Self-adjuvanting vesicular vaccine

Abstract

A self-adjuvanting extracellular vesicle (SAEV) is provided comprising an antigen and a STING activator, wherein the SAEV comprises a recombinant polypeptide which comprises the antigen and the STING activator, or the SAEV comprises a recombinant polypeptide comprising one of the antigen and the STING activator and the other of the antigen or the STING activator is co-loaded in the vesicle independently of the recombinant polypeptide. Methods of vaccination with a SAEV are also provided.

Description

SELF- AD JU V ANTING VESICULAR VACCINEField

[0001] The present invention generally relates to vaccines, and in particular, to vaccines based on extracellular vesicles.Background

[0002] Extracellular vesicles (EVs) are a heterogeneous group of lipid bound nanoparticles secreted by cells into the extracellular space. They act as mediators of many (patho) physiological processes. There are three main subtypes of EVs, including microvesicles (MVs), exosomes, and apoptotic bodies, each of which are differentiated based upon their size, content and function. The natural content, or cargo, of EVs consists of lipids, nucleic acids, and proteins.

[0003] The exploration of EVs for the delivery of therapeutic payloads to specific cells or tissues is ongoing. From a drug delivery perspective, EVs are similar to liposomes in that they are both are phospholipid based. However, EVs are assembled from a complex mixture of various lipids and surface and membrane proteins; some of these components aid tissue targeting, while others ensure minimal nonspecific interactions. Thus, despite their promising potential, the use of EVs in the clinical setting has been restricted due to their inherent complexity, size heterogeneity, and lack of standardized isolation and analysis methods.

[0004] It would be desirable to develop an EV-based therapeutic which overcomes one or more of the challenges associated with the use of EVs.Summary

[0005] Novel vaccines based on extracellular vesicles have now been developed which have advantageously been designed to be self adjuvanting, and as such, are capable of amplifying the immune response against a given antigen.

[0006] In its broadest sense, a self-adj uvanting extracellular vesicle (EV) for use as a vaccine is provided comprising an antigen and a STING activator. For example, a self-adj uvanting extracellular vesicle (SAEV) is provided comprising an antigen and a STING activator, wherein the SAEV comprises a recombinant polypeptide which comprises the antigen and the STING activator, or the SAEV comprises a recombinant polypeptide comprising one of the antigen or the STING activator and the otherof the antigen or the STING activator is co-loaded into the vesicle independent of the recombinant polypeptide.

[0007] In another aspect, a method of preparing a self-adjuvanting extracellular vesicle is provided comprising: i) introducing a transgene encoding a recombinant polypeptide comprising an EV anchoring polypeptide and a STING activator and / or an antigen into an EV-producing cell; ii) culturing the cell under suitable growth conditions to yield self-adjuvanting extracellular vesicles comprising the STING activator and the antigen, wherein the recombinant polypeptide comprises the STING activator and the antigen, or the recombinant polypeptide comprises one of the STING activator or the antigen and the other of the STING activator or the antigen is passively loaded into the SAEV independent of the recombinant polypeptide.

[0008] In another aspect, a method of vaccination in a mammal is provided. The method comprises administering to the mammal a self-adjuvanting extracellular vesicle comprising an antigen and a STING activator, wherein an immune response is induced in the mammal which is amplified in comparison to the immune response induced by the antigen alone.

[0009] In a further aspect, a method of vaccination in a mammal is provided comprising administering to the mammal an oncolytic virus comprising a transgene encoding an extracellular vesicular anchoring polypeptide and a STING activator, wherein the transgene is expressed in an extracellular self-adjuvanting vesicle and induces an immune response in the mammal which is amplified in comparison to the immune response induced by the oncolytic virus alone.

[0010] In another aspect, a method of vaccination is provided comprising administering to a mammal a transgene encoding an extracellular vesicular anchoring polypeptide and a STING activator and / or an antigen, wherein the transgene is expressed in an extracellular self-adjuvanting vesicle.

[0011] In another aspect, a transgene is provided encoding an antigen linked to an extracellular vesicle (EV)-anchoring moiety linked to a STING activator. A polypeptide encoded by the transgene is also provided, as well as an extracellular vesicle comprising the polypeptide.

[0012] These and other aspects of the invention are described herein in detail by reference to the following Figures in which:Brief Description of the Figures

[0013] Figure 1 illustrates A) a schematic representation of a vaccination study with VSVG- decorated exosomes comprising STING activator in accordance with an embodiment of the invention;B) western blotting and ELISA analysis showing expression of VSVG and Alix; C) results showing immune response to vaccination; D) schematic representation of the VSV challenge; and E) results from VSV titering in lungs after 96 h post-challenge.

[0014] Figure 2 illustrates A) a schematic representation of a tumour model study using an oncolytic virus expressing an exosomal dinucleotide cyclase in accordance with an embodiment of the invention; B) graphical illustration of survival data of the treated and untreated tumour model; and C) graphical illustration of tumour volume in the model on tumour rechallenge.

[0015] Figure 3 illustrates A) a transgene design for an adenoviral rotaviral vaccine in accordance with an embodiment of the invention; B) western blot analysis of cell lysates and vesicles;C) cyclic dinucleotide content of adenoviral transduced cells; D) a schematic of vaccination protocol;E) results of antibody detection in treated mouse sera; and F) a graphical illustration of prime and boost antibody responses.

[0016] Figure 4 illustrates amino acid sequences of 2A cleavage proteins.

[0017] Figure 5 illustrates polypeptide and nucleotide sequences for transgenes in accordance with embodiments of the invention, including: A) aDEC205 / CdaA#2 - polypeptide sequence;B) aDEC205 / CdaA#2 - nucleotide sequence; C) aDEC205 / CdaA#l - polypeptide sequence;D) aDEC205 / CdaA#l - nucleotide sequence; E) aDEC205 / CdaS - polypeptide sequence; andF) aDEC205 / CdaS - nucleotide sequence.

[0018] Figure 6 illustrates polypeptide and nucleotide sequences for transgenes in accordance with embodiments of the invention, including: A) VP7 Gl / G2-exoCyclase polypeptide sequence; B) VP7 Gl / G2-exoCyclase nucleotide sequence; C) VP7 G3 / G4-exoCyclase polypeptide sequence; and D) VP7 G3 / G4-exoCyclase nucleotide sequence.

[0019] Figure 7 illustrates: A) the timeline for a vaccination study using a transgene in accordance with an embodiment of the invention; B) IFN-y- secreting cells detected in mice 7 days post prime presented as spot forming units per million splenocytes. Bars show the mean+SD response from 5 mice; C) IFN-y secreting cells detected 7 days post boost. Presented at spot forming units per million Splenocytes. Bars show the mean+ SD response from 4-5 mice. A significantly higher response wasdetected in the ES07 0.5ug dose group compared to the ES01 group at the 0.5ug dose; and D) total IgG ELISA results 7 days post boost. Presented as mean+ SE from 5 mice.

[0020] Figure 8 illustrates the mRNA sequence of SARS-CoV-S spike protein linked with a flexible linker to mRNA encoding the cyclase, CdaS.Detailed Description

[0021] A self-adjuvanting extracellular vesicle (SAEV) is provided comprising an antigen and a STING activator, wherein the SAEV comprises a recombinant polypeptide comprising the antigen and the STING activator, or the SAEV comprises a recombinant polypeptide comprising one of the antigen and the STING activator and the other of the antigen or the STING activator is co-loaded in the vesicle independent of the recombinant polypeptide.

[0022] The term “extracellular vesicle” or “EV” as used herein encompasses any extracellular vesicle produced by cells, including but not limited to, microvesicles, exosomes, ectosomes, virus-like particles, macrovesicles, oncosomes, gesicles, and apoptotic bodies. Preferred extracellular vesicles are exosomes.

[0023] The present extracellular vesicles are genetically modified to promote an immunogenic response on administration to a mammal, and are further modified to enhance or boost the immunogenic response. Thus, the term “self-adjuvanting” refers to the ability of the vesicle to boost the immunogenic response, for example, boost or enhance the immune cell response, e.g. T-cell response, and / or boost or enhance an antibody response, as compared to the immunogenic response that occurs in the absence of such self-adjuvanting vesicles. In one embodiment, the immunogenic response resulting from a SAEV is boosted by at least about 10%, 15%, 20%, 25% or more of the immunogenic response that occurs in the absence of self-adjuvanting vesicles.

[0024] The self-adjuvanting extracellular vesicle or SAEV comprises a recombinant polypeptide that is incorporated within the vesicle when the vesicle is produced by a vesicle-producing cell. The recombinant polypeptide is expressed from a transgene introduced into vesicle-producing cells as will be described herein. The recombinant polypeptide comprises an EV anchoring polypeptide and a STING activator and / or an antigen.

[0025] The recombinant polypeptide of the SAEV comprises an EV-anchoring polypeptide which tethers the recombinant polypeptide to the EV membrane. The EV-anchoring polypeptide maybe an EV-directed transmembrane polypeptide, i.e. the portion of a transmembrane protein that spans the entirety of a phospholipid bilayer membrane of an EV, and which innately targets (is trafficked to) EV membranes. Proteins containing such EV-directed transmembrane domains can originate from viruses, bacteria or mammalian cells. Membrane spanning domains may be single pass or may pass through the membrane multiple times, such as two, three or four times (double, triple or quadruple pass). For example, tetraspanins such as TSPAN1 to TSP AN 33, are a family of membrane proteins found in all multicellular eukaryotes, and also referred to as the transmembrane 4 superfamily (TM4SF) proteins. They have four transmembrane alpha-helices and two extracellular domains, one short extracellular domain or loop, and one longer extracellular domain / loop. Although several protein families have four transmembrane alpha-helices, tetraspanins are defined by conserved amino acid sequences including four or more cysteine residues in the EC2 domain, with two in a highly conserved 'CCG' motif. Single pass and tetraspanin domains can be engineered via linker sequences to carry one or more STING activator and / or antigen.

[0026] Examples of proteins that specifically direct to, and are enriched in, EV membranes including single pass and tetraspanin domains, are set out in Table 1.Table 1: Examples of Proteins Comprising EV-directed Transmembrane Domains

[0027] The STING activator and / or antigen is included in the recombinant polypeptide, and their position within the recombinant polypeptide may vary.

[0028] In one embodiment, the STING activator and / or antigen are positioned in the recombinant polypeptide so as to be intravesicular.

[0029] In another embodiment, the STING activator and antigen are positioned in the recombinant polypeptide such that both the antigen and the STING activator are intravesicular. They may be adjacent within the recombinant polypeptide. Alternatively, the STING activator and antigen may be discretely positioned within the recombinant polypeptide, for example, with the targeting moiety and / or EV-anchoring moiety therebetween, or the STING activator and antigen may be locatedterminally within the recombinant polypeptide, one at the N-terminal end and the other at the C-terminal end of the recombinant polypeptide.

[0030] In another embodiment, the STING activator and antigen are positioned in the recombinant polypeptide such that the antigen is extravesicular and the STING activator is intravesicular. As will be described in more detail herein, this is done by providing a leader sequence such that the antigen is displayed on the EV-directed anchoring moiety, e.g. a transmembrane domain, linked to the STING activator.

[0031] Heterologous components of the SAEV, such as the antigen and anchoring moiety, anchoring moiety and STING activator, may be linked with a peptide linker. Examples of suitable peptide linkers include, but are not limited to, (G4Ser)n, (GnS)n, (G2S4)n and (EAAAK)n. In some cases, the peptide linker may be a self-cleaving peptide, for example, a peptide that induces ribosomal skipping thereby preventing formation of a peptide bond and resulting in expression of the STING activator, antigen or both (when both are present) separate from the EV-anchoring moiety and / or each other. Examples of self-cleaving peptides include, but are not limited to, 2A peptides such as P2A (porcine teschovirus-1), E2A (equine rhinitis A virus), F2A (foot-and-mouth disease virus 18), and T2A (thosea asigna virus 2A). The sequences of these exemplary self-cleaving peptides are shown in Fig. 4. As one of skill in the art will appreciate, sequences based on a 2A peptide sequence which vary from the native sequence but which retain the self-cleaving feature of the 2A peptide may also be used in the present recombinant polypeptide. For example, the 2A peptide may include the optional linker “GSG” (Gly- Ser-Gly) on the N-terminal end of a 2A peptide to enhance efficiency.

[0032] The STING activator is a moiety that activates stimulator of interferon genes (STING). STING activators include cyclic dinucleotides (CDNs), as well as dinucleotide cyclases that produce CDNs.

[0033] The term “dinucleotide cyclase”, also referred to herein as “cyclase”, refers to enzymes, including bacterial and mammalian enzymes, that catalyze the synthesis of cyclic dinucleotides (CDN) such as c-di-GMP, c-di-AMP, and cGAMP (e.g. 3’3’-cGAMP and 2’3’-cGAMP), as well as c-UAMP, c-di-UMP, c-UGM, c-CUMP, and c-AAGMP. Dinucleotide cyclases, thus, include, but are not limited to, i) di-adenylyl cyclases (DAC) proteins that synthesize c-di-AMP, such as DisA, CdaA, and CdaS; ii) proteins containing GGDEF domains (Pfam family: PF00990) that synthesize c-di-GMP; iii) CD-NTase enzymes that have the catalytic domain known as SMODS (PF18144) that synthesize 3’-5’ cGAMP, such as DncV, and iv) mammalian cyclases such as cGAS.

[0034] Dinucleotide cyclases in accordance with the present invention are those cyclases which remain constitutively functional in the cytoplasm of mammalian cells, e.g. bacterial or mammalian dinucleotide cyclases that survive at 37°C and which retain activity as depicted by an OD reading of at least about 0.5 at 630 nm, indicative of promoter activity / gene expression (e.g. SEAP activity) and correlates with the activation level of interferon signaling induced by cyclic dinucleotides. Activity may also be depicted by detection of cyclic dinucleotides using, for example, ELISA. Thus, dinucleotide cyclases in accordance with the invention retain at least about 20% of their endogenous activity, preferably at least about 30%, 40%, 50% or more of their endogenous (wildtype) activity when expressed in mammalian cells. Examples of suitable dinucleotide cyclases include c-di-GMP cyclases from Vibrio cholera such as VCA0848, and c-di-AMP cyclases such as CdaA from Listeria monocytogenes and MtbDisA from Mycobacterium tuberculosis.

[0035] It is noted that functionality of a dinucleotide cyclase may be reliant on its conformation. In some embodiments, the dinucleotide cyclase is functional when tethered to the EV-anchoring moiety, i.e. in an uncleaved state, e.g. CdaA. However, other dinucleotide cyclases require cleavage from the EV-anchoring moiety to be functional, e.g. DisA. Thus, for cyclases such as DisA, these cyclases are linked to the EV-anchoring moiety via a self-cleaving peptide in order that the DisA is released from the recombinant polypeptide to retain its function as a STING activator.

[0036] The antigen for incorporation into the SAEV may be any agent sufficient to induce an immune response in a mammal. Thus, in one embodiment, the antigen is a tumour-derived, or pathogen- derived (e.g. from parasites, viruses, bacteria or fungi) antigen. The antigen may be a protein, peptide, polysaccharide, lipid or nucleic acid. Tumour-derived antigens may be tumour-specific antigens or tumour-associated antigens. Tumour antigens for any cancer may be incorporated into the SAEV, including but not limited to, neuroblastoma, melanoma, osteosarcoma, renal cell cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, Burkitt lymphoma and colon cancer. Examples of tumourspecific antigens include alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, and epithelial tumour antigen (ETA). Tumor-associated antigens include, for example, differentiation antigens (such as melanocyte differentiation antigens), mutational antigens (such as p53), overexpressed cellular antigens (such as HER2), viral antigens (such as human papillomavirus proteins), and cancer / testis (CT) antigens that are expressed in germ cells of the testis and ovary but are silent in normal somatic cells (such as MAGE and NY-ESO-1). Examples of pathogen-derived antigens include cellsurface proteins, toxins and proteins, peptides or polysaccharides of the pathogen coat, capsule or cell wall, including viral glycoproteins and structural proteins.

[0037] Optionally, the recombinant polypeptide comprises an extracellular targeting moiety which is a moiety for directing the SAEVs to a target molecule on at least one target cell. The target cell may be a mammalian cell, including a human or non-human mammalian cell, such as an immune cell or a tumour cell. Examples of immune cells include, but are not limited to, T-cells, B-cells, natural killer (NK) cells, dendritic cells, macrophages, or neutrophils. The extracellular targeting moiety is generally linked to the EV-anchoring moiety.

[0038] A target molecule on such target cells may include a cell surface marker or a cell surface receptor. In one embodiment, the target molecule is a receptor ligand such as a TNF-a family receptor, an integrin, a C-type lectin receptor, a leptin, a carcinoembryonic antigen, a CD (cluster of differentiation) antigen, a carbonic anhydrase, FAP, MMP2, DEC205, DC40, CLEC9, CD3, CLEC9A, CEACAM5, CTLA4, CD7, CD 11c, CD 19, CD20, CD22, CD44, CD206, EGFR, fibroblast activating protein (FAP), CA9, MMP-2, PD-L1, SIRPa, a glycosaminoglycan, a polysaccharide, chondroitin sulfate, av-integrin, folate receptor, or a lipid. The target molecule may also be a tumor-associated antigen as set out above.

[0039] Thus, the targeting moiety is a moiety designed to direct the SAEV to the target cell by its recognition of the target molecule on the target cell. In one embodiment, the targeting moiety comprises an antibody or a functional fragment thereof, an scFv, a single domain antibody or a genetically engineered antibody mimetic protein that exhibits highly specific and / or high-affinity target protein binding such as a DARPin (Designed Ankyrin Repeat Protein). In one embodiment, the targeting moiety comprises anti-DEC205, anti-Clec9A, anti-CeacaM5, anti-FAP, anti-CEA, anti-CA9, anti-CTL4, anti-CD3, anti-CDl lc, anti-CD206, anti-CD19, anti-CD20, anti-CD22, anti-CD44, anti-CD7, SIRPa ectodomain, chlorotoxin, GE11 peptide, CTX, VAR2A, CD40 ligand, CD40-targeting peptide, CD206- targeting peptide, iRGD, LDLR (low density lipoprotein receptor) targeting peptide or PD1. The functional fragment may be a Fab' or a F(ab')2.

[0040] In one embodiment, the SAEV comprises a recombinant polypeptide comprising an extravesicular immune cell targeting moiety linked to an intravesicular cyclase via an EV anchoring moiety, and a second recombinant polypeptide comprising one or more antigens, such as tumour antigens, also directed into the SAEV by, for example, an anchoring moiety. In another embodiment, the SAEV comprises a recombinant polypeptide comprising an extravesicular antigen linked to an intravesicular cyclase via an EV anchoring moiety.

[0041] In another embodiment, the SAEV comprises a recombinant polypeptide comprising an extravesicular antigen linked to an EV anchoring moiety, additionally comprising STING activators, e.g. cyclic dinucleotides (CDNs) or dinucleotide cyclases, which are co-loaded within the SAEV independently of the recombinant polypeptide.

[0042] The SAEV is prepared by introducing a transgene encoding a recombinant polypeptide comprising the optional extracellular targeting moiety, the EV anchoring polypeptide and the STING activator and / or antigen to an EV-producing cell, i.e. any mammalian cell including mammalian tumour cells. In some embodiments, a second transgene may be introduced which encodes a targeting moiety, an antigen and / or a cyclase not incorporated in the first transgene.

[0043] As one of skill in the art will appreciate, the transgene, will also generally incorporate nucleic acid encoding an N-terminal signal or leader sequence of the extracellular targeting moiety. The signal or leader sequence generally comprises about 12-40 amino acids and, once translated, functions to translocate the extracellular targeting moiety for secretion. The signal peptide may be, e.g., the native signal peptide of the extracellular targeting moiety to be produced, a heterologous signal peptide, or a hybrid of the native and a heterologous signal peptide. Numerous signal peptides are used for production of secreted proteins, including but not limited to, murine immunoglobulin signal peptide (IgSP, EMBL Accession No. M13331), and leader sequences from other immunoglobulins, tissue plasminogen activator (tPA), insulin, Vesicular Stomatitis Virus glycoprotein (VSVG), IL-2, albumin, and chymotrypsin. Hybrid leader sequences have also been developed, for example, a leader sequence comprising an immunoglobulin signal peptide fused to a tissue-type plasminogen activator propeptide.

[0044] Nucleic acid encoding the extracellular targeting moiety of the transgene is coupled or linked to nucleic acid encoding the EV-anchoring domain which is capable of tethering or anchoring the extracellular targeting moiety to an extracellular vesicle (EV) such as an exosome, microvesicle, macrovesicle, oncosome, or the like. The leader sequence, extracellular targeting moiety and EV- anchoring domain may be homologous or heterologous sequences. The transgene also encodes a STING activator and / or antigen as described above.

[0045] The transgene is constructed using well-established methods, and may be constructed for delivery to EV-producing cells, either in vivo, ex vivo or in vitro, in a nucleic acid-based delivery system, e.g. mRNA or DNA construct, or a viral vector.

[0046] In a DNA construct, coding regions for the extracellular targeting moiety, an EV anchoring polypeptide and a STING activator and / or an antigen, are synthesized, or otherwise obtained,for example, commercially, and linked, as described, with DNA encoding a self-cleaving peptide where required to release the STING activator and / or antigen. The construct will incorporate a suitable promoter to regulate expression of the transgene, as well as a transcriptional stop sequence, e.g. a poly (A) addition sequence, at the terminal end thereof. Examples of suitable promoters for incorporation in the vaccine construct, include but are not limited to, CMV, EFla, CAG, PGK1, SV40, RSV, TRE, U6, UAS, Ubc, human beta actin, and CAG. The construct may also incorporate enhancer elements and / or transcriptional transactivators to enhance promoter activity when placed either upstream or downstream of the ORF.

[0047] In order to achieve extracellular expression of a component of the recombinant polypeptide, e.g. the antigen, the DNA construct is prepared to express the antigen N-terminal to the EV- anchoring domain. Alternatively, to achieve intravesicular expression of a component, e.g. the STING activator, the DNA construct is prepared to express the STING activator C-terminal to the EV-anchoring domain.

[0048] The DNA transgene construct is then generally adapted for administration. The transgene construct may be formulated for administration as a linear molecule, covalently-closed linear construct or mini-circle. Alternatively, the transgene construct may be incorporated into a vector such as a plasmid or cosmid using techniques well-known in the art. The resulting DNA construct may be incorporated within a delivery system, for example, biodegradable polymeric microparticles (e.g. chitosan, polylacticecoglycolides, polyethyleneimine, amine-functionalized polymethacrylates, cationic poly([3- amino esters), poloxamers and polyvinylpyrrolidone polymers), liposomes, lipid nanoparticles, and the like.

[0049] For use as an mRNA construct, a DNA transgene construct is prepared as described, and mRNA is synthesized therefrom by in vitro transcription of the cDNA template, typically plasmid DNA (pDNA), prepared as described using methods known in the art. Transcription of the cDNA template is conducted using RNA polymerase such as a bacteriophage RNA polymerase. For stability and efficient translation, the resultant mRNA strand will include a 5’ cap and 3’ poly(A) tail, as well as 5’ and 3’ untranslated regions (UTRs) flanking the coding region. The mRNA vaccine is then formulated for administration. In this regard, mRNA may be complexed with agents which prevent degradation, enhance uptake and promote translation. Examples of such adjuvants include, but are not limited to, cationic polypeptides (e.g. protamine), nanoemulsions, carrier peptides, liposomes, and immune activator proteins (e.g. CD70, CD40L, TLRs).

[0050] Viral vectors may also be utilized to administer the present transgene construct, including both DNA viral vectors and RNA viral vectors.

[0051] DNA viral vectors are adapted to expressibly incorporate the present DNA transgene construct, e.g. under the control of a viral promoter. Examples of suitable DNA viruses for use as vaccines include, but are not limited to, poxviruses such as vaccinia virus and modified vaccinia virus, adenoviruses, adeno-associated viruses, herpes simplex virus and cytomegalovirus, and including various serotypes thereof, both replication-competent and replication-deficient or replicationincompetent.

[0052] In one embodiment, a replication-incompetent adenovirus is prepared for use to deliver the present transgene construct. The transgene is incorporated within an E1 / E3 region which has been deleted from the adenovirus.

[0053] RNA viral vectors may also be adapted to expressibly incorporate an appropriate transcript of the present transgene construct, e.g. positive or negative strand. Examples of suitable RNA viruses for use to deliver the transgene include, but are not limited to, vesicular stomatitis viruses, retroviruses such as MoMLV, lentiviruses, Sendai viruses, measles-derived vaccines, Newcastle disease virus, alphaviruses such as Semliki Forest virus, flaviviruses, or an RNA replicon based on an RNA virus (i.e. derived from alphavirus, flavivirus, etc).

[0054] In one embodiment, the viral vector is an oncolytic viral vector. Examples of oncolytic DNA viruses include an adenovirus, herpes simplex virus (HSV), a parvovirus, or a poxvirus such as vaccinia virus (VACV) and myxoma virus (MYXV). Examples of oncolytic RNA viruses include Coxsackie virus, Maraba virus, measles virus (MV), Newcastle disease virus (NDV), poliovirus, reovirus, retroviruses, Seneca Valley virus (SVV), Semliki Forest virus (SF V), Vesicular stomatitis virus (VSV), and Sindbis virus (SBV). In addition to incorporating the present transgene construct, or transcript thereof, the selected oncolytic virus may be further modified to enhance its oncolytic activity, for example, to incorporate additional anti-cancer payloads, or to incorporate immune-suppressing blockers, e.g. a TGF-beta blocking agent. The viral vector may comprise an antigen, antigen-encoding nucleic acid or may be antigen-free.

[0055] The nucleic acid-based or viral vector vaccine may be administered directly to a mammal, or may be introduced into EV-producing cells to produce transgenic EV-producing cells which areadministered to a mammal. In both cases, SAEVs are produced in vivo which are therapeutically effective.

[0056] Alternatively, on introduction of the transgene to EV-producing cells in vitro, the cells are cultured under conditions suitable for growth and expression of the recombinant polypeptide encoded by the transgene. Conditions may vary with the EV-producing cells utilized as would be known by one of skill in the art. Once expressed, the polypeptide is incorporated within EVs in the cell due to the presence of the EV-anchoring domain in the recombinant polypeptide. EVs comprising the recombinant polypeptide (SAEVs) are isolated from cell culture using known techniques, for example, ultracentrifugation including differential ultracentrifugation, density gradient centrifugation and rate- zonal centrifugation techniques. Size exclusion chromatography, tangential flow filtration and affinitybased isolation techniques, e.g. immunoaffinity chromatography, may also be used alone or in combination with ultracentrifugation to isolate extracellular vesicles for therapeutic use.

[0057] As noted, the SAEV may comprise a recombinant polypeptide comprising one of the antigen and the STING activator and the other of the antigen or the STING activator may be co-loaded in the SAEV independently of the recombinant polypeptide. This may be accomplished in one embodiment by introducing a STING activator-encoding transgene into EV-producing tumour cells to produce SAEVs comprising recombinant STING activator, e.g. cyclase, and passively (naturally) loaded tumour antigen. Similarly, other antigens may be passively or naturally packaged into the SAEV, including pathogen-derived antigens, by introduction of a STING activator-encoding transgene into EV- producing cells that naturally express the antigen of interest, or which have been modified to express the antigen of interest. In another embodiment, antigen-encoding transgene is introduced into EV-producing cells which independently express a cyclase, either innately or due to modification of the EV-producing cells to express a cyclase, and thereby yield SAEVs comprising recombinant antigen and passively coloaded STING activator, e.g. cyclase or cyclic dinucleotide.

[0058] A method of vaccination via the present SAEVs is, thus, provided, which results in an enhanced immune response in comparison to vaccination with antigen alone. The nucleic acid-based or viral vector vaccine, or transgenic EV-producing cells, each of which generate SAEVs, or the isolated SAEVs themselves, are administered to a mammal to be treated or vaccinated in a therapeutically or prophylactically effective amount, i.e. an amount sufficient to generate in the mammal an immune response. The term “mammal” is used herein to refer to both human and non-human mammals. As one of skill in the art will appreciate, the amount required to generate an immune response will vary with anumber of factors, including, for example, the mode of administration, the type of vaccine, the particular transgene / antigens in the SAEV, the STING activator, and the mammal to be treated, e.g. species, age, size, etc. In this regard, for example, administration of a dosage in the range of about IxlO5to IxlO9pfus of viral vector in a mouse is sufficient to generate an immune response. The dose (e.g. priming dose) may optionally be followed by a boosting dose. A corresponding amount will generally be sufficient for administration to a human to generate an enhanced immune response, i.e. an immune response which is significantly amplified in comparison to that induced by the antigen alone, e.g. by at least about 10%, 20%, 30%, 40%, 50% or greater. Notably, the present method of vaccination is able to induce seroconversion at a significantly statistically lower dose. Thus, the present combination results in a synergistic effect.

[0059] A further method of vaccination is provided comprising administration to a mammal of an oncolytic virus comprising a transgene encoding a recombinant polypeptide comprising EV anchoring polypeptide and a STING activator, wherein the transgene is expressed in an extracellular vesicle. Administration of a dosage in the range of about IxlO5to IxlO9pfus of viral vector in a mouse is sufficient to generate an immune response, or a corresponding amount in a human. The immune response induced in the mammal is significantly enhanced in comparison to the immune response induced by the oncolytic virus alone, e.g. by at least about 10%, 20%, 30%, 40%, 50% or greater. Thus, this combination also results in a synergistic effect.

[0060] The present vaccine is administered to a mammal to induce an enhanced immune response in any one of several administrable routes including, but not limited to, oral administration, or parenteral administration such as intravenously, intramuscularly, intratumourally, intradermally, intranasally or by inhalation. For nucleic acid-based vaccines, other techniques such as administration by electroporation or using gene gun technology may be utilized. The prime and boosting vaccines, which may be the same or different vaccine type (e.g. both the prime and boosting vaccine may be a nucleic acid-based vaccine, or both may be a viral vectored vaccine, or the prime vaccine may be nucleic acid-based and the boosting vaccine may be a viral vector, or vice versa), may be administered by the same or different administrable routes.

[0061] As will be appreciated by one of skill in the art, the vaccine, and any pharmaceutically acceptable additives, are administered in a suitable carrier, such as saline or other suitable buffer.

[0062] Embodiments of the invention are described by reference to the following specific examples which are not to construed as limiting.Example 1 - VSV antigen-decorated exosomes transfected with dinucleotide cyclase

[0063] Exosomes comprising the extravesicular viral antigen, VSVG, and intravesicular dinucleotide cyclase, mtb DisA were prepared as follows.

[0064] HEK 293TREX cells (which have VSVG incorporated in their genome under the control of a doxocy cline inducible promoter) were transfected with a transgene, either pCDNA, or pCDNA-Mtb- DisA. Media was changed 4-6 hrs post transfection with media containing 2% EV-depleted FBS. Transfected cells were treated with doxocycline to induce VSVG expression 48h-post transfection, and exosomes (“gesicles”) were concentrated by ultracentrifugation 72h post transfection. VSVG is an antigenic protein that naturally trafficks into extracellular vesicles.

[0065] Zetaview analysis was performed to determine particle concentrations (3-5E10 / ml) and WB / CDN ELISA analysis confirmed the presence of VSVG and cyclic dinucleotides (produced on expression of the Mtb DisA cyclase) in the gesicles. Western blotting and ELISA analysis showed expression of VSVG and Alix (an exosome marker), and independent expression of the cyclase through the generation of cyclic dinucleotides (CDNs), respectively, passively loaded into exosomes used for vaccination. These results are shown in Fig. IB.

[0066] Mice were vaccinated with 3 doses of gesicles (~8ug of CDNs / dose) either including VSVG in the presence of Mtb DisA expression, or including only VSVG on days 0, 7 and 14 as illustrated in Fig. lA. Results from an in vitro neutralization assay demonstrate robust neutralization against VSV in animals vaccinated with exosomes containing both the viral antigen and cyclic dinucleotides provided by co-expressed cyclase. Serum from baseline (the day before vaccination), days 7, 21, 35, 43 and 52 were used for analysis as shown in Fig. 1C. Neutralization against VSV was shown to be greater than 50%, or 1-3 fold greater, in animals vaccinated with exosomes containing both the viral antigen and cyclic dinucleotides as compared to animals vaccinated with viral antigen alone.

[0067] On Day 80, mice were challenged with an intranasal dose of wtVSV as shown in Fig. ID. Lungs were harvested 96 h post-challenge, and VSV titres of homogenized lung tissue were determined. Titers are expressed as plaque forming units (PFU) per milligram of lung tissue in Fig. IE. Numbers in brackets represent the number of mice in each group with detectable plaques. There were no detectable plaques in mice treated with antigen and CDNs, while there were significant detectable plaques in naive and antigen only treated mice.

[0068] These results show the unexpectedly enhanced immune response to VSV challenge with treatment of exosomes decorated with extravesicular viral antigen combined with a co-expressed intravesicular STING activator (dinucleotide cyclase) which result in passive exosome loading of CDNs.Example 2 - Oncolytic viral activity enhanced with exosomes transfected with dinucleotide cyclase

[0069] Mice engrafted with MC38 colorectal carcinoma cells were treated with several strains of VSV or PBS as shown in Fig. 2A.

[0070] Viruses used were: 1) VSVD51-aDEC205 / CdaA#l, 2) VSVD51-aDEC205 / CdaA#2 (two versions of transgene having extra-vesicular anti-DEC205 ScFv and luminal CdaA cyclase linked by the CdaA transmembrane domain), 3) VSVD51-aDEC205 / CdaS (transgene having extra-vesicular anti- DEC205 ScFv and luminal CdaS cyclase linked by the CdaS transmembrane domain), 4) VSVD51-eGFP (virus expressing control protein) or 5) VSVD51-FreeCdaA (virus expressing CdaA cyclase ORF alone, i.e. not tethered to a transmembrane EV-anchoring moiety). Transgenes were inserted between G and L genes of VSV strain having a deltaM51 mutation in the matrix protein. Transgene and polypeptide sequences are shown in Fig. 5A-5F. Production of exosomes were confirmed using standard techniques.

[0071] Mice were monitored for tumour growth and euthanized when tumour volume endpoint was met. As shown in Fig. 2B, mice treated with oncolytic virus encoding transgenes comprising exosomes having an extravesicular targeting moiety (antiDEC205 ScFv, a dendritic cell marker) and a luminal (intravesicular) cyclase tethered by an anchoring transmembrane moiety exhibited increased survival (by at least 10%, and up to 25-50%) due to the presence of DC-targetted cyclase bearing tumour- derived SAEVS in combination with the oncolytic viral effect, while mice treated with oncolytic virus encoding non-exosomal cyclase exhibited significantly reduced survival (i.e. comparable to eGFP expressing control virus).

[0072] On Day 90, surviving mice were rechallenged with a subcutaneous injection of 5x10e5 MC38 cells and monitored for tumour growth. Naive mice were used as a control for tumour engraftment and growth, and exhibited significant tumour growth. All mice treated with VSVDM-exoCyclase viruses (oncolytic virus producing exosomal cyclase) rejected tumour challenge as shown in Fig. 2C exhibiting the enhancement of anti-tumoural immunity by exosomal cyclase expression in vivo. Thus, the exosomal cyclase enhanced the induction of neutralizing antibodies, and also lead to stable, long term neutralizing titre.Example 3 - Rotaviral antigen-decorated exosomes transfected with dinucleotide cyclase

[0073] A Rotaviral vaccine was prepared with a transgene comprising: a leader sequence followed by the VP7 sequence of the G1 serotype of human rotavirus, followed by the VP7 sequence of the G2 serotype of human rotavirus, displayed on a transmembrane domain (VSVG) followed by either the CdaA bacterial dinucleotide cyclase or luciferase as shown in Fig. 3A. These transgenes were inserted into an E1ZE3 deleted adenoviral vector. Transgene and polypeptide sequences are shown in Fig. 6A / B.

[0074] A549 cells were transduced with the adenoviral vectors: 1) Ad-EGFP; 2) Ad-Gl / G2exoLuciferase; or 3) Ad-Gl / G2exoCyclase at an MOI of 10 leading to the production of vesicles having rotaviral Ags (G1 and G2) displayed on their surface and a luminal cargo of either cyclase or luciferase. The cells were lysed at 48hrs for western blotting while vesicles were isolated from supernatant by ultracentrifugation, and then lysed for western blotting. Blots were probed with an anti- FLAG mAb to detect indicated bands. Blots were also probed for loading controls, betaActin (cell lysate) and flotillin- 1 (vesicles) as shown in Fig. 3B.

[0075] Cell lysates from Ad-transduced A549 cells were assayed for c-di-AMP content using a c-di-AMP specific ELISA. Levels of c-di-AMP detected in cell lysates are indicated in Fig. 3C.

[0076] Mice were orally vaccinated as shown in Fig. 3D with the Rotaviral vaccine. A dose range of IxlO5- IxlO7of each viral vector in saline was provided orally (sub-lingually) in lOul volume. Serum was collected at various time points. Mice were bled at day 12 post priming and serial dilutions were applied to a plate coated with recombinant G1 VP7 protein. The plate was washed and anti-Gl VP7 IgG were detected with labelled secondary Ab. Mean absorbance levels were graphed for each dilution series (Fig. 3E).

[0077] Sera collected post prime as well as post-boost were assayed for the presence of anti-Gl VP7 antibodies by ELISA. Reciprocal dilution data were graphed for both cyclase- and luciferase- inclusive vaccines at each dose provided (Fig. 3F), illustrating the significantly enhanced immune response resulting from the exosomal viral antigen combined with cyclase.

[0078] Similar results were obtained with a rotaviral vaccine prepared with a transgene in which the VP7 sequence of the G1 and G2 serotype were replaced with the G3 and G4 serotypes. Transgene and polypeptide sequences are shown in Fig. 6C / D.Example 4 - Spike-decorated exosomes transfected with dinucleotide cyclase

[0079] mRNA constructs were prepared comprising: i) mRNA encoding full-length SARS-CoV- S Spike protein, and ii) mRNA encoding full-length SARS-CoV-S spike protein linked with a flexible linker to mRNA encoding CdaS (see Fig. 8).

[0080] The mRNA constructs were prepared for use as a vaccine in a lipid nanoparticle comprising the following components:LIPID PERCENTAGESM-102 (amino lipid) 49.56% cholesterol 39.90%PEG-2000 1.17%DSPC (l,2-distearoyl-sn-glycero-3- 9.36% phospocholine)

[0081] Balb / C mice were vaccinated IM with 50uL of mRNA-LNP vaccines on day 0 (prime) and day 22 (boost) according to the timeline shown in Fig. 7A. There were 5 mice per vaccine group. Groups 1-4 were vaccinated with escalating doses (0.5ug-5ug) of the SARS-CoV-2-Spike vaccine, groups 5-8 were vaccinated with escalating doses (0.5ug-5ug) of the SARS-CoV-2-Spike-Cda-S vaccine as summarized in Table 2 below. The mice in group 9 did not receive any intervention. On day 9, 200pL of whole blood was taken from each mouse for IFN-y ELISPOT to assess the T-cell response, and on day 13 200pL of whole blood was taken for IgG ELISA analysis. The mice were sacrificed 7 days after the boosting vaccine (day 29). 200pL of whole blood was taken for ELISA analysis and the spleen was taken for T cell analysis including IFN-y ELISPOT.Table 2.

[0082] Peripheral blood mononuclear cells (PBMC) taken from whole blood or splenocytes isolated from the spleen were used for the IFN-y ELISPOT analysis. PBMC were separated using a Ficoll gradient and counted with a Coming cell counter. One hundred microliters containing the cells was plated onto a pre-coated IFN-y ELISPOT plate (CTL, Ohio USA). One hundred microliters of either media (as negative control), phorbol 12-myristate 13 -acetate (PMA) and ionomycin (ebiosciences) (as a positive control), or 1 pg / mL SPIKE pepmix (JPT peptides) was then added to the plates. The plates were incubated for 18-24 hours at 37°C with 5% CO2 before developing the ELISPOT plates. The ELISPOT plates were read using a CTL ELISPOT plate reader. The number of spots in the media alone wells was subtracted from the peptide stimulated wells and the results presented as the number of spot forming units (SFU) per million cells. A mouse was excluded if the background on the media alone wells was too high.

[0083] The anti-SPIKE antibody ELISA (Total IgG) was conducted fourteen days after the priming vaccination, and seven days after the boost vaccination. ELISA plates (96 well) were coated overnight at 4 °C with recombinant SARS-CoV-2 Spike Protein (CF, R&D Systems, 10500-CV-100) diluted to 0.5ug / ml in PBS, washed and then blocked. Serially diluted sera were applied to coated wells, incubated and washed 3X. HRP-conjugated anti-mouse IgG detection Abs were applied, incubated and washed 3X. TMB was added, stop solution was added and plates were read on a plate reader.Results

[0084] On administration of the vaccine, the mRNA was expressed, and the Spike protein trafficked into exosomes based on its own transmembrane domain to yield Spike-bearing exosomes in vivo with or without CdaS.

[0085] At prime, the T cell response to the spike protein was shown by IFN-y ELISPOT analysis to be enhanced using the vaccine with cyclase (see Fig. 7B). The T cell response was enhanced to different degrees across the dosages used, with the greatest enhancement seen with the 1 ug vaccine dosage.

[0086] After boost the T cell response was again shown to be enhanced using the vaccine with cyclase (see Fig. 7C), while antibody response was equivalent between the vaccines, with or without cyclase (see Fig. 7D).

Claims

CLAIMS1. A self-adjuvanting extracellular vesicle (SAEV) comprising an antigen and a STING activator, wherein the SAEV comprises a recombinant polypeptide which comprises the antigen and the STING activator, or the SAEV comprises a recombinant polypeptide comprising one of the antigen and the STING activator and the other of the antigen or the STING activator is co-loaded in the vesicle independent of the recombinant polypeptide.2 The SAEV of claim 1, wherein the STING activator is a cyclic dinucleotide or a dinucleotide cyclase.3 The SAEV of claim 1 or claim 2, which is a microvesicle, exosome, ectosome, virus-like particle, macrovesicle, oncosome, gesicle or apoptotic body.4 The SAEV of any one of claims 1-3, wherein the antigen is extravesicular and the STING activator is intravesicular.5 The SAEV of any one of claims 1-3, wherein the recombinant polypeptide comprises an intravesicular STING activator and is co-loaded with an antigen which is independent from the recombinant polypeptide.6 The SAEV of any one of claims 1-3, wherein the recombinant polypeptide comprises an extravesicular antigen and is co-loaded with STING activator which is independent from the recombinant polypeptide.7 The SAEV of any one of claims 1-6, wherein the antigen is a viral or tumour antigen.8 The SAEV of any one of claims 1-7, wherein the recombinant polypeptide comprises an extracellular vesicle (EV)-anchoring moiety.9 The SAEV of any one of claims 1-8, wherein the recombinant polypeptide comprises an extravesicular targeting moiety to target a particular cell type.10 The SAEV of claim 9, wherein the extravesicular targeting moiety targets an immune cell.11 The SAEV of any one of claims 1-10, comprising a second recombinant polypeptide that comprises an extravesicular targeting moiety to target a particular cell type.12 A method of preparing a self-adjuvanting extracellular vesicle comprising: i) introducing a transgene encoding a recombinant polypeptide comprising an EV anchoring polypeptide and a STING activator and / or an antigen into a cell; andii) culturing the cell under suitable growth conditions to yield self-adjuvanting extracellular vesicles comprising the STING activator and the antigen, wherein the recombinant polypeptide comprises the STING activator and the antigen, or the recombinant polypeptide comprises one of the STING activator or the antigen and the other of the STING activator or the antigen is co-loaded in vesicles independently of the recombinant polypeptide.

13. A method of vaccination comprising administering to a mammal a self-adjuvanting extracellular vesicle comprising an antigen and a STING activator as defined in any one of claims 1-11, wherein said self-adjuvanting extracellular vesicle induces an immune response which is amplified in comparison to the immune response induced by the antigen alone.

14. A method of vaccination comprising administering to a mammal an oncolytic virus comprising a a transgene that encodes an EV anchoring polypeptide and a STING activator, wherein the transgene is expressed in an extracellular self-adjuvanting vesicle and induces an immune response in the mammal which is amplified in comparison to the immune response induced by the oncolytic virus alone.

15. A method of vaccination comprising administering to a mammal a transgene encoding a recombinant polypeptide comprising an EV anchoring polypeptide, and a STING activator and / or an antigen, wherein the recombinant polypeptide is expressed in an extracellular vesicle.

16. The method of claim 15, wherein the transgene is administered in a nucleic acid-based vector or a viral vector.

17. The method of claim 15 or 16, wherein the recombinant polypeptide expresses antigen and a STING activator.

18. The method of claim 15 or 16, wherein the recombinant polypeptide expresses one of the antigen and the STING activator, and the other of the antigen and the STING activator is co-loaded in the vesicle independently of the recombinant polypeptide.

19. The method of 15 or 16, wherein the transgene encodes an EV-anchoring polypeptide and a STING activator, and a second transgene encoding a cell targeting moiety polypeptide is administered to the mammal.

20. A transgene encoding an antigen linked to an extracellular vesicle (EV)-anchoring moiety linked to a STING activator.

21. The transgene of claim 20, wherein the anchoring moiety is a transmembrane domain and the STING activator is a cyclase.

22. The transgene of claims 20 or 21, encoding an N-terminal leader or signal sequence.

23. The transgene of any one of claims 20-22, wherein the antigen comprises a transmembrane domain.

24. The transgene of any one of claims 20-23, wherein the antigen is a viral antigen.

25. A polypeptide encoded by a transgene as defined in any one of claims 20-24.

26. A self-adjuvanting extracellular vesicle (SAEV) comprising a polypeptide as defined in claim 25, wherein the antigen is extravesicular and the STING activator is intravesicular.

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