WO2015011478A1

WO2015011478A1 - Method of producing a preparation of membrane vesicles

Info

Publication number: WO2015011478A1
Application number: PCT/GB2014/052264
Authority: WO
Inventors: Kay GRÜNEWALD; Tzviya ZEEV-BEN-MORDEHAI
Original assignee: Isis Innovation Limited
Priority date: 2013-07-25
Filing date: 2014-07-24
Publication date: 2015-01-29
Also published as: GB201313249D0

Abstract

The invention relates to a method of producing a preparation of membrane vesicles comprising one or more membrane proteins, and to related methods, preparations and compositions.

Description

METHOD OF PRODUCING A PREPARATION OF MEMBRANE VESICLES

Field of the invention

The invention relates to a method of producing a preparation of membrane vesicles comprising one or more membrane proteins, and related methods, preparations and

compositions.

Background of the invention

Membrane proteins are a central subclass of the proteome. They are involved in many different biological process including cell signaling, cell adhesion, transport across the lipid bilayer, transduction of energy and immune response just to name a few. As such, membrane proteins are implicated in many disorders and thus are a key for diagnostics and therapeutics. Prerequisite to either basic research to understand membrane protein function or the clinical research of membrane proteins is the successful production of the protein of interest in a functional form. Membrane proteins are inherently a challenging target for production due to their requirement for a native lipidic environment. Remarkable achievements have been made in the past several years towards the production of membrane proteins. Most procedures developed involve isolating the protein by solubilizing the membrane with detergent followed by a purification step and subsequently reconstituting it into an artificial membrane. These procedures suffer from several drawbacks that compromise the yield of the production, limit the downstream applications and are costly. For solubilizing the protein, many different detergents are available but the choice of which to use - for e.g. making sure the target protein is not severely

affected/compromised - involves an exhaustive empirical screen. In the reconstitution step, lipids are introduced concomitantly with the removal of detergent. This step is usually characterized by low yields. More importantly, the topology of membrane proteins is crucial for their function but is very hard to control in the reconstitution experiments. Additionally, the biological relevance of such in- vitro model systems is inherently limited by the simplicity of its lipid composition compared to native membranes that are made from a wider range of lipids and possess local subdomains of varying compositions.

Membrane enveloped viruses are a good platform for displaying intact membrane proteins on their surface. In the pseudotyping approach, the native virus surface protein is replaced with the protein of interest. This gives rise to membrane proteins that are properly folded and oriented on cell-derived membranes. Vesicular stomatitis virus (VSV) is a favorable platform for the pseudotyping approach with reported success (Avinoam, Fridman et al. 2011). Pseudotyping VSV is based on a recombinant virus called VSVAG;. in this recombinant virus the gene for the native glycoprotein was deleted (Whitt 2010). Simpler systems, circumventing bio-safety issues, using virus-like particles (VLPs) are likewise an useful approach to display membrane proteins (Poliakov, Spilman et al. 2009; Irene, Eric et al. 2012). This approach is based on the observation that expression of the capsid or matrix proteins of many viruses leads to the assembly of particles that are structurally similar to authentic viruses. For production of VLPs, the protein of interest needs to be co-expressed in the same cell as the viral capsid/matrix protein or alternatively, it can be fused to the viral protein and the chimera is displayed on the membrane. An inherent limitation of the virus based and VLP technologies is the need for viral components. Additionally, integral membrane proteins with bulky cytoplasmic domains will not readily be packed in neither pseudotype virus nor VLPs due to sterical hindrance from the virus capsid or matrix proteins. Furthermore, in these cases the cytoplasmic domain of the membrane protein is potentially conformationally altered which limits the applications to studying the extracellular domain only.

Summary of the invention

The inventors have developed a simple method that provides high yields of membrane vesicles comprising one or more membrane proteins. Surprisingly, the membrane vesicles are produced by cells independently of a viral component and induced independently of a chemical vesiculant. The membrane vesicles produced by the method of the invention therefore provide a platform for intact membrane proteins with correct anchoring and topology in a membrane reflective of the true lipid composition of biological membranes. The membrane vesicles can be used for many applications including structural/functional studies, raising antibodies, protein- protein interaction assays as well as for diagnostic uses, biomarkers and therapeutics.

Accordingly, the inventions provides a method of producing a preparation of membrane vesicles comprising one or more membrane proteins, the method comprising (a) providing a population of cells into which the one or more membrane proteins have been introduced and (b) culturing the population of cells and thereby producing a preparation of extracellular membrane vesicles comprising the one or more membrane proteins; wherein step (b) is performed independently of a viral component and independently of a chemical vesiculant.

The invention also provides:

- a preparation of membrane vesicles produced by a method of the invention;

- a pharmaceutical composition comprising a preparation of membrane vesicles of the invention and a pharmaceutically acceptable carrier or diluent; - a vaccine composition comprising a preparation of membrane vesicles of the invention and an adjuvant;

- a method of vaccinating an individual against one or more membrane proteins, the

method comprising administering to the individual a preparation of the invention, a pharmaceutical composition of the invention or a vaccine composition of the invention;

method comprising: (a) producing a preparation of membrane vesicles comprising the one or more membrane proteins using a method of the invention; and (b) administering to the individual the preparation;

a method of determining the three-dimensional structure of one or more membrane proteins, the method comprising: (a) providing a preparation of membrane vesicles of the invention which comprises the one or more membrane proteins; and (b) determining the structure of the one or more membrane proteins;

a method of determining the three-dimensional structure of one or more membrane proteins, the method comprising: (a) producing a preparation of membrane vesicles comprising the one or more membrane proteins using a method of the invention; and (b) determining the structure of the one or more membrane proteins.

Brief description of the Figures

Figure 1. (A-C) In the pseudotyping approach, the native virus surface protein is replaced with the protein of interest. This approach was applied to two functionally related

transmembrane proteins named AFF and EFF on recombinant VSV (VSVAG, bullet shaped virus). While AFF was very efficiently incorporated into the VSVAG membrane (A) EFF was not (B) most likely due to the long cytoplasmic domain of EFF. (C) Typically, a small amount of vesicles highly enriched in the protein of interest (in area marked by black corners) was co- purified with the VSV pseudotyped virus. Inset, enlargement of the field marked with white frame. (D-G) Membrane protein enriched extracellular vesicles (MPEEVs) with AFF protein (D, F), EFF (E) and the herpesvirus glycoprotein B (gB) (G). The yield of MPEEVs is substantially improved compared to the small amount obtained as byproduct of the pseudotyping, compare (F) to (C). (H) As a control, vesicles collected from cells transfected with yellow fluorescence protein (YFP), i.e. a soluble, non-membrane protein, are shown. Proteins observed on the membrane are either serum proteins (from the cell culture medium) or endogenous membrane proteins. Interestingly, in the control experiment (H) the yield of vesicles was substantially reduced compared to the other preparations with membrane protein (D, E, G)). This indicates that membrane protein over-expression induces extracellular vesicle secretion. (I) SDS-PAGE of total protein from different vesicle preparations. The over-expressed protein's name is given on the top of the lane and the corresponding band marked by an asterisks. Additional protein bands are mainly from the serum (i.e. soluble, non-membrane proteins) in the cell growth medium as confirmed by mass spectrometry while not detecting other membrane proteins.

Detailed description of the invention

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a membrane vesicle" includes two or more such vesicles, reference to "a membrane protein" includes two or more such proteins, and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Methods of producing membrane vesicles

The present invention relates to a method of producing a preparation of membrane vesicles comprising one or more membrane proteins. The method comprises the step (a) of providing a population of cells into which the one or more membrane proteins have been introduced. The method further comprises the step (b) of culturing the population of cells and thereby producing a preparation of extracellular membrane vesicles comprising the one or more membrane proteins. The step of culturing the modified population of cells (i.e., the cells into which the one or more membrane proteins are introduced) and thereby producing a preparation of extracellular membrane vesicles is performed independently of a viral component and independently of a chemical vesiculant.

The term "producing" means increasing the total number of membrane vesicles comprising one or more membrane proteins over time. An unmodified or unprocessed population of cells may produce membrane vesicles comprising one or more membrane proteins. The method of the invention does not relate to the production of extracellular membrane vesicles in an unmodified or unprocessed population of cells. The population of cells used in the method of the invention are engineered to express, such as over-express, the one or more membrane proteins that are to be included in the membrane vesicles. In some instances, such as when the one or more membrane proteins are over-expressed, the membrane vesicles comprising one or more membrane proteins are produced in increased numbers, with a more homogenous protein population (i.e. with less background protein) and/or at a faster rate relative to the production of membrane vesicles in an unmodified or unprocessed population of cells.

Step (a) comprises providing a population of cells into which the one or more membrane proteins have been introduced. The population of cells may be a stable cell line engineered to express the one or more membrane proteins. Step (a) preferably comprises introducing the one or more membrane proteins into the population of cells. The term "introducing" means deliberately modifying, engineering or processing a population of cells in such a way that the cells express the one or more membrane proteins. The introduction of one or more membrane proteins into a population of cells may employ any available technique.

In one embodiment, one or more genes encoding the one or more membrane proteins of interest are introduced into the population by transfection. In other words, one or more genes encoding the one or more membrane proteins are introduced into the population by non- viral methods such as chemical-based (e.g., calcium phosphate, DEAE-Dextran, polymers, liposomes or nanoparticles), or physical methods (e.g., electroporation, nanoparticles, magnetofection).

In an alternative embodiment, one or more genes encoding the one or more membrane proteins are introduced into the population by transformation. In other words, one or more genes encoding the one or more membrane proteins are introduced into the population by non- viral methods such as chemical-based (e.g., calcium chloride), or physical methods (e.g., electroporation or heat shock).

In other embodiments, one or more genes encoding the one or more membrane proteins are introduced into the population by transduction. In other words, one or more genes encoding the one or more membrane proteins are introduced into the population by viral-based methods (i.e., using a viral vector). Suitable viral vectors are known in the art and include (but are not limited to) retroviruses, lentiviruses, adenoviruses, adeno-associated viruses and nanoengineered viruses. The viral vector is typically modified so that it is attenuated (i.e. modified to reduce its virulence).

In any of the above methods, nucleic acids sequences, expression constructs or vectors which encode the one or more membrane proteins may be introduced into the population. The cells may be transfected or transformed with one or more expression vectors. Suitable vectors include, but are not limited to, pHLsec, pCAGGS, pGE E and pSWITCH. Any population of cells may be used in the invention. The population of cells may naturally produce membrane vesicles in an unmodified or unprocessed state. The cells are preferably cell wall-free cells. Suitable populations of cells include higher eukaryotic cell lines such as mammalian cells, typically human cells. Particular examples of cells which may be modified by insertion of vectors or expression cassettes encoding the one or more membrane proteins include mammalian PC12, HEK293, HEK293A, HEK293T, CHO, BHK-21, HeLa, ARPE-19, RAW264.7 and COS cells. Yeast cells may also be used in accordance with invention.

Preferably, the cells selected are not only stable, but also allow for mature glycosylation and cell surface expression of the membrane protein. Preferably, the cells allow the production of large amounts of membrane vesicles. As such, the cells typically express the one or more membrane proteins at a high level. Populations of cells will be chosen to be compatible with the nucleic acids sequence, expression construct or vector used to transfect, transform or transduce the cells.

As discussed below, the one or more membrane proteins may be endogenous proteins, i.e. proteins expressed by the cells in their unmodified or unprocessed state. In the method of the invention, the population preferably over-expresses one or more endogenous membrane proteins. In this instance, over-expression relates to the increased expression of the one or more membrane proteins in a population of modified cells (i.e., transfected, transformed or transduced cells) when compared with a population of unmodified cells of the same type (i.e., non-transfected, non-transformed or non-transduced cells of the same type).

As discussed below, the one or more membrane proteins may be exogenous proteins, i.e. proteins which are naturally not expressed by the unmodified or unprocessed cells. In the method of the invention, the population of cells preferably over-expresses one or more exogenous membrane proteins. In this instance, over-expression relates to the increased expression of the one or more membrane proteins in a population of modified cells (i.e., transfected, transformed or transduced cells) when compared with the normal expression level in other unmodfied cells (i.e., other non-transfected, non-transformed or non-transduced cells).

Expression of membrane proteins may be measured using known methods, such as SDS- PAGE, western blotting, mass spectrometry and fluorescent- and colorimetric-based assays.

The term "culturing" typically means growing the population of cells in vitro under controlled conditions. In other words, cultured cells are typically grown outside of their natural environment in a conditioned media. Suitable media are known in the art. Suitable culture methods can be determined by a skilled practitioner based on his common general knowledge, taking into account, for example, the particular population of cells that are being used in the method of the invention. Cells may be cultured in standard conditions of 37°C, 5% C0₂ in medium, such as Dulbecco's Modified Eagle's Medium (DMEM) or Glasgow Minimum

Essential Medium (GMEM), supplemented with serum.

In the method of the invention, the population of cells secretes, releases or sheds membrane vesicles comprising the one or more membrane proteins into the culture medium. The secretion, release or shedding of membrane vesicles is typically stimulated (i.e., an increased rate and/or duration of secretion, release or shedding) by introducing the one or more membrane proteins into the population of cells. For example, where one or more membrane proteins are over-expressed in the population of cells, membrane vesicle secretion pathways may be used by cells as a mechanism to reduce the total amount of one or more membrane proteins within the cells.

The step of culturing the modified population of cells and producing a preparation of membrane vesicles is performed independently of a viral component. A viral component is typically a virus, a virus-like particle or a viral protein, such as a retroviral core protein. A viruslike particle is a virus lacking viral genetic material. Virus-like particles are non-infectious. Performed "independently of means a viral component is stimulating the production of membrane vesicles. In other words, a viral component is not driving the production of membrane vesicles. The production of membrane vesicles is not dependent on a viral component.

For example, a viral component is not used in step b) of the method of the invention to stimulate the secretion, release or shed of membrane vesicles comprising the membrane protein into the culture medium. However, viral components may be present during step b) of the method in, for example, an amount too low to stimulate the production of membrane vesicles or where the membrane proteins of interest are viral protein themselves. These viral components may, for example, be derived from a viral vector used in step a). In some embodiments of the invention, a viral component is present at a multiplicity of infection (MOI) of less than 1, less than 0.5 or less than 0.1. Preferably, step b) of the method is performed in the absence of a viral component. A viral component (i.e. a virus, virus-like particle or viral protein) may be used in the step (a) of introducing the one or more membrane proteins into a population of cells (see above). The one or more membrane proteins of interest may also comprise viral proteins (see below) or form together with the vesicle a virus like particle.

The step of culturing the modified cells and producing a preparation of membrane vesicles is performed independently of a chemical vesiculant. A chemical vesiculant is preferably formaldehyde, diotheritol (DTT) or N-ethyl maleimide (NEM). Performed "independently of means a chemical vesiculant is not simulating the production of membrane vesicles. In other words, a chemical vesiculant is not driving the production of membrane vesicles. The production of membrane vesicles is not dependent on a chemical vesiculant.

For example, chemical vesiculants are not used in step b) of the method of the invention to stimulate the secretion, release or shed of membrane vesicles comprising the one or more membrane proteins into the culture medium. However, chemical vesiculants may be present during step b) of the method in, for example, an amount too low to stimulate the production of membrane vesicles. In some embodiments of the invention, the chemical vesiculant

formaldehyde is present at a concentration of less than lOmM, less than ImM, less than 0.5mM or less than 0. ImM. In some embodiments of the invention, chemical vesiculants such as diothiotheritol or N-ethyl maleimide are present at a concentration of less than ImM, less than 0.5mM or less than 0. ImM.

Preferably, step b) of the method is performed in the absence of a chemical vesiculant. Chemical vesiculants may be used in the step (a) of introducing the one or more membrane proteins into a population of cells (see above).

Typically, the method of the invention further comprises isolating or substantially isolating from the modified population of cells the preparation of membrane vesicles comprising the one or more membrane proteins. The membrane vesicles may be isolated from or

substantially isolated from any cell type that secretes membrane vesicles. Preferably, the preparation of membrane vesicles are isolated from or substantially isolated from cell-wall free cells. In other embodiments, the preparation of membrane vesicles are isolated from or substantially isolated from mammalian cells such as human cells.

The term "isolated" means that all cells have been removed from the preparation of membrane vesicles. For example, membrane vesicles may be washed from cells to obtain a preparation of membrane vesicles. The preparation preferably does not comprise any cells. The term "substantially isolated" means that the vast majority of cells have been removed from the preparation of membrane vesicles. The preparation preferably comprises a significantly reduced amount of cells compared with an unmodified or unprocessed population of cells which produce membrane vesicles. Any remaining cells may be alive. Any remaining cells are preferably dead.

Methods of isolating membrane vesicles from cultured cells are well known in the art.

For example, membrane vesicles may be isolated from cells by centrifugation, for example by differential or ultra-centrifugation. Preferably, the step of isolation further comprises

purification by sucrose gradient. The membrane vesicles are typically present in the supernatant of centrifuged cells. Other methods of isolating membrane vesicles are contemplated by the invention. For example, simplified and shortened processes of membrane vesicle isolation have been developed.

Membrane vesicles

Membrane vesicles are small, substantially spherical membrane structures. The membrane vesicles produced in the method of the invention are typically "non-live" or not live. The membrane vesicles are typically dead.

The membrane vesicles comprising the one or more membrane proteins in the preparation are themselves not live cells. The preparation may contain some live cells. Live cells typically comprise a nucleus and undergo metabolic and respiratory metabolism. The membrane vesicles in the preparation do not comprise a nucleus. The membrane vesicles may not undergo metabolic and/or respiratory activity.

Membrane vesicles within the preparation may be of different sizes or shapes. The membrane vesicles are typically smaller than a typical mammalian cell, which may be, for example, 40μπι in diameter. For example, the membrane vesicles within the preparation may be ΙΟμπι or less in diameter, such as Ι μπι or less, 500nm or less, 300nm or less, 200nm or less, 150nm or less, lOOnm or less or 50nm or less. The membrane vesicles preferably have a diameter that is sufficient for the one or more membrane proteins to be orientated in their membrane in the same way as in live cells. The size and/or the shape of the membrane vesicles may be dependent on the one or more membrane proteins of interest.

The present invention relates to a preparation of membrane vesicles produced by the method of the invention. The preparation of membrane vesicles of the invention may comprise one or more different membrane proteins relative to a unmodified (i.e., naturally occurring) preparation of membrane vesicles. The preparation of membrane vesicles may comprise an increased ratio of one or more membrane proteins relative to a unmodified population of cells. For example, one or more membrane proteins in a preparation of membrane vesicles of the invention may be present in a ratio of more than 2, more than 4, more than 8, more than 10, more then 100, or more than 10000 relative to a preparation of unmodified preparation of membrane vesicles. The preparation of membrane vesicles of the invention does not relate to naturally occurring membrane vesicles produced by an unmodified or unprocessed population of cells.

The membrane vesicles in the preparation may be maintained under a wide range of temperatures. This allows the preparation of membrane vesicles, for example, to be stored at room temperature or below. The membrane vesicles are stable, for example, at 4°C . The membrane vesicles may be in a frozen form. For instance, the structure of the one or more membrane proteins in frozen membrane vesicles may be determined using transmission electron cryo microscopy (cryo EM) or electron cryo tomography (cryoET). The membrane vesicles may be stored below -70°€. Membrane proteins

The present invention relates to membrane vesicles comprising one or more membrane proteins. For example, the membrane vesicles of the invention may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more different membrane proteins. Any one or more membrane proteins may be used in the method of the invention.

In the context of the invention, a membrane protein is any protein that may be attached to or located in the vesicles' membrane. The one or more membrane proteins may be one or more proteins which are naturally attached to or located in cell membranes, such as channels and transporters. The one or more membrane proteins may be soluble proteins that have been modified to allow them to be attached to or located in the vesicles' membrane. The one or more membrane proteins may be portions or fragments of naturally occurring proteins, such as portions or fragments of channels or transporters. Combinations of these different types of membrane proteins may be used in accordance with the invention. The one or more membrane proteins are preferably one or more transmembrane proteins.

The membrane vesicles of the invention typically comprise one or more membrane proteins with correct anchoring and topology. This allows, for example, the provision of a preparation, a pharmaceutical composition or a vaccine comprising one or more biologically active membrane proteins, enabling the protein to penetrate across the plasma membrane of eukaryotic cells.

The one or more membrane proteins may be a therapeutic membrane protein. In other words, the one or more membrane proteins may be used to replace a protein that is deficient or abnormal; to augment an existing pathway; and/or provide a novel function or activity.

Alternatively, the protein may be used to interfere with a molecule or organism or deliver other compounds or proteins.

The membrane protein is preferably a transmembrane protein, an integral monotopic protein or a peripheral membrane protein. Transmembrane proteins are proteins that span biological membranes. Transmembrane proteins include β-sheet transmembrane proteins and a- helical transmembrane proteins. The transmembrane protein is preferably a G-protein coupled receptor (GPCR), an ion channel, a transporter, a glycophorin, an integrin, a cadherin, a selectin, a cluster of differentiation (CD) protein or a porin. The GPCR is preferably a metabotropic receptor or a non ligand-mediated GPCR.

Metabotropic receptors are ligand-gated peptide hormone and neurotransmitter receptors.

Preferred metabotropic receptors include, but are not limited to, muscarinic acetylcholine receptors, catecholamine receptors, serotonin receptors, GABAB receptors, metabotropic glutamate receptors and peptide hormone receptors. The peptide hormone receptor is preferably a neurotensin receptor, such as neurotensin receptor 1 (NTS1) or NTS2. The non ligand- mediated GPCR is preferably rhodopsin.

Ion channels are pore-forming proteins that help establish and control the voltage gradient across the plasma membrane of all living cells. Preferred ion channels include, but are not limited to, nicotinic acetylcholine receptors, potassium channels and calcium channels.

Preferred transporters include, but are not limited to, trans-activating regulatory protein (TAT) transporters, adenosine triphosphate (ATP) transporters, voltage-gated potassium channels (e.g. KcsA or KvAP), calcium ATPases and proton pumps.

Porins are β-sheet proteins that cross biological membranes and act as a pore through which molecules can diffuse. Preferred porins include, but are not limited to, Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF) and outer membrane porin G (OmpG).

An integral monotopic protein is permanently attached to a biological membrane from only one side. Preferred integral monotopic proteins include, but are not limited to, carnitine palmitoyltransferases, monoamine oxidases and fatty acid amide hydrolases.

A peripheral membrane protein is temporarily attached either to a biological membrane or to integral proteins therein by a combination of hydrophobic, electrostatic, and other non- covalent interactions. Preferred peripheral membrane proteins include, but are not limited to, enzymes, structural proteins, peripheral transporters of small hydrophobic molecules, electron carriers, polypeptide hormones, toxins and antimicrobial peptides. Preferred peripheral membrane enzymes include, but are not limited to, phospholipases (e.g. phospholipase C), cholesterol oxidases, glycosyltransferases, transglycosidases, signal peptidases and

lipooxygenases. Preferred peripheral membrane structural proteins include, but are not limited to, Annexins, synapsin I, synuclein and spectrin. Preferred peripheral transporters of small hydrophobic molecules include, but are not limited to, lipocalins, sterol carrier proteins and glycolipid transfer proteins. Preferred peripheral membrane electron carriers include, but are not limited to, cytochrome c, flavoproteins and cupredoxins. Preferred peripheral membrane polypetide hormones, toxins and antimicrobial peptides include, but are not limited to, bacterial toxins (e.g. lantibiotic peptides, Gramicidin S), defensins, neuronal peptides (e.g. tachykinin peptides) and apoptosis regulators (e.g. Bcl-2 family).

The one or more membrane proteins may be one or more fusion proteins. For instance, the one or membrane proteins may be viral or eukaryotic fusion proteins, including placental syntein. As discussed above, the one or more membrane proteins may be modified soluble proteins which artificially anchored to the membrane. For instance, proteins may be fused to part or all of a transmembrane protein. The one or more proteins may also be modified with cholesterol or a glycophosphatidylinositol (GPI) anchor.

The one or more membrane proteins may be a protein vaccine. For example, the one or more membrane proteins may be used to protect against a deleterious foreign agent; to treat an autoimmune disease; and/or to treat cancer. The one or more membrane proteins may be a bacterial or a fungal protein. As described above, the method of producing membrane vesicles comprising one or more membrane proteins is performed independently of a viral component (i.e., virus, virus-like particle or viral protein). However, the one or more membrane proteins that are introduced into the population of cells may be, or be derived from, a viral component. For example, the one or more membrane proteins that are introduced into the population of cells may be a viral protein or may be derived from a viral protein. The one or more membrane proteins may be selected from, but are not limited to in any way, the membrane proteins shown in Table 1 below.

Table 1

Membrane protein NCBI Accession

aquaporin-4 (AQP4) P_001641.1, P_004019.1 myelin-oligodendrocyte glycoprotein (MOG) P 996537.3, P 001163889.1,

P 996533.2, NP 001008230.1, NP 001008229.1, NP 996534.2, NP 002424.3, NP 996535.2,

NP_996532.2

acetylcholine receptor (AChR) - alpha subunit NP_000737.1 , NP_001177384.1 acetylcholine receptor (AChR) - beta subunit NP_000739.1

muscle specific tyrosine kinase (MuSK) NP_005583.1, NP_001159752.1,

NP 001159753.1

contactin associated protein-like 2 (CASPR2) NP_054860.1

metabotropic glutamate receptor 5 (mGluR5) NP_001137303.1, NP_000833.1 metabotropic glutamate receptor 1 (mGluRl) NP_000829.2, NP_001107801.1 N-Methyl-D-aspartate (NMD A) receptor NR1 NP_000823.4, NP_067544.1,

NP_015566.1, NP_001172019.1, NP_001172020.1

N-Methyl-D-aspartate (NMD A) receptor NR2A NP_001127879.1, NP_000824.1,

NP_001127880.1

N-Methyl-D-aspartate (NMD A) receptor NR2B NP_000825.2

leucine-rich glioma inactivated protein 1(LGI1) NP_005088.1

Contactin-2 (CNTN2) NP_005067.1

glutamic acid decarboxylase 1 (GADl) NP_038473.2, NP_000808.2 glutamic acid decarboxylase 2 (GAD2) NP_001127838.1, NP_000809.1

AMP A glutamate receptor 1 (GluAl) NP_000818.2, NP_001107655.1,

NP_001244948.1, NP_001244949.1, NP_001244950.1, NP_001244951.1, NP_001244952.1

AMPA glutamate receptor 2 (GluA2) NP_000817.2, NP_001077088.1,

NP 001077089.1

AMP A glutamate receptor 3 (GluA3) NP_015564.4, NP_000819.3,

NP_001243672.1

GAB A type B receptor subunit l(GABABRl) NP 001461.1, NP 068703.1,

NP 068704.2

GAB A receptor type B receptor subunit 2 NP_005449.5

(GABABR2)

Glycine receptor alpha 1 (GlyRAl) NP_000162.2, NP_001139512.1

Glycine receptor alpha 2 (GlyRA2) NP_002054.1, NP_001112357.1,

NP 001112358.1, NP 001165413.1

Glycine receptor alpha 3 (GlyRA3) NP_006520.2, NP_001036008.1

Glycine receptor alpha 4 (GlyRA4) NP_001019623.2, NP_001165756.1

Glycine receptor beta (GlyB) NP_000815.1, NP_001159532.1,

NP_001159533.1

Voltage-gated calcium channel (VGCC) NP_000713.2, NP_954856.1,

NP_954855.1, NP_000714.3, NP_001193846.1 , NP_001193845.1 , NP_000716.2, NP_060868.2, NP_001139270.1, NP_001005747.1, P_000717.2, NP_001005746.1,

P_001005505.1, P_001167522.1, P_758952.4, P_006021.2

Receptor protein tyrosine phosphatase sigma (RPTPo) P_002841.3, P_570924.2,

P_570923.2, P_570925.2

C. elegans protein EFF-1 P 001021990.1, P 001021989.1,

P_001021988.1, P_001021987.1

C. elegans protein AFF-1 P_495402.3

Herpes virus glycoprotein B (gB) AF097023_1, AAA60540.1

Herpes virus glycoprotein H (gH) AFH41178.1, AAA45938.1

Herpes virus glycoprotein L (gL) AFH78105.1, BAA01264.1

Herpes virus glycoprotein D (gD) AAB59754.1,

Herpes virus entry mediator/receptor AAB58354.1

Retrovirus Env protein CAB58990.1, CAB58982.1,

CAB58978.1, CAB59009.1

Retrovirus entry receptors (mCAT)

The polypeptide sequences of the membrane proteins listed above are identified by NCBI accession numbers. For some membrane proteins, multiple NCBI accession numbers are indicated which relate to different isoforms of the respective proteins. The method of the invention may involves the use of variants of these polypeptide sequences. For example, the method of the invention may use sequences which have at least 95%, at least 98%> or at least 99%), homology to any one of the polypeptide sequences identified in Table 1 based on amino acid identity over their entire sequence.

The above mentioned homology is calculated on the basis of amino acid identity

(sometimes referred to as "hard homology"). The UWGCG Package provides programs including GAP, BESTFIT, COMPARE, ALIGN and PILEUP that can be used to calculate homology or line up sequences (for example used on their default settings). The BLAST algorithm can also be used to compare or line up two sequences, typically on its default settings. Software for performing a BLAST comparison of two sequences is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm is further described below. Similar publicly available tools for the alignment and comparison of sequences may be found on the European Bioinformatics Institute website (http://www.ebi.ac.uk), for example the ALIGN and CLUSTALW programs. A BLAST analysis is preferably used for calculating identity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology

Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

The homologous sequences typically differ by at least 1, 2, 5, 10, 20 or more mutations

(which may be substitutions, deletions or insertions of amino acids). These mutations may be measured across any of the regions mentioned above in relation to calculating identity. The substitutions are preferably conservative substitutions. These are defined according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: ALIPHATIC Non-polar G A P

I L V

Polar - uncharged C S T M

N Q

Polar - charged D E

K R

AROMATIC H F W Y

Any of the one or more of the membrane proteins used in the method of the invention may be in the form of a dimer or a larger array. For example, AQP4 is known to form in a tetramer and then becomes clustered to higher order arrays. Any of the one or more membrane proteins may be further chemically-modified to form a derivative. Derivatives include polypeptides that have lipid extensions or have been glycosylated. Derivatives also include polypeptides that have been detectably labelled. Detectably labelled polypeptides have been labelled with a labelling moiety that can be readily detected. Examples of labelling moieties include, but are not limited to, radioisotopes or radionucleotides, fluorophores such as green fluorescent protein (GFP), electron-dense reagents, quenchers of fluorescence, enzymes, affinity tags and epitope tags. Preferred radioisotopes include, but are not limited to, tritium and iodine. Affinity tags are labels that confer the ability to specifically bind a reagent onto the labelled molecule. Examples include, but are not limited to, biotin, histidine tags and glutathione-S- transferase (GST). Labels may be detected by, for example, spectroscopic, photochemical, radiochemical, biochemical, immunochemical or chemical methods that are known in the art.

Any of the one or more membrane proteins may also comprise additional amino acids or polypeptide sequences. Any of the one or more membrane proteins may comprise additional polypeptide sequences such that they form fusion proteins. The additional polypeptide sequences may be fused at the amino terminus, carboxy terminus or both the amino terminus and the carboxy terminus. Alternatively, the additional polypeptide sequence may be within the coding region of the polypeptide. Examples of fusion partners include, but are not limited to, GST, maltose binding protein, alkaline phosphatates, thiorexidin, GFP or other fluorescent tags, biotin tags, histidine tags and epitope tags (for example, Myc or FLAG). CCRL2 polypeptides may be fused to a GTP-binding protein (G protein).

As outlined above, cells which produce membrane vesicles may be transfected, transformed or transduced with polynucleotide sequences that encode the one or more membrane proteins. For example, mammalian cells may be transfected with polynucleotide sequences that encode any one of the polypeptide sequences identified in Table 1, or additional isoforms or variants thereof. Polynucleotide sequences may be isolated and replicated using standard methods in the art. The gene encoding the one or more membrane proteins may be amplified using PCR involving specific primers. The amplified sequences may then be incorporated into a recombinant replicable vector such as a cloning vector. The vector may be used to replicate the polynucleotide in a compatible host cell. Thus polynucleotide sequences encoding the one or more membrane proteins may be made by introducing a polynucleotide encoding the one or more membrane proteins into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described above.

The polynucleotide sequence may be cloned into any suitable expression vector. In an expression vector, the polynucleotide sequence encoding a construct is typically operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell. Such expression vectors can be used to express a construct.

The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Multiple copies of the same or different polynucleotides, may be introduced into the vector.

The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide sequence and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene. Promoters and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed. For example, a cytomegalovirus (CMV) or chicken-beta-actin promoter may be typically used for constitutive expression of the polypeptide in mammalian cell lines.

Typically, the one or more membrane proteisn are derived from a cell transfected with the membrane protein, wherein the cell overexpresses the polynucleotide encoding the membrane protein. Increased levels of the membrane protein in the cell may lead to an increased level of the membrane protein in the preparation of membrane vesicles.

Pharmaceutical and vaccine compositions

The present invention provides a preparation of membrane vesicles comprising membrane proteins or parts thereof having the right topology relative to the membrane. The invention therefore also relates to a pharmaceutical composition comprising a preparation of membrane vesicles of the invention and a pharmaceutically acceptable carrier or diluent.

The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone;

disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.

Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.

Oral formulations include such normally employed excipients as, for example,

pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95%) of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.

Capsules, tablets and pills for oral administration to an individual or baby may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.

Pharmaceutical compositions suitable for delivery by needleless injection, for example, transdermally, may also be used.

The present invention further relates to a vaccine composition comprising a preparation of membrane vesicles of the invention and an adjuvant.

The term "adjuvant" means any material or composition capable of specifically or non- specifically altering, enhancing, directing, redirecting, potentiating or initiating an immune response.

Methods of vaccinating an individual

The present invention also relates to a method of vaccinating an individual against one or more membrane proteins, the method comprising administering to the individual a preparation of membrane vesicles of the invention, a pharmaceutical composition of the invention or a vaccine composition of the invention. In other words, the preparation of membrane vesicles of the invention, a pharmaceutical composition of the invention or a vaccine composition of the invention may be administered to the individual in order to generate an adaptive immune response (i.e., a humoral response and/or a cell-mediated response to a particular antigen).

The present invention also relates to the preparation of membrane vesicles of the invention, pharmaceutical composition of the invention or vaccine composition of the invention for use in (a) a method of treating or preventing a disease in an individual or (b) vaccinating an individual.

The present invention also relates to use of a preparation of membrane vesicles of the invention, pharmaceutical composition of the invention or vaccine composition of the invention in the manufacture of a medicament for (a) the treatment or prevention of disease in an individual or (b) vaccinating an individual.

Typically, the individual is human, but alternatively it may be another mammal such as a commercially farmed animal, such as a horse, a cow, a sheep or a pig, or may alternatively be a pet, such as a cat, a dog or a rodent (especially a rat or a mouse), or an experimental animal. The individual is typically a patient.

The preparation or composition may be administered to the individual by any suitable means. The preparation or composition can be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, intraarticular, topical or other appropriate administration routes. The formulation will depend upon factors such as the nature of the preparation or composition and the disease to be treated. The preparation or composition may be administered in a variety of dosage forms. It may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), parenterally, subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The preparation or composition may also be administered as a suppository. A physician will be able to determine the required route of administration for each particular individual.

A therapeutically or prophylactically effective amount of the preparation or composition is administered. The dose may be determined according to various parameters, especially according to the compound used; the age, weight and condition of the individual or baby to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular individual. A typical daily dose is from about 0.1 to 50mg per kg, preferably from about O. lmg/kg to lOmg/kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the individual to be treated, the type and severity of the disease and the frequency and route of administration. Preferably, daily dosage levels are from 5mg to 2g.

Methods for determining three-dimensional structure of membrane protein

The invention relates to a method of determining the three-dimensional structure of one or more membrane proteins, the method comprising (a) providing a preparation of membrane vesicles of the invention which comprises the one or more membrane proteins; and (b) determining the structure of the one or more membrane proteins.

The invention also relates to methods of determining the three-dimensional structure of one or more membrane proteins, the method comprising the step of providing a preparation of membrane vesicles of the invention which comprise the one or more membrane proteins and determining the structure of the one or more membrane proteins. Preferably, the membrane vesicles of the invention are provided in a frozen form.

The invention also preferably relates to a method of determining the three-dimensional structure of one or more membrane proteins, the method comprising (a) producing a preparation of membrane vesicles comprising one or more membrane proteins using a method of the invention, (b) preparing the membrane vesicles comprising the one or more membrane for electron microscopy, by e.g. negative staining, plunge freezing or high pressure freezing, and (c) determining the structure of the one or more membrane proteins. Freezing of the membrane vesicles allows the structure of the one or more of the membrane proteins to be determined (i.e., using transmission electron cryo microscopy, cryo TEM). Any method of preparing the membrane vesicles for freezing may be used. Suitable methods are known in the art. The method of the invention preferably comprises freezing the membrane vesicles so that the membrane vesicles are embedded in vitreous ice.

Any method can be used to determine the structure of the one or more membrane proteins. Suitable methods to determine the structure can be determined by a skilled practitioner based on his common general knowledge, taking into account, for example, the particular membrane proteins that are being characterised.

Typically, the structure of the one or more membrane proteins is determined using transmission electron microscopy (TEM), transmission electron cryo microscopy (cryo TEM), electron cryo tomography (cryoET), crystallography or electron microscopy (EM), including negative staining EM.

Cryo EM comprises determining the structure of a protein in a frozen-hydrated state (van Heel M, Gowen B, Matadeen R, Orlova EV, Finn R, Pape T, Cohen D, Stark H, Schmidt R, Schatz M, Patwardhan A (2000). "Single-particle electron cryo-microscopy: towards atomic resolution.". Q Rev Biophys. 33 : 307-69).

CryoET is a type of electron cryomicroscopy where tomography is used to obtain a 3D reconstruction of a sample from tilted 2D images at cryogenic temperatures (Koster et al. Journal of Structural Biology, Volume 120, Issue 3, December 1997, Pages 276-308).

Crystallography typically comprises determining the structure of a protein by x-ray diffraction measurement of the protein in a crystalline state (Rupp B (2009). Biomolecular Crystallography: Principles, Practice and Application to Structural Biology. New York: Garland Science.).

Two dimensional electron crystallography may be used determine the arrangement of atoms in the one or more membrane proteins using a transmission electron microscope

(Kuhlbrandt et al, (February 1994). "Atomic model of plant light-harvesting complex by electron crystallography". Nature 367 (6464): 614-21).

The invention is illustrated by the following Example. Example

1. Introduction

A novel technology has been developed that provides high yields of cell-derived, protein enriched extracellular vesicles (MPEEV). Crucially, the vesicles are cell-free and induced independently of a viral component. The basis for this approach is the utilization of the recently characterized biological process of membrane vesicle secretion (Poliakov, Spilman et al. 2009; Irene, Eric et al. 2012). Extracellular vesicle secretion seems to be a universal and evolutionary conserved process under both physiological and pathological conditions (Gyorgy, Szabo et al. 2011). Giant plasma membrane vesicles can also be induced by chemical agents like paraformaldehyde with dithiothreitol. However, these agents have severe effects on protein integrity and thus are used mainly to study membrane biophysics (Sezgin, Kaiser et al. 2012).

2. Method

The protocol that has been developed for preparing MPEEVs is unexpectedly simple: cells are transfected with the gene of the protein of interest and allowed time to over-express the protein and for accumulation of the MPEEVs in the growth medium. The MPEEVs can be separated from producer cells and cell debris by pipetting off the supernatant and particularly where purity is an important concern, differential centrifugation of the supernatant. Vesicles can be further concentrated for applications like microscopy by ultracentrifugation with sucrose cushion.

MPEEVs can be potentially isolated from a variety of cell-wall free cell types.

The success in MPEEVs production relies on the over-expression of the protein of interest. A high level of expression on the membrane is crucial. For that, plasmids using a strong promoter need to be used. The optimum time, post transfection, for collecting the MPEEVs will vary for different proteins and needs to be determined.

3. Results

Importantly, cryoEM imaging (Figure 1) confirmed that proteins retain their native topology. From these experiments, it is confirmed that MPEEVs are stable for at least several months in buffer at 4°C. 4. References

Avinoam, O., K. Fridman, et al. (2011). "Conserved eukaryotic fusogens can fuse viral

envelopes to cells." Science 332(6029): 589-592.

Gyorgy, B., T. G. Szabo, et al. (2011). "Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles." Cellular and molecular life sciences : CMLS 68(16): 2667-

2688.

Irene, T., L. Eric, et al. (2012). "Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy." Journal of Extracellular Vesicles 1.

Noad, R. and P. Roy (2003). "Virus-like particles as immunogens." Trends in microbiology

11(9): 438-444.

Poliakov, A., M. Spilman, et al. (2009). "Structural heterogeneity and protein composition of exosome-like vesicles (prostasomes) in human semen." The Prostate 69(2): 159-167.

Claims

1. A method of producing a preparation of membrane vesicles comprising one or more membrane proteins, the method comprising (a) providing a population of cells into which the one or more membrane proteins have been introduced and (b) culturing the population of cells and thereby producing a preparation of extracellular membrane vesicles comprising the one or more membrane proteins;

wherein step (b) is performed independently of a viral component and independently of a chemical vesiculant.

2. A method according to claim 1, wherein the viral component is a virus, virus-like particle or viral protein.

3. A method according to claim 1 or 2, wherein step (a) comprises introducing the one or more membrane proteins into the population of cells.

4. A method according to any one of the preceding claims, wherein the one or more membrane proteins are introduced into the population of cells by transfection, transformation or transduction.

5. A method according to any one of the preceding claims, wherein the method further comprises (c) isolating or substantially isolating from the population of cells resulting from step (a) of claim 1 the preparation of membrane vesicles comprising the one or more membrane proteins.

6. A method according to any one of the previous claims, wherein the cells are cell-wall free cells.

7. A method according to any one of the previous claims, wherein the cells over-express the one or more membrane proteins.

8. A method according to any one of the previous claims, wherein the one or more membrane proteins are selected from protein EFF-1, protein AFF-1, glycoprotein B (gB), glycoprotein H (gH), glycoprotein L (gL), aquaporin4 (AQP4), Glutamate receptor (GluD2; GD28), contactin associated protein-like 2 (Caspr2) and muscle specific tyrosine kinase (MuSK).

9. A method according to any one of the previous claims, wherein the membrane vesicles are about 2μπι or less in diameter.

10. A method according to any one of the previous claims, wherein the chemical vesiculant is formaldehyde, dithiotheritol (DTT) or N-ethyl maleimide (NEM).

11. A preparation of membrane vesicles produced by a method according to any one of the preceding claims.

12. A preparation according to claim 11, wherein the membrane vesicles are frozen.

13. A pharmaceutical composition comprising a preparation of membrane vesicles according to claim 11 and a pharmaceutically acceptable carrier or diluent.

14. A vaccine composition comprising a preparation of membrane vesicles according to claim 11 and an adjuvant.

15. A method of vaccinating an individual against one or more membrane proteins, the method comprising administering to the individual a preparation according to claim 11, a pharmaceutical composition according to claim 13 or a vaccine composition according to claim 14.

16. A method of vaccinating an individual against one or more membrane proteins, the method comprising:

(a) producing a preparation of membrane vesicles comprising the one or more membrane proteins using a method according to any one of claims 1 to 10; and

(b) administering to the individual the preparation.

17. A method of determining the three-dimensional structure of one or more

membrane proteins, the method comprising: (a) providing a preparation of membrane vesicles according to claim 11 or 12 which comprises the one or more membrane proteins; and

(b) determining the structure of the one or more membrane proteins.

18. A method of determining the three-dimensional structure of one or more

membrane proteins, the method comprising:

(b) determining the structure of the one or more membrane proteins.

19. A method according to claim 17 or 18, wherein the structure of the one or more membrane proteins is determined using transmission electron cryo microscopy (cryo EM), electron cryo tomography (cryoET), crystallography or electron micrscopy (EM).