Materials and methods for improved three-dimensional immunity

Abstract

Provided are improved immunogenic compositions, such as those comprising a nanodisc. In some aspects the nanodisc comprises a membrane scaffold protein (MSP), a phospholipid, and an immunogen, wherein the membrane scaffold protein and the immunogen are from different species. Provided is a method of inducing an improved immune response in a subject, the method comprising administering to the subject the immunogenic composition, wherein the MSP is from or is derived from the same species as the subject. Provided is a system for inducing an immune response in a subject, comprising the immunogenic composition, wherein the MSP is from or is derived from the same species as the subject. Provided is a system for active immunization to prevent a disease in a subject, the system comprising the immunogenic composition, wherein the MSP is from or is derived from the same species as the subject. Preferably, MSP is not immunogenic to subject.

Description

MATERIALS AND METHODS FOR IMPROVED THREE-DIMENSIONAL IMMUNITYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Applications 63 / 659,248 and 63 / 659,255, both filed on June 12, 2024, which are hereby incorporated by reference in their entirety.FIELD

[0002] Materials and methods for generating an enhanced immune response and breaking immune tolerance in a mammal, including the field of novel three-dimensional adjuvant and delivery platforms and systems.BACKGROUND

[0003] A challenge in vaccine development has been generating an enhanced immune response and breaking immune tolerance. There remains a need for novel materials, methods, and systems for making effective vaccines.SUMMARY

[0004] The inventors of the present invention appreciate that the identification of novel adjuvants, enhanced immunity to a desired immunogen, systems, and delivery platforms for generating an enhanced immune response and breaking immune tolerance and immune suppression, etc. is a challenge that was not previously met.

[0005] Provided are materials, methods, and systems for using three dimensional materials, including nanodiscs and the like, comprising a membrane scaffold protein (MSP), a phospholipid, and an immunogen(s) as an antigen delivery platform, system, or adjuvant. As an adjuvant / delivery platform, nanodiscs allow generation of an immune response against the immunogen in the nanodisc in an improved format and, for example not against the MSPs or other immunogens, such as those stabilizing the nanodisc. MSPs are endogenous proteins present in animal serum.

[0006] Provided is an immunogenic composition comprising: a nanodisc comprising a membrane scaffold protein (MSP), a phospholipid, and an immunogen.

[0007] In some embodiments, the phospholipid is part of a phospholipid bilayer.

[0008] In some embodiments, the immunogen is lipidated.

[0009] In some embodiments, the immunogen comprises a tag. In further embodiments, the tag is a His tag.

[0010] In some embodiments, the MSP is non-naturally occurring and / or non-human.

[0011] In some embodiments, the immunogen is antigenic.

[0012] In some embodiments, the nanodisc comprises more than one different immunogen.

[0013] In some embodiments, the immunogenic composition comprises more than one different nanodisc.

[0014] In some embodiments, the MSP is selected from the MSPs listed in Table 2 or comprises any of the amino acid sequences in SEQ ID NOs: 15-32.

[0015] Provided are methods for inducing an immune response in a subject, breaking an immune tolerance in a subject, and / or active immunization to prevent a disease in a subject, wherein the methods generally comprise providing a means for inducing the immune response, breaking the immune tolerance, and / or the active immunization in the subject, and administering the means to the subject. The means generally comprise any of the immunogenic compositions disclosed herein, wherein the means may be administered via a syringe.

[0016] Also provided are methods for inducing an immune response in a subject, breaking an immune tolerance in a subject, and / or active immunization to prevent a disease in a subject, wherein the methods generally comprise administering to the subject the immunogenic composition of any one of the preceding embodiments, wherein the MSP is from or is derived from the same species as the subject.

[0017] In some embodiments of the disclosed methods, the subject is a mammal, for example a human, a simian, a mouse, a hamster, a rat, a cow, or a llama.

[0018] The present disclosure provides systems for inducing an immune response in a subject, breaking an immune tolerance in a subject, and / or active immunization to prevent a disease in a subject. These systems generally comprise a first means for inducing the immune response, breaking the immune tolerance, and / or the active immunization in the subject, and a secondmeans for delivering the first means to the subject. The first means generally comprise any of the immunogenic compositions disclosed herein. The second means generally comprise a syringe.

[0019] Also provided is a system for inducing an immune response in a subject, comprising the immunogenic composition of any one of the preceding embodiments, wherein the MSP is from or is derived from the same species as the subject. In some embodiments, the system for inducing an immune response in a subject further comprises a delivery system. In further embodiments, the delivery system comprises a syringe.

[0020] Also provided are systems for inducing an immune response in a subject, breaking an immune tolerance in a subject, and / or active immunization to prevent a disease in a subject., wherein the systems generally comprise the immunogenic composition of any one of the preceding embodiments, wherein the MSP is from or is derived from the same species as the subject. In some embodiments, the system for active immunization to prevent a disease in a subject further comprises a delivery system. In some embodiments, the delivery system is a syringe.

[0021] In some embodiments of the disclosed systems, the immunogenic composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

[0022] In some embodiments of the disclosed systems, the subject is a mammal, for example a human, a simian, a mouse, a rat, a cow, or a llama.DESCRIPTION OF THE FIGURES

[0023] FIG. 1 shows an exemplary nanodisc. The nanodisc comprises a membrane scaffold protein (MSP) (1), a phospholipid (4), and an immunogen (3). Optionally, the nanodisc comprises immune peptides (2), which may be lipidated.

[0024] FIG. 2 A shows a top view of an apolipoprotein. The figure shows Helix 1 (1), Helix 2 (2), Helix 3 (3), Helix 4 (4), Helix 6 (6), Helix 7 (7), Helix 8 (8), Helix 9 (9), and Helix 10 (10).

[0025] FIG. 2B shows a side view of an apolipoprotein. The figure shows Helix 8 (8), Helix 9 (9), and Helix 10 (10).

[0026] FIG. 2C shows a phospholipid bilayer (1) comprising apolipoproteins (2), i.e., an empty nanodisc.

[0027] FIG. 2D shows a helical wheel diagram of helices in apolipoproteins showing amphipathic surfaces. The hydrophilic residues (1) are exposed to the outside and the hydrophobic residues (2) interact with the hydrophobic tail region of a lipid bilayer.

[0028] FIGS. 3A and 3B show a sequence alignment of apolipoprotein A- 1 from different species.

[0029] FIGS. 4A and 4B show exemplary designs of novel MSPs based on human apolipoprotein orthologues.DETAILED DESCRIPTION

[0030] To date the identification of novel adjuvants and delivery platforms for generating an enhanced immune response and breaking immune tolerance was a challenge that was not previously met. This was especially challenging for membrane proteins, that often lost their correct folding, immunogenicity and / or function when not embedded in a phospholipid bilayer. Provided are compositions, methods and systems that do not have the same limitations as previously described methods.

[0031] Nanodiscs are effective for multi-transmembrane immunogen targets, which are highly difficult to generate as stable recombinant proteins. Using a scaffold protein, nanodiscs can stabilize the structure of these types of immunogen targets to accurately present them to the immune system. However, current nanodiscs utilize a human membrane scaffold protein (human MSP) which is highly immunogenic in non-human systems. This generates significant off-target anti-nanodisc antibodies.

[0032] To address this, the present inventors found that development of nanodiscs utilizing non- human MSP compatible with the respective non-human system would be beneficial. For example, using nanodiscs utilizing mouse membrane scaffold proteins (mouse MSP) compatible with the mouse system would be beneficial for inducing an immune response against a nonmouse derived immunogen. Indeed, a superior feature of the present invention is match of the immunized species with the scaffold proteins, as desired.

[0033] Accordingly, this disclosure provides an immunogenic composition comprising a nanodisc comprising a membrane scaffold protein (MSP), a phospholipid, and an immunogen, wherein the MSP and the immunogen are from different species. Also provided are methods ofinducing an immune response and / or active immunization to prevent a disease in a subject by administering the immunogenic composition, wherein the MSP and the immunogen are from different species. Further yet provided are systems and kits comprising the immunogenic compositions and a delivery system.

[0034] Before the nanodiscs, immunogenic compositions, methods, and systems of the present disclosure are described, it is to be understood that both the foregoing summary and drawings, and the following detailed description may be exemplary and may not be restrictive of the aspects of the present disclosure as claimed. Certain details may be set forth to provide a better understanding of various features, aspects, and advantages of the invention. However, one skilled in the art will understand that these features, aspects, and advantages may be practiced without these details and / or in the absence of details not described herein. In other instances, well-known structures, methods, and / or processes associated with methods of practicing the various features, aspects, and advantages may not be shown or described in detail to avoid unnecessarily obscuring descriptions of other details of the invention.

[0035] For convenience, before further description of the present invention, certain terms employed in the specification, examples, and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as would be understood by a person of ordinary skill in the art.ABBREVIATIONS AND DEFINITIONS

[0036] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0037] The articles “a” and “an” are used herein to refer to one or more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “a cell” means one cell or more than one cell.

[0038] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±5%, preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0039] The term “adjuvant” as used herein refers to a pharmacological or immunological agent that modifies the effect of other agents (e.g., vaccines) while having few if any direct effects when given by itself. They are often included in vaccines to enhance the recipient’s immune response to a supplied antigen while keeping the injected foreign material at a minimum.

[0040] The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in immune-mediated reaction, or reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies raised to the antigen, and an increase in T-cell activity is typically manifested in increased cell proliferation, or cellular cytotoxicity, or cytokine secretion. An adjuvant may also alter an immune response, for example, by changing a primarily humoral or Th2 response into a primarily cellular, or Thl response.

[0041] As used herein, the term “complexed” relates to the non-covalent interaction of a molecule (e.g., immunogen, adjuvant, etc.) with a nanodisc.

[0042] As used herein, the term “conjugated” as used herein indicates a covalent bond association between a molecule (e.g., immunogen, adjuvant, etc.) and a nanodisc.

[0043] As used herein, the term “absorbed” refers to a molecule (e.g., immunogen, adjuvant, etc.) that is taken into and stably retained in the interior, that is, internal to the outer surface, of a nanodisc. The molecule may be partially absorbed such that at least a portion of the molecule is exposed or not positioned within the interior of the nanodisc.

[0044] As used herein, by “administering” is meant a method of giving a dosage of a pharmaceutical composition (e.g., an immunogenic composition, for example a vaccine) to a subject. The compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, by gavage, in cremes, or in lipid compositions. The preferred method of administration can vary depending on various factors (e.g., the components of the composition being administered and the severity of the condition being treated).

[0045] The term “immunogen” or “immunogenic composition” as used herein means a material used to provoke an immune response and may confer immunity after administration of the immunogen or immunogenic composition to a subject.

[0046] As used herein, the term “antigen” is defined herein as a molecule containing one or more epitopes that will stimulate a host’s immune system to make a cellular antigen-specific immune response, and / or a humoral antibody response. Antigens can be peptides, proteins, polysaccharides, saccharides, lipids, nucleic acids, and combinations thereof. The antigen can be derived from a virus, bacterium, parasite, plant, protozoan, fungus, tissue or transformed cell such as a cancer or leukemic cell and can be a whole cell or immunogenic component thereof, e.g., cell wall components. An antigen may be an oligonucleotide or polynucleotide which expresses an antigen. Antigens can be natural or synthetic antigens, for example, haptens, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens (see, e.g., Bergmann, et al., Eur. J. Immunol., 23:2777-2781 (1993); Bergmann, et al., J. Immunol., 157:3242-3249 (1996); Suhrbier, Immunol, and Cell Biol., 75:402-408 (1997)).

[0047] The term "epitope" as used herein means a portion of an antigen to which an antibody specifically binds. Epitopes usually consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope can be composed of contiguous and / or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3 -dimensional space through the folding of the protein molecule.

[0048] “Membrane scaffold proteins” (MSPs) as used herein are endogenous proteins or are non- naturally occurring (i.e., artificial) proteins that self-assemble with phospholipids, phospholipid mixtures, or in the absence of phospholipids, into nanometer size membrane bilayers. A subset of these nanometer size assemblies are discoidal in shape and are referred to as “nanodiscs” or “nanodisc structures.” These nanodisc structures preserve the overall bilayer structure of normal membranes while providing a system that is soluble in solution and can be assembled or affixed to a variety of surfaces.

[0049] Nanodiscs can have diameters of about 2 nm to about 500 nm, about 2 nm to about 200 nm, about 2 nm to about 100 nm, about 2 nm to about 50 nm, about 2 nm to about 20 nm, or about 2 nm to about 10 nm. The amino acid sequences of specifically exemplified MSPs are given in Table 2 and SEQ ID NOs: 15-32.

[0050] As used herein, “subject” means any animal, preferably a mammal, most preferably a human, to whom will be or has been treated by or subjected to a method according to an embodiment of the invention. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, simians, monkeys, llamas, humans, etc., more preferably a human.

[0051] As used herein, the term “enhanced” when used with respect to an immune response, means stimulating, evoking, increasing, improving, or augmenting any response of a mammal's immune system. The immune response may be a cellular response (z.e., cell-mediated, such as cytotoxic T lymphocyte mediated) or a humoral response (z.e., antibody mediated response), and may be a primary or secondary immune response. Examples of enhancement of immune response include increased CD4+ or CD8+ T cell activity and generation of cytolytic T cells. The enhancement of immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, release of cytokines (for example IL-2 production), regression of tumors, survival of tumor bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity.

[0052] As used herein, the term “CD4+ or CD8+ T cell activity” refers to a T cell immune response that is characterized by observing a high proportion of immunogen-specific CD4+ T cells or CD8+ T cells within the population of total responding T cells following vaccination. The total immunogen-specific T-cell response can be determined by an IFN-gamma Enzyme- Linked ImmunoSpot (ELISPOT) assay. The immunogen-specific CD4+ or CD8+ T cell immune response can be determined by an intracellular cytokine staining (ICS) assay.

[0053] The immunogenic compositions disclosed herein may enable the breaking of immune tolerance. As used herein, the term “immune tolerance” is the state where the immune system becomes unresponsive to specific substances or tissues that would normally trigger an immune response. This unresponsiveness prevents the immune system from attacking the body's owncells or tissues, or “self-antigens.” This entrained limitation on immune function against such self-antigens protects the host against adverse autoimmune reactions. It also presents a major obstacle for presently available immunotherapy protocols, such as cancer immunotherapy protocols.

[0054] The term “His tag,” as used herein, refers to a stretch of amino acids comprising multiple histidine residues. The His tag may comprise 2, 3, 4, 5, 6, 7, 8 or more histidine residues. In some embodiments, the His tag is fused to the N- or C-terminus of a protein; alternatively, it can be fused at any suitable location within the protein. In specific embodiments, the His tag is fused to the N- or C-terminal of an immunogen, such as any of the immunogens disclosed herein.

[0055] As used herein, the term “lipidated” refers to a peptide or protein comprising one or more lipidated amino acids. The term “lipidated amino acid” refers to an amino acid, typically a lysine or cysteine, which has a lipid moiety attached. Lipidation can include carboxyl- or aminoterminal lipidation, or main-chain lipidation.

[0056] In some embodiments, the noted immune peptides are synthetic peptides. A “synthetic peptide” refers to a polymer of amino acid residues that has been generated by chemically coupling a carboxyl group or C-terminus of one amino acid to an amino group or N-terminus of another. Chemical peptide synthesis typically starts at the C-terminus of the peptide and ends at the N-terminus. Various methods for generating synthetic peptides are well known in the art.

[0057] In some embodiments of any of the compositions or methods described herein, a range is intended to comprise every integer or fraction or value within the range.

[0058] Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of’ and / or “consisting essentially of’ such features.IMMUNOGENIC COMPOSITIONS

[0059] Provided is an immunogenic composition comprising a nanodisc. The nanodisc is formed by a membrane scaffold protein (MSP) and a phospholipid and includes an immunogen. In some embodiments, the nanodisc further comprises an immune peptide. The immunogenic composition may comprise a pharmaceutically acceptable carrier or an adjuvant.Membrane Scaffold Protein

[0060] Membrane scaffold proteins (MSP) may be engineered for greater stability, size homogeneity, and useful functionalities in the resultant nanoscale lipoprotein particle, i.e., nanodisc. The disclosed nanodiscs formed from these MSPs can be used to solubilize membrane proteins and complexes in a functionally stable monodisperse phospholipid-bilayer associated form.

[0061] The disclosed MSPs are modeled after apolipoprotein, which under certain conditions, can self-assemble with phospholipid to form discoidal structures having diameters of 2 nm to 500 nM. The MSPs may be engineered to alter the size of the MSP with respect to membrane particle formation and / or to increase the stability and monodispersity of the self-assembled nanoparticle by altering the sequence of the parent molecule.

[0062] Accordingly, in some embodiments, the MSP is non-naturally occurring and / or nonhuman. In further embodiments, the MSP is engineered.

[0063] In some embodiments, the MSP is an apolipoprotein.

[0064] In some embodiments, the MSP is any of apolipoprotein Al (ApoA-I), apolipoprotein A- II (ApoA-II), apolipoprotein A4 (ApoA4), apolipoprotein Cs (ApoCs), or apolipoprotein protein E (ApoE). In some embodiments, the MSP is an apolipoprotein mimetic, such as an MSP derived from ApoA-I.

[0065] In some embodiments, the apolipoprotein is apolipoprotein- Al (apo A-I) or is derived from apo A-I.

[0066] In some embodiments, the MSP is from or is derived from the same species as the subject in which the nanodisc is inteded to be used. In further embodiments, the MSP is not immunogenic to the subject in which the nanodisc is intended to be used.

[0067] In some embodiments, the MSP is selected from the MSPs listed in Table 2 or comprises any of the amino acid sequences in SEQ ID NOs: 15-32.

[0068] In some embodiments, the nanodisc comprises more than one MSP selected from the MSPs listed in Table 2 or comprises any of the amino acid sequences in SEQ ID NOs: 15-32, such as two or more of the noted MSPs.

[0069] The following Tables illustrate how MSPs may be formed.TABLE 1TABLE 1 (Continued)TABLE 2Phospholipids

[0070] The disclosed nanodiscs may be formed by an MSP and a phospholipid. In some embodiments, the nanodiscs comprise more than one phospholipid. In some embodiments, the phospholipids may be part of a phospholipid bilayer.

[0071] In some embodiments, the phospholipids may be amphipathic lipids. Exemplary amphipathic lipids include any lipid molecule which has both a hydrophobic and a hydrophilic moiety, such as a phospholipid.

[0072] Exemplary phospholipids include, but are not limited to, small alkyl chain phospholipids, phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine 1 -myristoyl-2-palmitoylphosphatidylcholine, 1 -palmitoyl-2- myristoylphosphatidylcholine, 1 -palmitoyl-2-stearoylphosphatidylcholine, 1 -stearoyl-2- palmitoylphosphatidylcholine, dioleoylphosphatidylcholine dioleophosphatidylethanolamine, dilauroylphosphatidylglycerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerols, diphosphatidylglycerols such as dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, egg sphingomyelin, milk sphingomyelin, palmitoyl sphingomyelin, phytosphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, dipalmitoylphosphatidylglycerol salt, phosphatidic acid, galactocerebroside, gangliosides, cerebrosides, dilaurylphosphatidylcholine, ( 1 ,3)-D-mannosyl-( 1 ,3)diglyceride, aminophenylglycoside, 3-cholesteryl-6'-(glycosylthio)hexyl ether glycolipids, and cholesterol and its derivatives.

[0073] In some embodiments, the phospholipids include phosphatidylcholine (POPC), phosphatidylglycerol (POPG), and phosphatidylethanolamine (POGE), and any combination thereof. In some embodiments, the phospholipids include a 3:1:1 mixture of POPC:POPG:POPE by weight.Immunogen and Immune Peptides

[0074] The disclosed nanodiscs are associated with (e.g., complexed, conjugated, absorbed, or partially absorbed) an immunogen. The immunogen is selected to induce an immune response

[0075] The immunogen may be heterologously expressed in mammalian cells and purified to homogeneity. In some embodiments, the purification is conducted in detergent micelles using affinity and size-exclusion chromatography.

[0076] In some embodiments, the immunogen is engineered. In some embodiments, the immunogen is non-naturally occurring.

[0077] In some embodiments, the immunogen is lipidated, such as with any of the phospholipids disclosed herein.

[0078] In some embodiments, the immunogen comprises a tag. In further embodiments, the tag is a His tag.

[0079] In some embodiments, the immunogen is a membrane protein. In some embodiments, the membrane protein is hydrophobic or partially hydrophobic. In further embodiments, the immunogen is a receptor, transporter, enzyme, or a pathogen antigen. In some embodiments, the pathogen is a virus or bacteria.

[0080] In some embodiments, the nanodisc is self-assembling, such that the MSP, phospholipid, and immunogen may be admixed to form the nanodisc. For example, the purified immunogen may be reconstituted into nanodiscs by mixing it with detergent solubilized lipids (3: 1: 1 mixture of POPC:POPG:POPE by weight) and membrane scaffold proteins. In some embodiments, the mixing is conducted at room temperature for 30 minutes. This is followed by incubation with Biobeads® (SM-2 Resin, BIO-RAD Catalog 1523920, California). In some embodiments, the incubation is at room temperature for 2.5 hours.

[0081] In further embodiments, the immunogen retains its folding and / or function in the nanodisc.

[0082] In some embodiments, the immunogen is antigenic. In some embodiments, the immunogen is antigenic when in the nanodisc. For example, the immunogen may be positioned in the nanodisc such that a region of the immunogen capable of inducing an immune response is exposed.

[0083] In some embodiments, the nanodisc comprises more than one different immunogen.

[0084] In some embodiments, the immunogenic composition comprises more than one different nanodisc.

[0085] In some embodiments, the nanodisc further comprises an immune peptide. In some embodiments, the immune peptide improves the effectiveness of the immunogenic composition. In further embodiments, the immune peptide is engineered. In yet further embodiments, the immune peptide comprises the amino acid sequence of SEQ ID NO: 33.

[0086] In some embodiments, the immune peptide is lipidated, such as with any of the phospholipids disclosed herein. As example, the nanodisc may comprise phosphatidyl choline (POPC) and phosphatidyl glycerol (POPG). As such, in an exemplary embodiment, the immune peptide may include a POPC and POPG. This lipidated immune peptide may be used in addition to, or in place of the regular lipid mixture described above to generate immune peptide decorated nanodiscs. The total lipid to peptide ratio used to form the lipidated peptide may be from 50: 1 to 2: 1 by weight, such as 20: 1 to 5: 1, by weight, or 10:2 by weight.Pharmaceutically Acceptable Carrier

[0087] In some embodiments, the immunogenic composition comprises a pharmaceutically acceptable carrier. “Pharmaceutically acceptable” means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, appropriate salts and buffers, dispersion media, antibacterial and antifungal agents, and the like. Such pharmaceutically acceptable carriers are well known in the art (see Remington’s Pharmaceutical Sciences, 18thedition, A.R. Gennaro, Ed., Mack Publishing Company (1990); Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and E. Hovgaard, Eds., Taylor & Francis (2000); and Handbook of Pharmaceutical Excipients, 3rdedition, A. Kibbe, Ed., Pharmaceutical Press (2000)).

[0088] Suitable pharmaceutically acceptable forms for injectable use include sterile aqueous solutions or dispersions and sterile powders for immediate preparation of sterile injectable solutions or dispersions. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, the maintenance of the preferred particle size in the case of dispersions, and the use of surfactants. Prevention of the action of microorganisms can be carried out with various antibacterial and antifungal agents such as parabens, chlorobutanol, phenols, sorbic acid, thimerosal, and the like. In many cases, it may be preferable to include an isotonic agent, such as sugar or sodium chloride. Prolonged absorption of the injectable composition may be brought about by the use of agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0089] Sterilized injection solutions are prepared by incorporating a dose amount of the nanodiscs in the appropriate solvent with the various other components listed above, as needed, followed by filtration sterilization. For sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and lyophilization techniques that yield powders of the active ingredient and any additional desired ingredients from the presterilized filtered solution.Adjuvant

[0090] In some embodiments, the immunogenic composition comprises an adjuvant.

[0091] The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in an immune response, such as manifested by a significant increase in the titer of antibodies raised to the antigen.

[0092] Suitable adjuvants include, but are not limited to 1018 ISS, 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, PLG microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA), which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox. Quil or Superfos.

[0093] Additional suitable adjuvants include cytokines. Several cytokines have been directly linked to influencing dendritic cell migration to lymphoid tissues {e.g., TNF-alpha), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T-lymphocytes e.g., GM-CSF, IL-1 and IL-4) and acting as immunoadjuvants {e.g., IL- 12).Nanodiscs

[0094] Nanodiscs are prepared by mixing the MSP and phospholipid in solution, such as at a MSP:phospholipid ratio of 2:200, or 1:200, or 1: 100, or 1:50, or 1:30. The mixture may be incubated at ambient or elevated temperatures {i.e., 20°C and 37°C, respectively) for 2 to 24hours. The mixture is purified via dialysis, affinity chromatography, and / or size-exclusion chromatography .

[0095] The immunogen may be incorporated into nanodiscs by mixing with detergent solubilized lipids (3: 1: 1 mixture of POPC:POPG:POPE by weight) and membrane scaffold proteins at room temperature for 30 minutes. The nanodisc comprising the immunogen may be purified using and of dialysis, affinity chromatography, and / or size-exclusion chromatography.

[0096] To decorate nanodiscs with immune peptides, a lipidated form of the immune peptide is designed. The lipid-peptide conjugate may then be mixed with phosphatidyl choline (POPC) and phosphatidyl glycerol (POPG) to prepare a lipid mixture (5:5:2 of POPC:POPG:lipid-peptide by weight), along with detergents to solubilize the mixture. The lipid-detergent mixture containing the lipid-peptide conjugate may be added to the nanodisc in addition to the immunogen, or alternative to the immunogen to generate immune peptide decorated nanodiscs.

[0097] In some embodiments, the nanodiscs have a molar ratio of phospholipid / MSP from 2 to 250 (e.g., 10 to 200, 20 to 100, 20 to 50, 30 to 40).

[0098] In some embodiments, the nanodisc is 500 nm or less in size / diameter. In further embodiments, the nanodisc is 50 nm or less in size / diameter. In yet further embodiments, the nanodisc is 2-200 nm in size / diameter. In yet further embodiments, the nanodisc is 2-20 nm in size / diameter. In some embodiments, the nanodisc is 2-10 nm in height. In further embodiments, the nanodisc is 4-8 nm in height. In yet further embodiments, the nanodisc is 4-7 nm in height. See US Patent No. 7,691,414. In some embodiments, the nanodisc is about 5.5 nm in height.METHODS

[0099] Provided is a method of inducing an immune response in a subject, the method comprising administering to the subject the immunogenic composition of any one of the preceding embodiments, wherein the MSP is from or is derived from the same species as the subject. Also provided is a method for active immunization to prevent a disease in a subject, the method comprising administering to the subject the immunogenic composition of any one of the preceding embodiments, wherein the MSP is from or is derived from the same species as the subject. In some embodiments of the methods, the MSP is not immunogenic to the subject.Immune Response Stimulation

[0100] The nanodiscs in a pharmaceutically acceptable carrier, i.e., a pharmaceutically acceptable composition or immunogenic composition, may be administered in various dosage forms such as injections, drug release capsules and the like. The disclosed nanodiscs and immunogenic compositions comprising the nanodiscs are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. For example, one dosage amount can either added to 1000 ml of subcutaneous infusion or injected into the proposed injection site (e.g., "Remington"). Alternatively, one dosage amount can be dissolved in 0.1 ml to 5 ml of isotonic NaCl solution and either added to 1000 ml of subcutaneous infusion or injected into the proposed injection site.

[0101] In some embodiments, the nanodiscs are about 0.0001 to 5.0 milligrams, or about 0.001 to 2.0 milligrams, or about 0.01 to 0.5 milligram to per dose of the immunogenic composition. Multiple doses may be administered, such as one dose daily or weekly for 2 to 20 total doses. An exemplary doses schedule includes one dose weekly for six weeks. Serum samples are collected for antibody titer evaluations at the end of the dose schedule and / or a set time after one or more of the individual doses in the dose schedule.

[0102] The immunogenic composition may be administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

[0103] In some embodiments, the subject is a mammal, for example a human, a simian, a mouse, a rat, a cow, or a llama.

[0104] The immune response generated by the method may comprise an enhanced antibody response against the antigenic protein. For example, the immune response can be characterized by the presence of a high proportion of responders, such as more than 50%, 60%, 70%, 80%, 90%, or 100% of subjects tested.

[0105] The immune response generated by the method may comprise an enhanced CD8+ T cell response against the antigenic protein in the subject, such as a response characterized by the presence of a high proportion of CD8+ responders, such as more than 50%, 60%, 70%, 80%,90%, or 100% of subjects tested as determined by an ICS assay, with a median total cytokine response of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or more.

[0106] The enhanced CD8+ T cell response may comprise an increase or induction of poly functional CD8+ T cells specific to the antigenic protein. Such polyfunctional CD8+ T cells express more than one cytokine, such as two or more of IFN-gamma, IL-2, and TNF-alpha.

[0107] The immune response generated by the method may comprise an enhanced CD4+ T cell response against the antigenic protein in the subject, such as a response characterized by the presence of a high proportion of CD4+ responders, such as more than 50%, 60%, 70%, 80%, 90%, or 100% of subjects tested as determined by an ICS assay, with a median total cytokine response of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or more.

[0108] The enhanced CD4+ T cell response may comprise an increase or induction of polyfunctional CD4+ T cells specific to the antigenic protein. Such polyfunctional CD4+ T cells express more than one cytokine, such as two or more of IFN-gamma, IL-2, and TNF-alpha.

[0109] The immune response may comprise any or all of an enhanced CD4+ T cell response, an enhanced antibody response, and an enhanced CD8+ T cell response, against the antigenic protein in the subject.Assessing the Immune Response

[0110] An immune response may be assessed by an of: a higher antibody titer in a subject post administration of the immunogenic composition (e.g., a greater concentration of antibodies circulating in the subject’s blood), an increase in the antibody avidity / affinity to their target antigen or enhanced neutralization of the target antigen, faster antibody production, an extended antibody response (e.g., longer antibody half-life in the subject), and / or by improvement in the antibody effector functions, such as improved complement activation or increased antibodydependent cellular cytotoxicity. Also useful for evaluating the immune response is examination of germinal center formation via meso scale discovery (MSD) assays or enzyme-linked immunosorbent assays (ELISA).

[0111] Further methods for assessment of an immune response include intracellular cytokine staining (ICS) assays. An ICS assay is a functional immunology assay that uses flow cytometry to assess cytokine production by individual cells. It involves stimulating immune cells withspecific antigens or mitogens, inducing cytokine production, and then fixing and permeabilizing the cells to allow intracellular staining of the cytokines. This technique is used to determine which cells are producing specific cytokines and to assess the strength of an immune response.

[0112] Peripheral blood mononuclear cells are tested for the presence of polyfunctional CD4+ or CD8+ T cells by ICS. After short term (5 hour) stimulation with an immunogenic composition of the present disclosure, cells are surface stained for phenotypic markers such as CD3, CD4, CD8, CD27, CD45RA, fixed, permeabilized and stained further for the presence of intracellular cytokines such as INF-y, TNF-a and IL-2. Flow cytometry analysis using Flow-Jo software is used to detect single / multiple cytokine secreting central memory (CD27+CD45RA-), CD4+ or CD8+ T cells at baseline, and post- vaccination time points. Cytokine release may also be measured by other means known in the art, e.g., using Western blot, ELISA, or immunohistochemical assays to detect the presence of released cytokines in a sample containing CD4+ and / or CD8+ T-cells.

[0113] Breakage of an immune tolerance may be measured by assessing the levels of B cells that produce autoantibodies against specific antigens, such as self-antigens. Such levels may be measured using an ELISA or immunofluorescence to detect the presence of specific antigens, such as nuclear proteins, autoantibodies against cyclic citrullinated peptides (CCP), or rheumatoid factors. Breakage of an immune tolerance may also be measured by simple blood tests for immunoglobulin levels.SYSTEMS

[0114] Provided is a system for inducing an immune response in a subject, comprising the immunogenic composition of any one of the preceding embodiments, wherein the MSP is from or is derived from the same species as the subject. In further embodiments, the MSP is not immunogenic to the subject. In some embodiments, the system for inducing an immune response in a subject further comprises a delivery system. In further embodiments, the delivery system comprises a syringe.

[0115] Also provided is a system for active immunization to prevent a disease in a subject, the system comprising the immunogenic composition of any one of the preceding embodiments, wherein the MSP is from or is derived from the same species as the subject. In further embodiments, the MSP is not immunogenic to the subject. In some embodiments, the system foractive immunization to prevent a disease in a subject further comprises a delivery system. In some embodiments, the delivery system is a syringe.

[0116] In embodiments of the systems, the immunogenic composition may be administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

[0117] In embodiments of the systems, the subject may be a mammal, for example a human, a simian, a mouse, a rat, a cow, or a llama.KITS

[0118] As used herein, the term “kit” refers to any grouping of articles for storage, transport, or delivery of a material. In the context of an immunogenic compositions described herein, such kits may include systems that allow for the storage of the immunogenic compositions, transport of the immunogenic compositions, administration of the immunogenic composition, and / or supporting materials (e.g., written instructions for use of the materials of the kit, etc.). For example, kits may include one or more enclosures (e.g., boxes) containing the necessary agents (e.g., the immunogenic composition, etc.) and / or supporting materials. The necessary agents may be provided in a container. In some embodiments, the kit may include two or more separate containers that each contain a portion of the necessary agents.

[0119] Thus, provided herein is a kit comprising: a container comprising any one of the immunogenic compositions disclosed herein. In some embodiments, the kit further comprises supporting materials such as instructions for use of the materials in the kit. In some embodiments, the kit may further comprise additional containers having diluents, such as a pharmaceutically acceptable carrier or saline solution for intravenous administration.

[0120] In further embodiments, the supporting materials provide instructions for administration of the immunogenic composition, such as by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration. In some embodiments, the kit further comprises a delivery system. In some embodiments, the delivery system is a syringe.

[0121] In some embodiments, the kit is configured for use in a mammal, for example a human, a simian, a mouse, a rat, a cow, or a llama.EXAMPLESExample 1: Method for preparing nanodiscs

[0122] A target membrane protein immunogen was first heterologously expressed in mammalian cells and purified to homogeneity in detergent micelles using affinity and size-exclusion chromatography. The purified membrane protein immunogen was then reconstituted into nanodiscs by mixing it with detergent solubilized lipids (3: 1: 1 mixture of POPC:POPG:POPE by weight) and membrane scaffold proteins at room temperature for 30 minutes, followed by incubation with Biobeads® (SM-2 Resin, BIO-RAD Catalog 1523920, California) at room temperature for 2.5 hours. The immunogen-containing nanodiscs were further purified using affinity and size-exclusion chromatography.Example 2: Method for incorporating immune peptide in the nanodiscs

[0123] To decorate nanodiscs with immune peptides, a lipidated form of immune peptide PADRE (Palmitoyl-GSSGAKFVAAWTLKAAA (SEQ ID NO: 33)) was designed and synthesized using Fmoc solid-phase peptide synthesis, which allowed for a simple immune peptide incorporation into nanodiscs. The lipid-PADRE conjugate was mixed with phosphatidyl choline (POPC) and phosphatidyl glycerol (POPG) to prepare a lipid mixture (5:5:2 of POPC:POPG: lipid-PADRE by weight), along with detergents to solubilize the mixture. The lipid-detergent mixture containing lipid-PADRE conjugate was used in place of the regular lipid mixture described above to generate immune peptide PADRE decorated nanodiscs.Example 3: Method for immunization using nanodiscs

[0124] Mice are immunized with nanodiscs comprising a non-mouse immunogen and mouse MSPs, such as obtained as described in Examples 1-2. An immunization schedule of 1 dose weekly for 6 weeks via a subcutaneous / intradermal or intrapleural injection route, wherein the dose comprises 10 pg of the antigen in a total volume of 120 pl of PBS. Serum samples are collected for antibody titer evaluations.Example 4: Method for immunization using nanodiscs

[0125] Rats are immunized with nanodiscs comprising a non-rat immunogen and rat MSPs, such as obtained as described in Examples 1-2. An immunization schedule of 1 dose weekly for 6 weeks via a subcutaneous / intradermal or intrapleural injection route, wherein the dose comprises10 pg of the antigen in a total volume of 120 pl of PBS. Serum samples are collected for antibody titer evaluations.Example 5: Method for immunization using nanodiscs

[0126] Chickens are immunized with nanodiscs comprising a non-chicken immunogen and chicken MSPs, such as obtained as described in Examples 1-2. An immunization schedule of 1 dose weekly for 6 weeks via a subcutaneous / intradermal or intrapleural injection route, wherein the dose comprises 10 pg of the antigen in a total volume of 120 pl of PBS. Serum samples are collected for antibody titer evaluations.Example 6: Method for immunization using nanodiscs

[0127] Hamsters are immunized with nanodiscs comprising a non-hamster immunogen and hamster MSPs, such as obtained as described in Examples 1-2. An immunization schedule of 1 dose weekly for 6 weeks via a subcutaneous / intradermal or intrapleural injection route, wherein the dose comprises 10 pg of the antigen in a total volume of 120 pl of PBS. Serum samples are collected for antibody titer evaluations.Example 7: Method for immunization using nanodiscs

[0128] Cows are immunized with nanodiscs comprising a non-bovine immunogen and bovine MSPs, such as obtained as described in Examples 1-2. An immunization schedule of 1 dose weekly for 6 weeks via a subcutaneous / intradermal or intrapleural injection route, wherein the dose comprises 10 pg of the antigen in a total volume of 120 pl of PBS. Serum samples are collected for antibody titer evaluations.Example 8: Method for immunization using nanodiscs

[0129] Monkeys are immunized with nanodiscs comprising a non-simian immunogen and simian MSPs, such as obtained as described in Examples 1-2. An immunization schedule of 1 dose weekly for 6 weeks via a subcutaneous / intradermal or intrapleural injection route, wherein the dose comprises 10 pg of the antigen in a total volume of 120 pl of PBS. Serum samples are collected for antibody titer evaluations.Example 9: Method for immunization using nanodiscs

[0130] Llamas are immunized with nanodiscs comprising a non-llama immunogen and llama MSPs, such as obtained as described in Examples 1-2. An immunization schedule of 1 dose weekly for 6 weeks via a subcutaneous / intradermal or intrapleural injection route, wherein the dose comprises 10 pg of the antigen in a total volume of 120 pl of PBS. Serum samples are collected for antibody titer evaluations.Example 10: Method of testing for an immune response

[0131] Serum titer of circulating antibodies may be measured by ELISA. For example, ELISA plates are coated with the immunogen that was included in the immunogenic composition administered to the subject, and the plates are incubated overnight at 4° C. The plates are then blocked with 1% BSA in PBS for 2 hours, after which 100 pL of 4-fold serial dilutions of serum from the subject is added to each well of the ELISA plate and incubated for 1 hour at room temperature. Wells are then incubated with rabbit anti-mouse IgG-HRP (1:5000 dilution; HRP is horse radish peroxidase) for 1 h at room temperature, followed by addition of the HRP substrate, TMB (3,3',5,5'-tetramethylbenzidine). The enzymatic reaction is stopped by adding 2N H2SO4, and the absorbance at 450 nm (OD450) is measured using a microplate reader. The highest dilution with twice the absorbance of background is considered as the end-point dilution titer.Example 11: Method of testing for an immune response

[0132] Testing for an enhanced immune response may involve assessing the levels and activity of key immune cells and molecules. This can be achieved using blood tests, tissue samples, and in vitro assays, such as assays that measure antibody levels, T cell responses, and cytokine production.

[0133] Peripheral blood mononuclear cells are tested for the presence of polyfunctional CD4+ or CD8+ T cells by ICS. After short term (5 hour) stimulation with an immunogenic composition of the present disclosure, cells are surface stained for phenotypic markers such as CD3, CD4, CD8, CD27, CD45RA, fixed, permeabilized and stained further for the presence of intracellular cytokines such as INF-y, TNF-a and IL-2. Flow cytometry analysis using Flow-Jo software is used to detect single / multiple cytokine secreting central memory (CD27+CD45RA-), CD4+ or CD8+ T cells at baseline, and post- vaccination time points. Cytokine release may also bemeasured by other means known in the art, e.g., using Western blot, ELISA, or immunohistochemical assays to detect the presence of released cytokines in a sample containing CD4+ and / or CD8+ T-cells.EMBODIMENTS OF THE DISCLOSURE

[0134] The present disclosure relates to an immunogenic composition comprising a nanodisc comprising a membrane scaffold protein (MSP), a phospholipid, and an immunogen, wherein the MSP and the immunogen are from different species.

[0135] The present disclosure also relates to an immunogenic composition comprising a nanodisc means. In any of the embodiments comprising a nanodisc means, the nanodisc means comprises a phospholipid. In any of the embodiments comprising a nanodisc means, the nanodisc means comprises an immunogen. In any of the embodiments comprising a nanodisc means, the nanodisc means comprises a MSP.

[0136] In any of the embodiments of the immunogenic compositions, more than one different nanodisc or nanodisc means may be included.

[0137] In any of the embodiments of the immunogenic compositions, the phospholipid is part of a phospholipid bilayer.

[0138] In any of the embodiments of the immunogenic compositions, the immunogen is lipidated.

[0139] In any of the embodiments of the immunogenic compositions, the nanodisc or nanodisc means comprises an immune peptide.

[0140] In any of the embodiments of the immunogenic compositions, the immune peptide comprises the amino acid sequence of SEQ ID NO: 33.

[0141] In any of the embodiments of the immunogenic compositions, the immune peptide is lipidated.

[0142] In any of the embodiments of the immunogenic compositions, the immune peptide comprises phosphatidylcholine (POPC) and phosphatidylglycerol (POPG).

[0143] In any of the embodiments of the immunogenic compositions, the immunogen comprises a tag.

[0144] In any of the embodiments of the immunogenic compositions, the immunogen comprises a tag, wherein the tag is a His tag.

[0145] In any of the embodiments of the immunogenic compositions, the immunogen is antigenic.

[0146] In any of the embodiments of the immunogenic compositions, the MSP is non-naturally occurring and / or non-human.

[0147] In any of the embodiments of the immunogenic compositions, the MSP is selected from the MSPs listed in Table 2 or comprises any of the amino acid sequences in SEQ ID NOs 15-32.

[0148] In any of the embodiments of the immunogenic compositions, the MSP and the immunogen are from different species.

[0149] The present disclosure further relates to a method comprising administering to a subject an immunogenic composition according to any of the embodiments disclosed herein, wherein the MSP is from or is derived from the same species as the subject.

[0150] In any of the embodiments of the method, an immune response may be induced in the subject, an immune tolerance may be broken in the subject, the method may provide active immunization to prevent a disease in the subject, or any combination thereof.

[0151] In any of the embodiments of the method, the immunogenic composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

[0152] The present disclosure further yet provides a system comprising the immunogenic composition according to any of the embodiments disclosed herein, and a delivery system, wherein the MSP that is from or is derived from the same species as the subject.

[0153] In any of the embodiments of the system, the system is configured to induce an immune response in the subject, break an immune tolerance in the subject, provide active immunization to prevent a disease in the subject, or any combination thereof.

[0154] In any of the embodiments of the system, the delivery system comprises a syringe.

[0155] In any of the embodiments of the system, the immunogenic composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

[0156] All publications and patents referred to herein are incorporated by reference. Various modifications and variations of the described subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention are obvious to those skilled in the art and are intended to be within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. An immunogenic composition comprising: a nanodisc comprising a membrane scaffold protein (MSP), a phospholipid, and an immunogen, wherein the MSP and the immunogen are from different species.

2. The immunogenic composition of claim 1 , wherein the phospholipid is part of a phospholipid bilayer.

3. The immunogenic composition of claim 1, wherein the immunogen is lipidated.

4. The immunogenic composition of claim 1 , wherein the immunogenic composition comprises more than one different nanodisc.

5. The immunogenic composition of claim 1, wherein the nanodisc further comprises an immune peptide.

6. The immunogenic composition of claim 5, wherein the immune peptide comprises the amino acid sequence of SEQ ID NO: 33.

7. The immunogenic composition of claim 5, wherein the immune peptide is lipidated.

8. The immunogenic composition of claim 7, wherein the immune peptide comprises phosphatidylcholine (POPC) and phosphatidylglycerol (POPG).

9. An immunogenic composition comprising: a nanodisc means.

10. The immunogenic composition of claim 9, wherein the nanodisc means comprises a phospholipid that is part of a phospholipid bilayer.

11. The immunogenic composition of claim 9, wherein the nanodisc means comprises an immunogen that is lipidated.

12. The immunogenic composition of claim 9, wherein the nanodisc means comprises a membrane scaffold protein (MSP).

13. The immunogenic composition of claim 1 or 11, wherein the immunogen comprises a tag.

14. The immunogenic composition of claim 13, wherein the tag is a His tag.

15. The immunogenic composition of claim 1 or 11, wherein the immunogen is antigenic.

16. The immunogenic composition of claim 1 or 12, wherein the MSP is non-naturally occurring and / or non-human.

17. The immunogenic composition of claim 1 or 12, wherein the MSP is selected from the MSPs listed in Table 2 or comprises any of the amino acid sequences in SEQ ID NOs 15- 32.

18. The immunogenic composition of claim 12, wherein the MSP and the immunogen are from different species.

19. A method of inducing an immune response in a subject, the method comprising administering to the subject the immunogenic composition of any one of claims 1-18, wherein the MSP is from or is derived from the same species as the subject.

20. A method for active immunization to prevent a disease in a subject, the method comprising administering to the subject the immunogenic composition of any one of claims 1-18, wherein the MSP is from or is derived from the same species as the subject.

21. A system comprising the immunogenic composition of any one of claims 1-18 and a delivery system, wherein the nanodisc or nanodisc means comprises an MSP that is from or is derived from the same species as the subject, and wherein the system is configured to induce an immune response in a subject and / or active immunization to prevent a disease in the subject.

22. The system of claim 21, wherein the delivery system comprises a syringe.

23. The system of claim 21, wherein the immunogenic composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.-SO-

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