be from the genus Escherichia. In certain aspects, the gram-negative bacterium may be from the genus Pseudomonas.

Method of Making Bacterial Vesicles

[00101] The bacterial vesicles described in the foregoing sections may be produced using methods disclosed herein.

[00102] In certain aspects, the present disclosure provides a method for generating non- naturally occurring artificial outer membrane vesicles (aOMVs) from a gram -negative bacterium. The method may include: a) disrupting a spheroplast generated from the gram -negative bacterium to generate vesicles comprising outer membrane and vesicles comprising inner membrane; b) exposing the vesicles to an ionic surfactant to disrupt vesicles comprising inner membrane and to an alkaline pH to open the vesicles comprising outer membrane thereby generating outer membrane sheets; c) purifying the outer membrane sheets; and d) applying energy or force to the purified outer membrane sheets sufficient to convert the outer membrane sheets into OMVs, thereby generating the non-naturally occurring OMVs. In certain aspects, aOMVs may be generated by a method depicted in Fig. 1.

[00103] In certain aspects, generating the spheroplast from the gram -negative bacterium may involve incubating the gram-negative bacterium with an enzyme having muramidase activity under conditions sufficient for removal of peptidoglycan layer in cell wall of the gram-negative bacterium, thereby converting the gram-negative bacterium into the spheroplast. In certain aspects, the enzyme having muramidase activity may be a murein hydrolase. In certain aspects, the murein hydrolase may be a glycosidase (e.g., N-acetylmuramide glycanhydrolase (also known as lysozyme)) or a glucosaminidases. In certain aspects, the murein hydrolase may be an endopeptidase or an amidase. Lysozymes are commercially available and conditions for removal of the peptidoglycan may be manufacturer’s protocol which may optionally be modified to optimize results. In certain aspects, the lysozyme may be hen egg white lysozyme (HEWL).

[00104] In certain aspects, disrupting the spheroplast to generate the vesicles may involve mechanical, electrical or chemical methods. Examples of the methods include cytolysis using osmosis, electroporation, sonication, homogenization, detergent treatment, freeze-thawing, extrusion, mechanical degradation, and chemical treatment, but are not limited thereto. In a mechanical degradation method, a solution of spheroplasts is shaken together with metal, ceramic or sufficiently hard plastic balls. In certain aspects, disrupting the spheroplasts may include applying a shear force to the spheroplasts. Shear force may be applied by extruding the spheroplasts. Extrusion may include forcing the spheroplasts through pores smaller than the size of the spheroplasts. In the context of extrusion, spheroplast may be forced to sequentially pass through a series of filters having decreasing pore sizes.

For example, spheroplasts are sequentially passed through three filters with respective pore sizes of 10 pm 5 pm l pm to form vesicles.

[00105] In certain aspects, disrupting the spheroplasts may include applying acoustic energy to the spheroplasts. Acoustic energy may be applied via a sonication device. Sonication conditions may be adjusted for the desired disruptive energy. For example, low temperature, low energy, and/or short duration for sonication may be used when disrupting spheroplasts to generate vesicles. Sonication can be performed with different degree of intensity, including low energy sonication over periods of 1 minute to 3 hours. In certain aspects, sonication may be performed using an ultrasonic probe-type device. In certain aspects, an ultrasonic bath may be used for sonication. The duration of sonication may be adjusted based on the type of device being used to perform the sonication. For example, an ultrasonic probe-type device may provide about 1000 times higher energy than an ultrasonic bath. In certain aspects, ultrasonic probe-type device may be used for disrupting the spheroplasts. [00106] Following disruption of the spheroplasts to generate vesicles, such as, vesicles that have the outer membrane enclosing cytosolic contents and vesicles that have the inner membrane enclosing cytosolic contents (or both), these vesicles may be isolated from any remaining spheroplasts. Separation of these vesicles from spheroplasts may be performed using differences in size, density, buoyancy, etc. In certain aspects, centrifugation or fdtration may be performed to isolate the vesicles.

[00107] The isolated vesicles may then be exposed to an ionic surfactant to disrupt vesicles comprising inner membrane and to an alkaline pH to open up the vesicles comprising the outer membrane. In certain aspects, the steps of exposing the vesicles to an ionic surfactant and to alkaline pH may be performed as a single step by using an alkaline solution comprising the ionic surfactant. In other aspects, exposing the vesicles to the ionic surfactant to disrupt vesicles comprising inner membrane may be performed first followed by exposing the vesicles comprising the outer membrane to the alkaline pH. In such aspects, the vesicles comprising the outer membrane may be separated from the disrupted inner membrane vesicles prior to exposing the vesicles comprising the outer membrane to the alkaline pH.

[00108] Any suitable ionic surfactant may be used for disrupting vesicles comprising inner membrane. In certain aspects, the ionic surfactant may be a detergent. In certain aspects, the detergent may be sodium lauroyl sarcosinate, also known as sarkosyl, deoxycholate, sodium dodecyl sulfate (SDS), cetyl trimethylammonium bromide (CTAB), or a combination thereof. In certain aspects, the conditions used for disruption of vesicles comprising inner membrane may involve use of 0.25%-2% sarkosyl for about 20 min, which can solubilize the inner membrane without substantially affecting the outer membrane.

[00109] In certain aspects, the alkaline pH used for opening vesicles comprising outer membrane may be a pH of 9-14, e.g., pH 9-13, pH 10-14, pH 11-14, pH 9-12, or pH 10-12. An alkaline solution for opening vesicles comprising outer membrane may be prepared using sodium carbonate (Na₂CC>₃), sodium hydroxide (NaOH), ammonia (N¾), calcium hydroxide (Ca(OH)2), potassium hydroxide (KOH), sodium hydrogen carbonate (NaHCCfi), or magnesium hydroxide (Mg(OH)2). The duration of incubation of the vesicles comprising outer membrane in an alkaline solution may be adjusted based on the number of vesicles, total volume of the solution, and the like. In certain aspects, the duration of incubation may be 10 min-3 hrs. The incubation may be performed at room temperature (about 25°C), 4°C, or 37°C. The incubation time may be decreased when performing incubation at a higher temperature and/or higher alkaline pH. The incubation time may be increased when performing incubation at a lower temperature and/or lower pH.

[00110] Outer membrane sheets generated from opening of vesicles comprising outer membrane may be separated from whole vesicles (i.e., unopened) vesicles by utilizing any suitable separation method. In certain aspects, purifying the outer membrane sheets may involve centrifugation, e.g., centrifugation (such as, density gradient centrifugation or density gradient ultracentrifugation), filtration, or another suitable method, such as size exclusion, dialysis, tangential flow filtration and the like. In certain aspects, purifying the outer sheet membranes may include performing centrifugation by collecting the outer membrane sheets (e.g., by centrifugation to obtain a pellet comprising the sheets) and fractionating the outer membrane sheets using density gradient centrifugation. In certain aspects, the density gradient may be formed by layering iodixanol or sucrose. For example, membrane sheets may be separated using an iodixanol gradient, such as, a density gradient formed by 10%, 30%, and 50% iodixanol. Outer membrane sheets present in a layer formed between 10% and 30% iodixanol after ultracentrifugation may be collected to provide purified outer membrane sheets. In case of OMVs comprising a recombinant protein, e.g., SARS-COV-2 spike protein as disclosed herein, the outer membrane sheets present in a layer formed between 30% and 50% iodixanol after ultracentrifugation may be collected to provide purified outer membrane sheets comprising the protein in the outer membrane.

[00111] In certain aspects, the method of generating the non-naturally occurring aOMVs may be involve applying energy or force to the purified outer membrane sheets sufficient to convert the outer membrane sheets into aOMVs. Suitable sources of energy include mild sonication, shear force, acoustic force, freeze-thaw, and the like. In certain aspects, the purified outer membrane sheets may be sonicated for a duration of time sufficient to convert the outer membrane sheets into aOMVs. In certain aspects, the purified outer membrane sheets may be sonicated by applying energy 100-1000 times less than that applied for disrupting spheroplasts. In certain aspects, mild sonication may include using an ultrasonic bath for converting the outer membrane sheets into aOMVs.

[00112] The methods of the present disclosure provide for increased yield of aOMVs as compared to prior art methods for isolating the naturally secreted OMVs or the methods for generating extruded OMVs. Per ml of bacterial culture, the methods of the present disclosure provide a yield of aOMVs that is at least about 2X, 3X, 4X, or even 5X higher than the number of OMVs isolated per ml of the same bacterial culture. In addition, the aOMVs compositions produced by the methods of the present disclosure have fewer contaminants as compared to those found in OMV preparations. For example, the number of aOMVs present per total amount of proteins is at least 2X, 3X, 4X, or even 5X higher than the number of OMVs per total amount of proteins. Furthermore, the aOMVs have less contamination from non-outer membrane components, such as, periplasm, inner membrane, and cytosol. Additional features of the aOMVs produced by the disclosed methods are provided in the preceding sections.

[00113] In a particular aspect, the aOMVs of the present disclosure may be prepared from an E. coli or P. aeruginosa. In a particular aspect, the aOMVs may be prepared by incubating a gram-negative bacterium (e.g., E. coli or P. aeruginosa) as a suspension in sucrose (e.g., 5%-30%), lysozyme, and EDTA (at e.g., pH 8.0) which results in removal of peptidoglycan layer. The resulting spheroplasts may be sonicated and subjected to centrifugation at different speeds to separate whole cells from membranes. The separated membranes may be freeze-thawed and incubated in a detergent (e.g., Sarkosyl). The outer membranes may be separated by centrifugation (e.g., at 40,000 x g) and incubated in high pH solution and fractionated to isolate membrane layers formed between 10% and 30% iodixanol. The isolated membrane may be sonicated to produce aOMVs.

[00114] In certain aspects, the step of incubating the gram -negative bacterium with a divalent ion chelator under conditions sufficient to render the outer membrane sensitive to chemical or enzymatic disruption may include incubating the bacterium with a divalent cation chelating agent, such as, an agent containing ethylenediamine (e.g., ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(P-ami noethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)) or Porphine (e.g., porphyrins). In certain aspects, the step of incubating the gram-negative bacterium with a surfactant under conditions sufficient to render the outer membrane sensitive to chemical or enzymatic disruption may include incubating the bacterium with an ionic or a non-ionic detergent. In certain aspects, a non-ionic detergent, such as, Tween or Triton, e.g., Tween-20 or Triton X-100 may be used.

[00115] Any gram -negative or gram -positive bacteria may be used for generating the vesicles using the methods described herein. In certain aspects, the gram-negative or gram-positive bacteria may be those listed in the preceding sections.

Methods for Producing Recombinant SARS-CoV-2 Proteins

[00116] The SARS-CoV-2 proteins disclosed herein, e.g., spike protein, SI protein, or fragments thereof, such as RBD and fusions thereof, e.g., may be expressed such that they are localized to the outer membrane and exposed on the outer membrane of the gram-negative bacterium and OMVs derived therefrom.

[00117] The SARS-CoV-2 proteins disclosed herein may alternatively or additionally be expressed such that the protein is secreted by a host cell genetically modified to secrete the protein or expressed intracellularly. The SARS-CoV-2 protein secreted by the genetically modified host cell may be purified from the culture medium. The SARS-CoV-2 protein expressed intracellularly by the genetically modified host cell may be purified by lysing the host cell. The protein may be a soluble protein, i.e., not localized in a membrane. In certain embodiments, the soluble SARS-CoV-2 protein may be produced by non-recombinant methods (e.g., chemical synthesis).

[00118] Where the SARS-CoV-2 protein is produced using recombinant techniques, the methods can involve any suitable construct and any suitable host cell, which can be a prokaryotic or eukaryotic cell, usually a bacterial or yeast host cell, more usually a bacterial cell. Methods for introduction of genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate methods and the like. The method for transfer can be selected so as to provide for stable expression of the introduced SARS-CoV-2 protein-encoding nucleic acid. The SARS- CoV-2 protein-encoding nucleic acid can be provided as an inheritable episomal element (e.g., plasmid) or can be genomically integrated.

[00119] The present disclosure provides nucleic acids (including isolated nucleic acids) that comprise a nucleotide sequence encoding a SARS-CoV-2 protein of the present disclosure. In some embodiments, the nucleotide sequence encoding the SARS-CoV-2 protein is operably linked to a transcriptional control element, e.g., a promoter. The promoter is in some cases constitutive. The promoter is in some cases inducible. In some cases, the promoter is suitable for use (e.g., active in) a prokaryotic host cell. In some cases, the promoter is suitable for use (e.g., active in) a eukaryotic host cell.

[00120] In some instances, a nucleic acid comprising a nucleotide sequence encoding a

SARS-CoV-2 protein of the present disclosure is present in an expression vector. The present disclosure provides a recombinant expression vector (e.g., an isolated recombinant expression vector) that comprises a nucleotide sequence encoding a SARS-CoV-2 protein of the present disclosure. In some embodiments, the nucleotide sequence encoding the SARS-CoV-2 protein is operably linked to a transcriptional control element, e.g., a promoter. The promoter is in some cases constitutive. The promoter is in some cases inducible. In some cases, the promoter is suitable for use (e.g., active in) a prokaryotic host cell. In some cases, the promoter is suitable for use (e.g., active in) a eukaryotic host cell.

[00121] Suitable vectors for transferring SARS-CoV-2 protein-encoding nucleic acid can vary in composition. Integrative vectors can be conditionally replicative or suicide plasmids, bacteriophages, and the like. The constructs can include various elements, including for example, promoters, selectable genetic markers (e.g., genes conferring resistance to antibiotics (for instance kanamycin, erythromycin, chloramphenicol, or gentamycin)), origin of replication (to promote replication in a host cell, e.g., a bacterial host cell), and the like. The choice of vector will depend upon a variety of factors such as the type of cell in which propagation is desired and the purpose of propagation. Certain vectors are useful for amplifying and making large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Still other vectors are suitable for transfer and expression in cells in a whole animal. The choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially.

[00122] In one example, the vector is an expression vector based on episomal plasmids containing selectable drug resistance markers and elements that provide for autonomous replication in different host cells (e.g., in both E. coli and P. aeruginosa). One example of such a “shuttle vector” is the plasmid pFPIO (Pagotto et al. (2000) Gene 244:13-19).

[00123] Constructs (recombinant vectors) can be prepared by, for example, inserting a polynucleotide of interest into a construct backbone, typically by means of DNA ligase attachment to a cleaved restriction enzyme site in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination or site-specific recombination. Typically, homologous recombination is accomplished by attaching regions of homology to the vector on the flanks of the desired nucleotide sequence, while site-specific recombination can be accomplished through use of sequences that facilitate site-specific recombination (e.g., cre-lox, att sites, etc.). Nucleic acid containing such sequences can be added by, for example, ligation of oligonucleotides, or by polymerase chain reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence.

[00124] Vectors can provide for extrachromosomal maintenance in a host cell or can provide for integration into the host cell genome. Vectors are amply described in numerous publications well known to those in the art, including, e.g., Short Protocols in Molecular Biology, (1999) F. Ausubel, et al., eds., Wiley & Sons. Vectors may provide for expression of the nucleic acids encoding the SARS- CoV-2 protein, may provide for propagating the subject nucleic acids, or both.

[00125] Examples of vectors that may be used include but are not limited to those derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. For example, plasmid vectors such as pBR322, pUC 19/18, pUC 118, 119 and the M13 mp series of vectors may be used. pET21 is also an expression vector that may be used. Bacteriophage vectors may include lgtl0, lgtl 1, lgtl8-23, lZAR/R and the EMBL series of bacteriophage vectors. Further vectors that may be utilized include, but are not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and the charomid 9 series of vectors.

[00126] For expression of a SARS-CoV-2 protein, an expression cassette may be employed. Thus, the present disclosure provides a recombinant expression vector comprising a subject nucleic acid. The expression vector provides transcriptional and translational regulatory sequences, and may provide for inducible or constitutive expression, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. These control regions may be native to an SARS-CoV-2 protein, or may be derived from exogenous sources. In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoters can be either constitutive or inducible, and can be a strong constitutive promoter (e.g., T7, and the like). [00127] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding proteins of interest. A selectable marker operative in the expression host may be present to facilitate selection of cells containing the vector. In addition, the expression construct may include additional elements. For example, the expression vector may have one or two replication systems, thus allowing it to be maintained in organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. In addition, the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

[00128] It should be noted that SARS-CoV-2 protein of the present disclosure may comprise additional elements, such as a detectable label, e.g., a radioactive label, a fluorescent label, a biotin label, an immunologically detectable label (e.g., a hemagglutinin tag, a poly-Histidine tag) and the like. Additional elements of SARS-CoV-2 protein can be provided to facilitate isolation (e.g., biotin tag, immunologically detectable tag) through various methods (e.g., affinity capture, etc.).

[00129] Isolation and purification of SARS-CoV-2 protein can be accomplished according to methods known in the art. For example, SARS-CoV-2 protein can be isolated from a lysate of cells genetically modified to express a SARS-CoV-2 protein, or from a synthetic reaction mix, by immunoaffinity purification, which generally involves contacting the sample with an anti-SARS-CoV-2 protein antibody, washing to remove non-specifically bound material, and eluting specifically bound SARS-CoV-2 protein. Isolated SARS-CoV-2 protein can be further purified by dialysis and other methods normally employed in protein purification methods. In one example, the SARS-CoV-2 protein can be isolated using metal chelate chromatography methods.

[00130] Any of a number of suitable host cells can be used in the production of SARS-

CoV-2 protein. In general, the SARS-CoV-2 protein described herein may be expressed in prokaryotes or eukaryotes, e.g., bacteria such as Escherichia coli or P. aeruginosa in accordance with conventional techniques. Thus, the present disclosure further provides a genetically modified in vitro host cell, which contains a nucleic acid encoding a SARS-CoV-2 protein.

[00131] SARS-CoV-2 protein can be prepared in substantially pure or substantially isolated form (i.e., substantially free from host cell polypeptides) or substantially isolated form. The SARS-CoV-2 protein can be present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components). Purified SARS-CoV-2 protein can be provided such that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, e.g., less than 90%, usually less than 60% and more usually less than 50% of the composition is made up of other expressed polypeptides. Methods for Inducing Immune Response

[00132] Compositions disclosed herein may be used for producing an immune response to SARS-CoV-2 in a mammalian subject. In certain embodiments, the method for producing an immune response to SARS Co-V-2 in a mammalian subject may involve administering a composition disclosed herein to the subject in an amount effective to induce an immune response to the SARS CoV-2 in the mammalian subject.

[00133] The composition may be administered to the subject in an amount effective to induce the immune response in the mammalian subject. The immune response induced by the administering may be generation of antibodies against SARS Co-V-2 and/or activation of T cells, e.g., Thl.

[00134] In certain aspects, the administering produces reduced inflammatory response as compared to that produced by administering naturally occurring OMVs released by the gram -negative bacterium or exOMVs produced from the bacterium. The reduced inflammatory response may include production of a lower level of pro-inflammatory cytokines as compared to that produced by administering naturally occurring OMVs released by the gram -negative bacterium. The reduced inflammatory response may include production of a lower level of tumor necrosis factor alpha (TNF-a) and/or interleukin-6 (IL-6) as compared to that produced by administering naturally occurring OMVs released by the gram -negative bacterium or exOMVs produced from the bacterium.

[00135] In certain aspects, the administering produces reduced toxic response as compared to that produced by administering naturally occurring OMVs released by the gram -negative bacterium or exOMVs produced from the bacterium. The toxic response may include reduction in number of leukocytes, reduction in number of platelets, and/or reduction in body weight.

[00136] The aOMVs provide an immune response superior to that provided by traditional adjuvants, such as, an adjuvant comprising aluminum, e.g., aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, or potassium aluminum sulfate (Alum); Incomplete Freund's adjuvant (IF A); and/or cytosine phosphoguanine (CpG).

Immunization

[00137] Immunogenic compositions used as vaccines may include an immunologically effective amount of the active component, e.g., aOMVs with SARS-CoV-2 spike protein, e.g., SI or a fragment thereof present on the surface or aOMVs and recombinant SARS-CoV-2 spike protein, e.g., S 1 or a fragment thereof. Immunogenic compositions used for prevention of COVID-19 may also include other compatible components, as needed. By "immunologically effective amount" is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective to elicit treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of the individual to be treated (e.g., non-human primate, primate, human, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating clinician's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[00138] Dosage regimen may be a single dose schedule or a multiple dose schedule (e.g., including booster doses) with a unit dosage form of the composition administered at different times. The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the compositions of the present invention in an amount sufficient to produce the desired effect, which compositions are provided in association with a pharmaceutically acceptable excipient (e.g., pharmaceutically acceptable diluent, carrier or vehicle). The vaccine may be administered in conjunction with other immunoregulatory agents.

[00139] The compositions are administered in an amount effective to elicit an immune response, particularly a humoral immune response, in the subject. Amounts for the immunization of the mixture generally range from about 0.001 mg to about 1.0 mg per 70 kilogram patient, more commonly from about 0.001 mg to about 0.2 mg per 70 kilogram patient. Dosages from 0.001 up to about 10 mg per patient per day may be used, particularly when the composition is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ. Substantially higher dosages (e.g. 10 to 100 mg or more) are possible in oral, nasal, or topical administration. The initial administration of the mixture can be followed by booster immunization of the same of different mixture, with at least one booster, more usually two boosters.

Routes of Administration

[00140] The present disclosure contemplates the administration of the disclosed compositions in any appropriate manner. Suitable routes of administration include parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection), intraperitoneal, intracistemal, intraarticular, intraperitoneal, intracerebral (intraparenchymal) and intracerebro ventricular), oral, nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), sublingual and inhalation.

[00141] The present disclosure contemplates methods wherein the compositions of the present disclosure is administered to a subject at least twice daily, at least once daily, at least once every 48 hours, at least once every 72 hours, at least once weekly, at least once every 2 weeks, or once monthly. Combination Therapy

[00142] The present disclosure contemplates the use of the compositions provided herein in combination with one or more active therapeutic agents or other prophylactic or therapeutic modalities. In such combination therapy, the various active agents frequently have different mechanisms of action. Such combination therapy may be especially advantageous by allowing a dose reduction of one or more of the agents, thereby reducing or eliminating the adverse effects associated with one or more of the agents; furthermore, such combination therapy may have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder, or condition.

[00143] In certain aspects, the therapeutic agent may be an agonist of STimulator of

Interferon Genes, STING. In certain aspects, the STING agonist may be a ligand of STING. In certain aspects, the ligand of STING may be 5,6-dimethylxanthenone-4-acetic acid (DMXAA) or MK-1454, a synthetic cyclic dinucleotide (CDN).

[00144] In certain aspects, a therapeutic agent such as a STING agonist may be loaded into the OMVs (e.g., aOMVs or exOMVs) by applying energy (e.g., by sonication) to a mixture of purified outer membrane sheets (obtained as described in the preceding sections) and the therapeutic agent to form OMVs loaded with the therapeutic agent. A separation or a fractionation step may be used to separate unloaded OMVs and free therapeutic agent from OMVs loaded with the therapeutic agent. The fractionation step may involve separating the mixture using a density gradient formed by using 10%, 30%, and 50% iodixanol and collecting the layer formed between 30% and 50% or 10% and 30% iodixanol to obtain OMVs loaded with the therapeutic agent. aOMVs loaded with the STING ligand DMXAA induce an IFN-b response that is significantly higher (more than 5 times) than that produced by aOMVs alone or DMXAA alone indicating a strong synergy between aOMV and DMXAA in induction of the IFN-b response (data not shown). Thus, the loading of a STING agonist into the aOMV would enhance the immune response against the viral component expressed on the aOMV surface.

[00145] As used herein, “combination” is meant to include therapies that can be administered separately, for example, formulated separately for separate administration (e.g., as may be provided in a kit), and therapies that can be administered together in a single formulation (i.e., a “co formulation”).

[00146] In certain embodiments, compositions of the present disclosure are administered or applied sequentially, e.g., where one agent is administered prior to one or more other agents. In other embodiments, the compositions are administered simultaneously, e.g., where two or more compositions are administered at or about the same time; the two or more compositions may be present in two or more separate formulations or combined into a single formulation (i.e., a co-formulation). Regardless of whether the two or more compositions are administered sequentially or simultaneously, they are considered to be administered in combination for purposes of the present disclosure.

[00147] The compositions of the present disclosure can be used in combination with other agents useful in the treatment, prevention, suppression or amelioration of the diseases, disorders or conditions set forth herein, including those that are normally administered to subjects suffering from infections. Lor example, the compositions disclosed herein may be administered as a treatment for COVID-19 in combination with other anti-viral therapeutics, such as, Remdesivir, anti-SARS-CoV-2 antibodies, and the like.

EXAMPLES

[00148] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.

Example 1: Isolation and characterization of E. coli aOMYs

Methods

Preparation of aOMVs

[00149] A uropathogenic Escherichia coli strain was acquired to produce aOMVs. The bacterial culture was pelleted, resuspended in 20% sucrose in 20 mM Tris, pH 8.0 (4 mL per g cells), lysozyme (600 pg per g cells), and 0.1 M EDTA (0.2 mL per g cells) were added. The resulting spheroplasts were pelleted, and then sonicated in ice-cold 10 mM Tris, pH 8.0. The cells were pelleted at 8,000 x g for 5 min, and then whole membranes were pelleted from the supernatants at 40,000 x g for 60 min. The membranes were resuspended in distilled water, freeze-thawed, and incubated in 0.5% Sarkosyl (sodium N-lauroylsarcinosinate; 20 min, 25°C). The outer membrane was pelleted (40,000 x g for 90 min), and incubated with high pH solution (200 mM Na2C03, pH 14.0) for 1 hour at 25°C. The pellets were applied to 4 mL of 50% iodixanol (Axis-Shield PoC AS), followed by addition of 4 mL of 30% iodixanol and 2 mL of 10% iodixanol to ultracentrifuge tube. The layers formed between 10% and 30% iodixanol after ultracentrifugation at 100,000 x g for 2 hours was collected. Finally, the samples were sonicated for 30 min, and considered aOMVs (Figure 1).

Quantification of aOMVs

[00150] Protein concentration of aOMVs was determined with a Bradford dye assay

(Bio-Rad Laboratories). aOMV particle concentration were assessed by ZetaView analyzer (Particle Metrix GmbH). Measurements were assessed in triplicates and each individual data was obtained from two stationary layers with five times measurements in each layer. Sensitivity of camera was configured at 70 in all measurements. Data were analyzed using ZetaView analysis software version 8.2.30.1.

Preparation of OMVs

[00151] E. coli cultures were pelleted at 6,000 ^c g, 4 °C for 20 min, twice, and then the supernatant fraction was filtered through a 0.45-pm vacuum filter and was concentrated by ultrafiltration Vivaflow 200 module (Sartorius) with a 100 kDa cut-off membrane. The retentate was again filtered through a 0.22-pm vacuum filter to remove any remaining cells. The resulting filtrate was subjected to ultracentrifiigation at 150,000 ^c g, 4 °C for 3 h and resuspended in PBS. These extruded OMVs are considered similar to natural OMVs since the cells are not treated with detergent to generate the OMVs.

Transmission electron microscopy

[00152] Formvar/carbon Cu copper grids (Electron Microscopy Sciences) were glow discharge-treated before aOMVs were loaded. Then aOMVs were washed two times in distilled water and then fixed using 2.5% glutaraldehyde dissolved PBS. After two further washes in filtered water, the samples were stained using 2% uranyl acetate for 1.5 min. Negative-stained samples were examined on a digitized LEO 912AB Omega electron microscope (Carl Zeiss SMT) at 120 kV with a Veleta CCD camera (Olympus-SiS).

SDS-PAGE

[00153] E. coli lysates, OMVs, and aOMVs were separated by 10% SDS-PAGE and whole protein bands were stained by Coomassie brilliant blue G-250 dye (Thermo Fisher Scientific). For Western blot analysis, separated gel by 10% SDS-PAGE was transferred to a polyvinylidene difluoride membrane. The blocked membrane was then incubated with anti-OmpA antibody (lab-made), anti -lipid A antibody (Abeam), or anti-FtsZ antibody (Antibodies-online, Inc.). After incubation with horseradish peroxidase-conjugated secondary antibody, the immunoreactive bands were visualized with a chemiluminescent substrate.

RNA and DNA analysis

[00154] RNA from aOMVs or OMVs was isolated using miRCURY™ RNA isolation kit for biofluids (Exiqon) according to manufacturer’s protocol. DNA was isolated using Qiamp DNA Blood Mini kit (Qiagen) according to manufacturer’s protocol. One microliter of isolated RNA or DNA were analyzed for its quality, yield, and nucleotide length with capillary electrophoresis using Agilent RNA 6000 Nanochip and Agilent High sensitivity DNA chip, respectively, on an Agilent 2100 Bioanalyzer® (Agilent Technologies).

LC-MS/MS analysis

[00155] Two biological replicate aOMVs or OMVs (30 pg) were digested with trypsin using the fdter-aided sample preparation (FASP) method and C18 spin columns desalting according to manufacturer’s instructions. All fractions were dried on Speedvac and reconstituted in 3% acetonitrile and 0.2% formic acid and analyzed on Orbitrap Fusion Tribrid mass spectrometer interfaced with Easy- nLC 1200 (Thermo Fisher Scientific, Waltham, MA). Peptides were trapped on the Acclaim Pepmap 100 Cl 8 trap column (100 pm x 2 cm, particle size 5 pm; Thermo Fischer Scientific) and separated on the in- house packed C18 analytical column (75 pm x 30 cm, particle size 3 pm) using the gradient from 5% to 33% B in 160 min, from 33% to 100% B in 5 min, solvent A was 0.2% formic acid and solvent B was 80% acetonitrile and 0.2% formic acid. Precursor ion mass spectra were recorded at 120000 resolution, the most intense precursor ions were selected, fragmented using HCD at collision energy setting of 30 and the MS/MS spectra were recorded at 30000 resolution with the maximum injection time of 125 ms and the isolation window of 1.0 Da. Charge states 2 to 7 were selected for fragmentation, dynamic exclusion was set to 45 s with 10 ppm tolerance.

[00156] Results

[00157] aOMVs were prepared in higher yield than naturally produced OMVs or OMVs prepared by extrusion (Data not shown). aOMVs presented similar shape and size as OMVs, as shown by a transmission electron microscopy image (Data not shown). aOMV proteins had mostly similar size with outer membrane proteins, especially OmpA and LPS (Data not shown), which are major components on outer membrane. In addition, cytosolic proteins such as FtsZ were completely removed in aOMVs. RNA peaks were not detected in aOMVs in contrast to OMVs. And DNA contents in aOMVs were mostly removed. Data not shown.

[00158] 177 and 181 proteins were identified from OMVs and aOMVs, respectively. 112 proteins were identified in both vesicle preparations, whereas 65 and 69 proteins were uniquely identified in OMVs and aOMVs, respectively. Based on the relative protein abundance, 13 proteins did not change markedly in abundance among 112 proteins. However, 20 and 79 proteins were relatively increased and decreased in aOMVs as compared to OMVs, respectively. In the GO term subcellular localization analysis, aOMV proteome showed features distinct from OMV proteome. aOMV proteome was enriched with cell outer membrane proteins, whereas OMV proteome was enriched with cytosol and inner membrane proteins. In the GO term biological process analysis, aOMV proteome was enriched for proteins involved in ion transport. By contrast, OMV proteome was enriched for proteins involved translation and ribosomal subunit assembly. Data not shown.

Example 2: Less toxicity and immunological properties of E. coli aOMYs in vitro and in vivo

Methods

RAW 264.7 cytokines

[00159] RAW 264.7 (1 x 10⁵), a mouse macrophage cell line, were seeded into 24-well plates. Various dose of aOMVs and OMVs were applied to the cells to induce pro-inflammatory cytokines (TNF-a and IL-6) for 15 h. Supernatant concentrations of cytokines were measured by ELISA kit (R&D systems).

Mice experiments

[00160] Mice (wild-type mice of the C57BL/6 genetic background, 6 weeks old) were intraperitoneally (i.p.) injected with OMVs (5 x 10⁹) or aOMVs (5 x 10⁹, 2 x 10¹⁰). Mice were sacrificed at 6 h following anesthetization with i.p. injection of xylazine chloride (Bayer) and ketamine hydrochloride (Pfizer). Rectal temperature was measured by thermometer (Bioseb). Peritoneal fluid (PF) and blood were collected from mice, and then cytokines in the supernatant were analyzed by DuoSet ELISA Development kit (R&D Systems). Leukocytes and platelets in blood were counted using the light microscopy following incubation with 1% hydrochloride and Rees-Ecker diluting fluid (Thermo Fisher Scientific), respectively.

Dendritic cell uptake and cytokines

[00161] Bone marrow cells were harvested from the femur and the tibia of mice

(C57BL/6). The cells were differentiated into dendritic cells in 10% FBS/RPMI supplemented with nutrients and 20 ng/mL GM-CSF for one week. Separately, isolated aOMVs were labeled with DiO, followed by incubation with dendritic cells, and examined for uptake by dendritic cells using FACS. For the uptake inhibitor treatment, dendritic cells pretreated with dynasore (Sigma Aldrich) for 1 h were sequentially incubated with DiO-labeled aOMVs for 6 h. For cytokine analysis, differentiated dendritic cells (l x 10⁵) were seeded into 24-well plates. Various doses of aOMVs and OMVs were applied to the cells to induce TNFa, IL-6, IL-12p70, and IL-4 for 24 h. Supernatant concentrations of cytokines were measured by ELISA kit (R&D systems).

Antibody titer against E. coli lysates or OMV proteins

[00162] 5 x 10⁹ of OMVs or aOMVs were intraperitoneally injected to mice (wild-type

C57BL/6 genetic background, 6 weeks old) once a week for three weeks. Blood samples were taken from mice 3 days after each injection and assayed for their antibodies specific for E.coli lysates or OMV proteins. The mouse serum was 1:500 diluted in 1% BSA/PBS and placed in 96-well plates coated with 200 ng of E.coli lysates or OMV proteins. After incubation for 2 h, immunological changes were measured with a peroxidase -conjugated anti-mouse antibody.

Splenocyte cytokines

[00163] Seven days after the three injections of OMVs or aOMVs (5 x 10⁹), CD4+ T cells from spleen were isolated from the mice. The cells (5 x 10⁵) were incubated for 72 h with 100 ng/mL of OMVs, followed by ELISA to quantitatively analyze TNF-a, IFN-g, and IL-4.

Results

[00164] aOMVs did not induce IL-6 from macrophages, as compared to OMVs (Figure

2). TNF-a secretion from cells incubated with aOMVs was significantly lower than from cells incubated OMVs. There were no significant changes in body weight and temperature in aOMV-injected mice (Figure 3). Also, aOMVs did not cause increase in pro -inflammatory cytokines (Figure 4) and induced decrease in leukocyte and platelet number to a lesser extent than OMVs (Figure 5).

[00165] Secretion of TNF-a, IL-6, and IL-12p70, cytokines that induce T helper type 1 response, increased with an increase in the dose of aOMVs from bone marrow-derived dendritic cells (Figure 6, Panels A-D). Higher levels of TNF-a and IFN-g were secreted from splenic CD4+ T cells of aOMV-immunized group, compared to the sham group (Figure 7, Panels A-C). However, there was no change in the level of IL-4 between aOMV-immunized and sham group.

Example 3: Isolation and characterization of P. aeruginosa aOMVs

Methods

Preparation of aOMVs

[00166] A P. aeruginosa PAOl strain was acquired from ATCC company. The bacterial culture was pelleted, resuspended in 20% sucrose in 20 mM Tris, pH 8.0 (4 mL per g cells) and lysozyme (600 pg per g cells) were added. The resulting spheroplasts were pelleted, and then sonicated in ice-cold 10 mM Tris, pH 8.0. The cells were pelleted at 8,000 x g for 5 min, and then whole membranes were pelleted from the supernatants at 40,000 x g for 60 min. The membranes were resuspended in distilled water, freeze-thawed, and incubated in 0.5% Sarkosyl (sodium N- lauroylsarcinosinate; 20 min, 25°C). The outer membrane was pelleted (40,000 x g for 90 min), and incubated with high pH solution (200 mM Na₂CC>₃, pH 14.0) for 1 hour at 25°C. The pellets were applied to 4 mL of 50% iodixanol (Axis-Shield PoC AS), followed by addition of 4 mL of 30% iodixanol and 2 mL of 10% iodixanol to ultracentrifuge tube. The layers formed between 10% and 30% iodixanol after ultracentrifugation at 100,000 x g for 2 hours was collected. Finally, the samples were sonicated for 30 min, and considered aOMVs.

Quantification of aOMVs

[00167] Protein concentration of aOMVs was determined with a Bradford dye assay

Generation of OMVs

[00168] P. aeruginosa cultures were pelleted at 6,000 ^c g, 4 °C for 20 min, twice, and then the supernatant fraction was filtered through a 0.45-mih vacuum filter and was concentrated by ultrafiltration Vivaflow 200 module (Sartorius) with a 100 kDa cut-off membrane. The retentate was again filtered through a 0.22-pm vacuum filter to remove any remaining cells. The resulting filtrate was subjected to ultracentrifugation at 150,000 * g, 4 °C for 3 h and resuspended with PBS. These OMVs are considered similar to natural OMVs since the cells are not treated with detergent to generate the OMVs.

Transmission electron microscopy

[00169] Formvar/carbon Cu copper grids (Electron Microscopy Sciences) were glow discharge-treated before aOMVs were loaded. Then aOMVs were washed two times in distilled water and then fixed using 2.5% glutaraldehyde dissolved PBS. After two further washes in filtered water, the samples were stained using 2% uranyl acetate for 1.5 min. Negative-stained samples were examined on a digitized LEO 912AB Omega electron microscope (Carl Zeiss SMT) at 120 kV with a Veleta CCD camera (Olympus-SiS).

RNA and DNA analysis

[00170] RNA from aOMVs or OMVs was isolated using miRCURYTM RNA isolation kit for biofluids (Exiqon) according to manufacturer’s protocol. DNA was isolated using Qiamp DNA Blood Mini kit (Qiagen) according to manufacturer’s protocol. One microliter of isolated RNA or DNA were analyzed for its quality, yield, and nucleotide length with capillary electrophoresis using Agilent RNA 6000 Nanochip and Agilent High sensitivity DNA chip, respectively, on an Agilent 2100 Bioanalyzer® (Agilent Technologies).

LC-MS/MS analysis:

[00171] Two biological replicate aOMVs or OMVs (30 pg) were digested with trypsin using the filter-aided sample preparation (FASP) method and C18 spin columns desalting according to manufacturer’s instructions. All fractions were dried on Speedvac and reconstituted in 3% acetonitrile and 0.2% formic acid and analyzed on Orbitrap Fusion Tribrid mass spectrometer interfaced with Easy- nLC 1200 (Thermo Fisher Scientific, Waltham, MA). Peptides were trapped on the Acclaim Pepmap 100 Cl 8 trap column (100 pm x 2 cm, particle size 5 pm; Thermo Fischer Scientific) and separated on the in- house packed C18 analytical column (75 pm x 30 cm, particle size 3 pm) using the gradient from 5% to 33% B in 160 min, from 33% to 100% B in 5 min, solvent A was 0.2% formic acid and solvent B was 80% acetonitrile and 0.2% formic acid. Precursor ion mass spectra were recorded at 120000 resolution, the most intense precursor ions were selected, fragmented using HCD at collision energy setting of 30 and the MS/MS spectra were recorded at 30000 resolution with the maximum injection time of 125 ms and the isolation window of 1.0 Da. Charge states 2 to 7 were selected for fragmentation, dynamic exclusion was set to 45 s with 10 ppm tolerance.

Results

[00172] aOMVs were prepared in higher yield than naturally produced OMVs and extruded OMVs (Data not shown). aOMVs presented similar shape and size with OMVs, but were clearer as visualized by transmission electron microscopy (Data not shown). RNA contents in aOMVs were almost removed in contrast to OMVs (Data not shown). DNA peaks were not detectable in both aOMVs and OMVs (Data not shown).

[00173] A total of 496 and 227 proteins from OMVs and aOMVs, respectively (Data not shown) were identified. 173 proteins were identified in both vesicle preparations, whereas 323 and 54 proteins were uniquely identified in OMVs and aOMVs, respectively. Based on the relative protein abundance, 28 proteins did not change markedly in abundance among 173 proteins. However, 93 and 52 proteins were relatively increased and decreased in aOMVs, respectively, as compared to their level in naturally occurring OMVs and exOMVs. In the GO term subcellular localization analysis, aOMV- enriched proteome showed distinct features from OMV-enriched proteome (Data not shown). aOMV proteome was enriched with cell outer membrane proteins, whereas OMV proteome was enriched with cytosol and inner membrane proteins. In the GO term biological process analysis, aOMV proteome was enriched with biological processes including cell motility and ion transport (Data not shown). By contrast, OMV proteome was enriched with biological processes including translation and metabolism.

Example 4: Less toxicity and immunological properties of P. aeruginosa aOMVs in vitro

Methods

MH-S cytokines

[00174] MH-S (l x 10⁵), a mouse lung macrophage cell line, were seeded into 24-well plates. Various dose of aOMVs and OMVs were applied to the cells to induce pro-inflammatory cytokines (TNF-a and IL-6) for 15 h. Supernatant concentrations of cytokines were measured by ELISA kit (R&D systems).

Antibody titer against P. aeruginosa lysates

[00175] 5 x 10⁹ of OMVs or aOMVs were intraperitoneally injected to mice (wild-type

C57BL/6 genetic background, 6 weeks old) once a week for three weeks. Blood samples were taken from mice 3 days after each injection and assayed for their antibodies specific for P. aeruginosa lysates. The mouse serum was 1:500 diluted in 1% BSA/PBS and placed in 96-well plates coated with 200 ng of E. coli lysates. After incubation for 2 h, immunological changes were measured with a peroxidase- conjugated anti-mouse antibody.

Splenocyte cytokines

[00176] Seven days after the three injections of OMVs or aOMVs (5 x 10⁹), CD4+ T cells from spleen were isolated from the mice. The cells (5 x 10⁵) were incubated for 72 h with 1 pg/mL of P. aeruginosa proteins, followed by ELISA to quantitatively analyze TNF-a, IFN-g, IL-4, and IL-17.

Results

[00177] aOMVs were observed to increase TNF-a and IL-6 from macrophages to a lesser extent than OMVs (Figure 8), showing that aOMVs might be much safer vaccine candidate.

[00178] The P. aeruginosa lysate-specific antibodies in the mouse blood started to from

7 days after the first injection of aOMVs, and were amplified by the second and the third injection, with a peak at 7 days after the third injection (Figure 9). And, higher levels of TNF-a, IFN-g, and IL-17 (Th-1 and Th-17-re lated) were secreted from splenic CD4+ T cells of aOMV-immunized group, compared to the sham group (Figure 10, Panels A-D). Interestingly, IFN- g was dramatically increased in aOMV- than OMV-immunized group. However, there was no change in the level of IL-4 in aOMV or OMV- immunized compared to sham group.

Example 5: Isolation and characterization of aOMVs derived from spike protein SI -overexpressed bacteria

Methods

[00179] Display of spike protein SI on the bacterial surface: E. coli BL21 (DE3) was used for all experiments. Plasmid pET-28a(+) contains a fusion of the signal sequence and first nine amino acids of Lpp, the sequence for five outer membrane -spanning domains of OmpA, and the full sequence for SARS-CoV-2 spike protein SI (Figure 11). For SI overexpression, bacterial cultures were inoculated at a ratio of 1/50 from overnight cultures and grown in 200 mL LB medium, supplemented with 50 pg/mL kanamycin. When cultures reached ant optical density of OD600 = 0.6, protein expression was induced by addition of 0.1 mM IPTG (Thermo Fisher Scientific). [00180] Preparation of aOMYs: The bacteria was cultured for 3 hours with 0.1 mM

IPTG as described above. The culture was pelleted, resuspended in 20% sucrose in 20 mM Tris, pH 8.0 (4 mL per g cells), lysozyme (600 pg per g cells), and 0.1 M EDTA (0.2 mL per g cells) were added. The resulting spheroplasts were pelleted, and then sonicated in ice-cold 10 mM Tris, pH 8.0. The cells were pelleted at 8,000 x g for 5 min, and then whole membranes were pelleted from the supernatants at 40,000 x g for 60 min. The membranes were resuspended in distilled water, freeze-thawed, and incubated in 0.5% Sarkosyl (sodium N-lauroylsarcinosinate; 20 min, 25°C). The outer membrane was pelleted (40,000 x g for 90 min), and incubated with high pH solution (200 mM Na2C03, pH 14.0) for 1 hour at 25°C. The pellets were applied to 4 mL of 50% iodixanol (Axis-Shield PoC AS), followed by addition of 4 mL of 30% iodixanol and 2 mL of 10% iodixanol to ultracentrifuge tube. The layers formed between 30% and 50% iodixanol after ultracentrifugation at 100,000 x g for 2 hours was collected finally, the samples were sonicated for 30 min, and considered aOMVs (figure 1).

[00181] Quantification of aOMVs: Protein concentration of aOMVs was determined with a Bradford dye assay (Bio-Rad Laboratories). aOMV particle concentration were assessed by ZetaView analyzer (Particle Metrix GmbH). Measurements were assessed in triplicates and each individual data was obtained from two stationary layers with five times measurements in each layer. Sensitivity of camera was configured at 70 in all measurements. Data were analyzed using ZetaView analysis software version 8.2.30.1.

[00182] Transmission electron microscopy: Lormvar/carbon Cu copper grids (Electron

Microscopy Sciences) were glow discharge-treated before aOMVs were loaded. Then aOMVs were washed two times in distilled water and then fixed using 2.5% glutaraldehyde dissolved PBS. After two further washes in filtered water, the samples were stained using 2% uranyl acetate for 1.5 min. Negative- stained samples were examined on a digitized LEO 912AB Omega electron microscope (Carl Zeiss SMT) at 120 kV with a Veleta CCD camera (Olympus-SiS).

[00183] SDS-PAGE: Bacterial lysates and aOMVs were separated by 10% SDS-PAGE and the separated gel was transferred to a polyvinylidene difluoride membrane. The blocked membrane was then incubated with anti-His tag antibody (Thermo Lisher Scientific) to confirm overexpression of SI. After incubation with horseradish peroxidase-conjugated secondary antibody, the immunoreactive bands were visualized with a chemiluminescent substrate.

Results

[00184] Bacterial aOMVs presented nano-sized spherical structures, and no bacterial structures were observed (figure 12). Also, SI protein complex was well overexpressed on aOMVs, and more enriched as compared to cell lysates (500 fold; figure 13). Example 6: Immunological properties of E. coli aOMV^sl in vivo

Methods

[00185] Antibody titer against SI or aOMV proteins: Various doses of aOMVSl (10, 100, 1000 ng) were subcutaneously injected to mice (wild-type C57BL/6 genetic background, 6 weeks old) once a week for three weeks. Blood samples were taken from mice 3 days after each injection and assayed for their antibodies specific for recombinant SI or aOMV proteins. The mouse serum was 1:500 diluted in 1% BSA/PBS and placed in 96-well plates coated with 200 ng of recombinant SI or aOMV proteins. After incubation for 2 h, immunological changes were measured with a peroxidase-conjugated anti-mouse antibody.

Results

[00186] The SI or aOMV-specific antibodies in the mouse blood were induced after the first boosting of aOMV^sl, and were amplified by the second boost, with a peak at 7 days after the last injection (Figures 14A and 14B).

[00187] Fig. 22 shows the level of IFN-gamma secreted from mouse splenic CD4⁺ T cells upon ex vivo treatment with S 1 after the mice were immunized with 5 x 10⁹ of SyB V^S1. * * * , P<0.001; versus sham group; one way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3. SyBV^sl is an alternate term for aOMV^sl.

[00188] Fig. 23 shows the percentage of various adaptive immune cell types in spleens, after mice were subcutaneously immunized with SyBV^sl three times at regular intervals of one week.

[00189] Bone marrow-derived dendritic cell (BMDC) cytokines: SyBV^sl were lyophilized and then stored at room temperature, 4 degrees or -20 degrees for one month. Separately, bone marrow cells were harvested from the femur and the tibia of mice (C57BF/6). The cells were differentiated into dendritic cells in 10% FBS/RPMI supplemented with nutrients and 20 ng/mF GM- CSF for one week. For cytokine analysis, differentiated dendritic cells (1 x 10⁵) were seeded into 24-well plates. Various conditions of SyBV^sl (5 xlO⁹) were applied to the cells to induce IFN-beta for 24 h. Supernatant concentrations of cytokines were measured by EFISA kit (R&D systems).

[00190] Fig. 24 is a graph of IFN-beta in the supernatants of mouse BMDCs treated with lyophilized SyBV^sl (5 x 10⁹) for 24 h. *, P<0.05; **, P<0.01; one way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3. Fyophilized SyBV^sl were kept at RT, 4°C, or -20°C for 1 month.

[00191] High level of IFN-g was secreted from splenic CD4⁺ T cells of SyBV^sl immunized group, compared to the sham (Figure 22), showing Sl-specific adaptive response. Also, B cell, T cell and memory cell were mostly observed in spleen immunized with SyBV^sl (Figure 23). Regarding stability of SyBV^sl, there were no change of immunogenic activity (Figure 24) and morphology of vesicles regardless of lyophilization and storage condition.

[00192]

Example 7: Isolation and characterization of aOMYs derived from receptor binding domain (RBD)- overexpressing bacteria

Methods

[00193] Display of RBD on the bacterial surface: E. coli BL21 (DE3) was used for all experiments. Plasmid pET-28a(+) contains a fusion of the signal sequence and first nine amino acids of Lpp, the sequence for five outer membrane -spanning domains of OmpA, and the full sequence for SARS-CoV-2 RBD protein (Figure 15A). For RBD overexpression, bacterial cultures were inoculated at a ratio of 1/50 from overnight cultures and grown in 200 mL LB medium, supplemented with 50 pg/mL kanamycin. When cultures reached ant optical density of OD600 = 0.6, protein expression was induced by addition of 0.1 mM IPTG (Thermo Fisher Scientific). Finally, aOMVs were isolated from the engineered bacteria as described in Figure 1.

[00194] SDS-PAGE: Bacterial aOMVs were separated by 10% SDS-PAGE and the separated gel was transferred to a polyvinylidene difluoride membrane. The blocked membrane was then incubated with anti -RBD antibody (Elabscience) to confirm overexpression of RBD. After incubation with horseradish peroxidase -conjugated secondary antibody, the immunoreactive bands were visualized with a chemiluminescent substrate.

[00195] Antibody titer against RBD proteins: SyBVRBD (5 x 10⁹) were subcutaneously injected to mice (wild-type C57BL/6 genetic background, 6 weeks old) once a week for three weeks. Blood samples were taken from mice 3 days after each injection and assayed for their antibodies specific for recombinant RBD proteins. The mouse serum was 1:500 diluted in 1% BSA PBS and placed in 96- well plates coated with 200 ng of recombinant RBD proteins. After incubation for 2 h, immunological changes were measured with a peroxidase-conjugated anti-mouse antibody.

[00196] Splenocyte cytokines: Seven days after the three injections of SyBV^RliD (5 x

10⁹), CD4+ T cells from spleen were isolated from the mice. The cells (5 x 10⁵) were incubated for 72 h with 1 pg/mL of RBD, followed by ELISA to quantitatively analyze IFN-g.

Results

[00197] RBD protein complex was well overexpressed on aOMVs as compared to control aOMVs prepared from bacteria not treated with IPTG (Figure 15B).

[00198] Figure 25 A is a graph showing the levels of RBD protein-specific antibodies measured in the course of three subcutaneous immunization of SyBV¹*™ (5 x 10⁹, at weekly intervals). *, P<0.05; versus sham group; one way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=5. SyBV^RliD is an alternate name for aOMV^RliD.

[00199] Figure 25B shows the level of IFN -gamma secreted from mouse splenic CD4⁺ T cells upon ex vivo treatment with RBD after the mice were immunized with 5 x 10⁹ of SyBV^RliD. ***, P<0.001; versus sham group; one way ANOVA with Tukey’s multiple comparison test. Error bars indicate SEM. N=3.

[00200] The RBD-specific antibodies in the mouse blood started after the first boosting of SyBV™⁰, and were amplified by the second boosting (Figure 25A). Also, high level of IFN-g was secreted from splenic CD4⁺ T cells of SyBV^RBD-immunized group, compared to the sham (Figure 25B), showing RBD-specific adaptive response induced by SyBV^RliD

Example 8: Immunization with recombinant RBD proteins together with E. coli aOMYs

Methods

[00201] Antibody titer against RBD proteins: Mice (wild-type C57BL/6 genetic background, 6 weeks old) were subcutaneously injected once a week for three weeks with two doses of RBD (1 and 10 pg) in combination with E. coli aOMVs (1 pg). 1 pg of aOMVs was used. aOMVs and recombinant RBD proteins were isolated separately and then mixed together just before immunization. Blood samples were taken from mice 3 days after each injection and assayed for their antibodies specific for RBD proteins. The mouse serum was 1:500 diluted in 1% BSA/PBS and placed in 96-well plates coated with 200 ng of RBD proteins. After incubation for 2 h, immunological changes were measured with a peroxidase-conjugated anti-mouse antibody.

[00202] Splenocvte cytokines: Seven days after the three injections of two doses of RBD

(1 and 10 pg) in combination with SyBV (5 x 10⁹), CD4⁺ T cells from spleen were isolated from the mice. The cells (5 x 10⁵) were incubated for 72 h with 1 pg/mL of RBD, followed by ELISA to quantitatively analyze IFN-g.

Results

[00203] The RBD-specific antibodies in the mouse blood were significantly increased in mouse immunized with RBD plus aOMVs. 10 pg RBD was more effective in inducing RBD-specific antibodies 1 pg RBD. See Figure 16.

[00204] High level of IFN -g was secreted from splenic CD4⁺ T cells of mice immunized with RBD plus SyBV, and RBD 10 pg group have shown more increased cytokine profile than 1 pg group (Figure 26). Example 9: Optimization of extruded 0MV^S1 isolation protocol

Methods

[00205] Preparation of extruded OMV^sl with ultracentrifugation method: Engineered E. coli cultures were pelleted at 6,000 ^c g, 4 °C for 20 min, twice, and then the supernatant fraction was filtered through a 0.45-pm vacuum filter and was concentrated by ultrafiltration Vivaflow 200 module (Sartorius) with a 100 kDa cut-off membrane. The retentate was again filtered through a 0.22-pm vacuum filter to remove any remaining cells. The resulting filtrate was subjected to ultracentrifugation at 150,000 x g, 4 °C for 3 h and resuspended in PBS. The suspension was incubated with high pH solution (200 mM Na₂CC>₃, pH 14.0) for 1 hour at 25°C. The pellets were applied to 4 mL of 50% iodixanol (Axis-Shield PoC AS), followed by addition of 4 mL of 30% iodixanol and 2 mL of 10% iodixanol to ultracentrifuge tube. The layers formed between 30% and 50% iodixanol after ultracentrifiigation at 100,000 x g for 2 hours was collected. Finally, the samples were sonicated for 30 min (Figure 17).

[00206] Preparation of extruded OMV^sl with PEG precipitation method: Engineered E. coli cultures were pelleted at 6,000 ^c g, 4 °C for 20 min, twice, and then the supernatant fraction was filtered through a 0.45-pm vacuum filter and was concentrated by ultrafiltration Vivaflow 200 module (Sartorius) with a 100 kDa cut-off membrane. The retentate was were mixed with 50% PEG (Sigma Aldrich) to make a final PEG concentration of 10%. After incubation for 2 h at 4°C, the samples were centrifuged at 3,000 x g for 10 min for pelleting. PEG of 50% was added once again to the vesicle pellets, followed by incubation for 2 h at 4°C and centrifugation at 3,000 x g for 10 min. The resulting pellets were incubated with high pH solution (200 mM Na₂CC>₃, pH 14.0) for 1 hour at 25°C. And then the mixed solution was incubated with 50% PEG (Sigma Aldrich) to make a final PEG concentration of 10%. After incubation for 2 h at 4°C, the samples were centrifuged at 3,000 x g for 10 min for pelleting. Finally, the samples were sonicated for 30 min (Figure 17).

[00207] Transmission electron microscopy: Formvar/carbon Cu copper grids (Electron

Microscopy Sciences) were glow discharge-treated before OMVs were loaded. Then OMVs were washed two times in distilled water and then fixed using 2.5% glutaraldehyde dissolved PBS. After two further washes in filtered water, the samples were stained using 2% uranyl acetate for 1.5 min. Negative- stained samples were examined on a digitized LEO 912AB Omega electron microscope (Carl Zeiss SMT) at 120 kV with a Veleta CCD camera (Olympus-SiS).

[00208] SDS-PAGE: OMVs were separated by 10% SDS-PAGE and the separated gel was transferred to a polyvinylidene difluoride membrane. The blocked membrane was then incubated with anti -His tag antibody (Thermo Fisher Scientific) to confirm overexpression of SI. After incubation with horseradish peroxidase -conjugated secondary antibody, the immunoreactive bands were visualized with a chemiluminescent substrate. Results

[00209] The existence of pure vesicles isolated with ultracentrifugation or PEG precipitation was confirmed by electron microscopy (Figure 18). Sonication step was not necessary for vesicular formation (Figure 17). Instead, outer membrane sheets were stored at 4°C overnight to slowly induce vesiculation to form OMVs. Also, SI protein complex was well expressed on extruded OMVs by PEG precipitation regardless of sonication (Figure 19).

Example 10: Optimization of aOMV^sl isolation protocol

Methods

[00210] Preparation of aOMV^sl with PEG precipitation method: Outer membranes isolated as described above (Figure 1) were incubated with high pH solution (200 mM Na2C03, pH 14.0) for 1 hour at 25°C. And then the mixed solution was incubated with 50% PEG (Sigma Aldrich) to make a final PEG concentration of 10%. After incubation for 2 h at 4°C, the samples were centrifuged at 3,000 x g for 10 min for pelleting. PEG of 50% was added once again to the vesicle pellets, followed by incubation for 2 h at 4°C and centrifugation at 3,000 x g for 10 min. The resulting pellets were sonicated for 30 min (Figure 20).

[00211] SDS-PAGE: Bacterial aOMVs were separated by 10% SDS-PAGE and the separated gel was transferred to a polyvinylidene difluoride membrane. The blocked membrane was then incubated with anti-S 1 antibody (Arigo biolaboratories) to confirm overexpression of S 1. After incubation with horseradish peroxidase-conjugated secondary antibody, the immunoreactive bands were visualized with a chemiluminescent substrate.

Results

[00212] Also, SI protein complex was well expressed on artificially produced aOMVs by PEG precipitation regardless of sonication (Figure 21).

[00213] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:

1. A composition comprising

(i) non-naturally occurring artificial outer membrane vesicles (aOMVs) generated from a gram negative bacterium, wherein the aOMVs are deficient in one or more of the following components present in the gram -negative bacterium: periplasmic proteins, inner membrane proteins, nucleic acids, cytoplasmic proteins, and ribosomes; and/or wherein the aOMVs are enriched in outer membrane proteins; and wherein the gram-negative bacterium is genetically modified for expression of at least one SARS-CoV-2 protein in the outer membrane; or

(ii) non-naturally occurring aOMVs generated from a gram -negative bacterium, wherein the aOMVs are deficient in one or more of the following components present in the gram -negative bacterium: periplasmic proteins, inner membrane proteins, nucleic acids, cytoplasmic proteins, and ribosomes; and/or wherein the aOMVs are enriched in outer membrane proteins; and a recombinant SARS-CoV-2 protein; or

(iii) non-naturally occurring (aOMVs) generated from a gram -negative bacterium, wherein the aOMVs are deficient in one or more of the following components present in the gram -negative bacterium: periplasmic proteins, inner membrane proteins, nucleic acids, cytoplasmic proteins, and ribosomes; and/or wherein the aOMVs are enriched in outer membrane proteins, wherein the gram negative bacterium is genetically modified for expression of at least one SARS-CoV-2 protein in the outer membrane; and a recombinant SARS-CoV-2 protein.

2. The composition of claim 1, wherein the aOMVs are deficient in one or more of the following components present in the gram-negative bacterium: periplasmic proteins, inner membrane proteins, nucleic acids, cytoplasmic proteins, and ribosomes.

3. The composition of claim 1, wherein the aOMVs are enriched in outer membrane proteins.

4. The composition of claim 1, wherein the aOMVs are deficient in one or more of the following components present in the gram-negative bacterium: periplasmic proteins, inner membrane proteins, nucleic acids, cytoplasmic proteins, and ribosomes and are enriched in outer membrane proteins.

5. The composition of any one of claims 1-4, wherein the gram -negative bacterium is genetically modified for displaying a SARS-CoV-2 protein on the outer membrane, wherein optionally the SARS-CoV-2 protein is a SARS-CoV-2 spike protein.

6. The composition of claim 5, wherein the SARS-CoV-2 spike protein is conjugated to an outer membrane protein A (OmpA) protein.

7. The composition of claim 6, wherein the OmpA protein is conjugated to a lipoprotein signal peptide.

8. The composition of claim 7, wherein the OmpA protein is conjugated to a lipoprotein signal peptide via an N-terminal fragment of E. coli lipoprotein (Lpp).

9. The composition of any one of claims 5-8, wherein the SARS-CoV-2 spike protein comprises S 1 protein or a fragment thereof.

10. The composition of claim 9, wherein the fragment of SI protein comprises receptor binding domain (RBD).

11. The composition of any one of claims 1-4, wherein the composition comprises a recombinant SARS-CoV-2 protein, wherein the SARS-CoV-2 protein is optionally a SARS-CoV-2 spike protein.

12. The composition of claim 11, wherein the recombinant SARS-CoV-2 spike protein comprises S 1 protein or a fragment thereof.

13. The composition of claim 12, wherein the fragment of SI protein comprises receptor binding domain (RBD).

14. The composition of any one of claims 1-13, wherein the aOMVs have a diameter of 50 nm - 150 nm.

15. The composition of any one of claims 1-14, wherein the gram-negative bacterium is genetically modified for decreased production of lipopolysaccharides (LPS).

16. The composition of any one of claims 1-15, wherein the gram -negative bacterium is genetically modified for increased expression of at least one endogenous outer membrane protein.

17. The composition of any one of claims 1-15, wherein the gram -negative bacterium is Escherichia coli (E. coli).

18. The composition of any one of claims 1-16, wherein the gram -negative bacterium is Pseudomonas aeruginosa ( P . aeruginosa).

19. A method of producing an immune response to SARS-CoV-2 in a mammalian subject, the method comprising administering the composition of any one of claims 1-18 to the subject in an amount effective to induce an immune response to the SARS-CoV-2 in the mammalian subject.

20. The method of claim 19, wherein the administering produces reduced inflammatory response as compared to that produced by administering naturally occurring OMVs released by the gram -negative bacterium.

21. The method of claim 20, wherein the reduced inflammatory response comprises production of a lower level of pro-inflammatory cytokines as compared to that produced by administering naturally occurring OMVs released by the gram -negative bacterium.

22. The method of claim 20, wherein the reduced inflammatory response comprises production of a lower level of tumor necrosis factor alpha (TNF-a) and/or interleukin-6 (IL-6) as compared to that produced by administering naturally occurring OMVs released by the gram -negative bacterium.

23. The method of claim 19, wherein the administering produces a reduction in toxic response as compared to the toxic response produced by administering naturally occurring OMVs released by the gram -negative bacterium.

24. The method of claim 23, wherein the toxic response comprises one or more of a reduction in number of leukocytes, a reduction in number of platelets, and a reduction in body weight.

25. The method of any one of claims 19-24, wherein the immune response comprises generation of antibodies against SARS-CoV-2.

26. The method of any one of claims 19-25, wherein the immune response comprises activation of T cells against SARS-CoV-2.

27. The method of any one of claims 19-26, wherein the aOMVs are generated from Escherichia coli ( E . coli).

28. The method of any one of claims 19-26, wherein the aOMVs are generated from Pseudomonas aeruginosa ( P . aeruginosa).

29. A method for generating the non-naturally occurring artificial outer membrane vesicles (aOMVs) from a gram-negative bacterium according to any one of claims 1-28, the method comprising: a) disrupting a spheroplast generated from the gram-negative bacterium to generate vesicles comprising outer membrane and vesicles comprising inner membrane; b) exposing the vesicles to an ionic surfactant to disrupt vesicles comprising inner membrane and to an alkaline pH to open the vesicles comprising outer membrane thereby generating outer membrane sheets; c) purifying the outer membrane sheets; and d) applying energy to the purified outer membrane sheets sufficient to convert the outer membrane sheets into aOMVs, thereby generating the non-naturally occurring aOMVs.

30. The method of claim 29, comprising generating the spheroplast from the gram-negative bacterium by incubating the gram-negative bacterium with lysozyme under conditions sufficient for removal of peptidoglycan layer in cell wall of the gram-negative bacterium, thereby converting the gram negative bacterium into the spheroplast.

31. The method of claim 29 or 30, wherein disrupting the spheroplast to generate the vesicles comprises applying shear force to the spheroplast.

32. The method of claim 29 or 30, wherein disrupting the spheroplast to generate the vesicles comprises applying acoustic energy to the spheroplast.

33. The method of any one of claims 29-32, wherein step b) comprises exposing the vesicles to the ionic surfactant to disrupt vesicles comprising inner membrane, isolating the vesicles comprising the outer membrane, and exposing the vesicles comprising the outer membrane to the alkaline pH.

34. The method of any one of claims 29-33, wherein the alkaline pH comprises a pH of 11-

14.

35. The method of any one of claims 29-34, wherein step c) comprises density centrifugation.

36. The method of any one of claims 29-35, wherein step d) comprises applying shear force to the purified outer membrane sheets.

37. The method of any one of claims 29-35, wherein step d) comprises applying acoustic energy to the purified outer membrane sheets.

38. A method for generating the non-naturally occurring artificial outer membrane vesicles (aOMVs) from a gram-negative bacterium according to any one of claims 1-28, the method comprising: a) disrupting a spheroplast generated from the gram-negative bacterium to generate vesicles comprising outer membrane and vesicles comprising inner membrane; b) exposing the vesicles to an ionic surfactant to disrupt vesicles comprising inner membrane; c) purifying the vesicles comprising outer membrane; and d) exposing the vesicles comprising outer membrane to an alkaline pH to open the vesicles thereby generating outer membrane sheets; e) purifying the outer membrane sheets; and f) applying energy to the purified outer membrane sheets sufficient to convert the outer membrane sheets into aOMVs, thereby generating the non-naturally occurring aOMVs.

39. The method of claim 38, comprising generating the spheroplast from the gram-negative bacterium by incubating the gram-negative bacterium with lysozyme under conditions sufficient for removal of peptidoglycan layer in cell wall of the gram-negative bacterium, thereby converting the gram negative bacterium into the spheroplast.

40. The method of claim 38 or 39, wherein disrupting the spheroplast to generate the vesicles comprises applying shear force to the spheroplast.

41. The method of claim 38 or 39, wherein disrupting the spheroplast to generate the vesicles comprises applying acoustic energy to the spheroplast.

42. The method of any one of claims 38-41, wherein the alkaline pH comprises a pH of 11-

14.

43. The method of any one of claims 38-42, wherein step e) comprises density gradient centrifugation or polyethylene glycol (PEG) precipitation.

44. The method of any one of claims 38-43, wherein step f) comprises applying shear force to the purified outer membrane sheets.

45. The method of any one of claims 38-43, wherein step f) comprises applying acoustic energy to the purified outer membrane sheets.

46. A composition comprising:

(i) non-naturally occurring extruded outer membrane vesicles (exOMVs) generated from a gram-negative bacterium, wherein the gram-negative bacterium is genetically modified for localizing a SARS-CoV-2 protein on the outer membrane; or

(ii) exOMVs generated from a gram-negative bacterium and a recombinant SARS-CoV-2 protein; or

(iii) non-naturally occurring exOMVs generated from a gram -negative bacterium and a recombinant SARS-CoV-2 protein, wherein the gram -negative bacterium is genetically modified for expression of a SARS-CoV-2 protein on the outer membrane.

47. The composition of claim 46, wherein the gram -negative bacterium is genetically modified for displaying a SARS-CoV-2 protein on the outer membrane, wherein optionally the SARS- CoV-2 protein is a SARS-CoV-2 spike protein.

48. The composition of claim 47, wherein the SARS-CoV-2 spike protein is conjugated to an outer membrane protein A (OmpA) protein.

49. The composition of claim 48, wherein the OmpA protein is conjugated to a lipoprotein signal peptide.

50. The composition of claim 49, wherein the OmpA protein is conjugated to a lipoprotein signal peptide via an N-terminal fragment of E. coli lipoprotein (Lpp).

51. The composition of any one of claims 47-50, wherein the SARS-CoV-2 spike protein comprises S 1 protein or a fragment thereof.

52. The composition of claim 51, wherein the fragment of S 1 protein comprises receptor binding domain (RBD).

53. The composition of claim 46, wherein the composition comprises a recombinant SARS- CoV-2 protein, wherein optionally the SARS-CoV-2 protein is a SARS-CoV-2 spike protein.

54. The composition of claim 53, wherein the recombinant SARS-CoV-2 spike protein comprises S 1 protein or a fragment thereof.

55. The composition of claim 54, wherein the fragment of S 1 protein comprises receptor binding domain (RBD).

56. The composition of any one of claims 46-55, wherein the exOMVs have a diameter of 50 nm - 150 nm.

57. The composition of any one of claims 46-56, wherein the gram -negative bacterium is genetically modified for decreased production of lipopoly saccharides (LPS).

58. The composition of any one of claims 46-57, wherein the gram -negative bacterium is genetically modified for increased expression of at least one endogenous outer membrane protein.

59. The composition of any one of claims 46-58, wherein the gram-negative bacterium is Escherichia coli ( E . coli).

60. The composition of any one of claims 46-58, wherein the gram-negative bacterium is Pseudomonas aeruginosa ( P . aeruginosa).

61. The composition of any one of claims 46-60, wherein the extruded vesicles are treated with high pH to remove intra-vesicular content.

62. A method of producing an immune response to SARS-CoV-2 in a mammalian subject, the method comprising administering the composition of any one of claims 46-60 to the subject in an amount effective to induce an immune response to the SARS-CoV-2 in the mammalian subject.

63. The method of claim 61, wherein the immune response comprises generation of antibodies against SARS-CoV-2.

64. The method of claim 61 or 62, wherein the immune response comprises activation of T cells against SARS-CoV-2.

65. A method for generating the non-naturally occurring extruded outer membrane vesicles (exOMVs) from a gram -negative bacterium according to any one of claims 46-63, the method comprising: a) serially extruding gram-negative bacteria through fdters comprising pores of sequentially smaller size; b) concentrating the filtrate; and c) resuspending the filtrate to provide the exOMVs.

66. The method of claim 65, wherein concentrating the filtrate comprises centrifugation.

67. The method of claim 66, wherein centrifugation comprises ultracentrifugation.

68. The method of claim 66, wherein centrifugation comprises density gradient centrifugation.

69. The method of claim 65, wherein concentrating the filtrate comprises precipitation.

70. The method of claim 69, wherein precipitation comprises polyethylene glycol (PEG) precipitation.

71. The method of any one of claims 65-70, wherein the exOMVs are exposed to a high pH solution to open the exOMVs.

72. The method of claim 71, wherein the exOMVs exposed to the high pH solution are purified.

73. The method of claim 72, wherein the purification of the exOMVs exposed to the high pH solution comprises density gradient centrifugation or precipitation.

74. The method of any one of claims 65-73, wherein the alkaline pH comprises a pH of 11- 14.

75. The method of any one of claims 71-74, wherein further comprising applying energy to the exOMVs exposed to the reclose the opened exOMVs.

76. The method of claim 75, wherein applying energy comprises applying shear force to the opened exOMVs.

77. The method of claim 75, wherein applying energy comprises applying acoustic energy to the opened exOMVs.