Extracellular vesicle based vaccine platform

Abstract

The invention is based on fusion proteins comprising immunogenic antigens and partial cell adhesion proteins expressed in secreted extracellular vesicles for use in inducing immune responses in mammals. The invention is applicable as a novel platform technology for the development of vaccines, for methods of vaccination, for example against diseases or the generation of immune responses in mammals. The invention is for example useful as a platform technology for the generation of novel antibodies. The invention provides the cell adhesion protein based fusion proteins, recombinant cells, extracellular vesicles, and methods and uses thereof, for vaccination and vaccine development against for example infectious diseases.

Description

EXTRACELLULAR VESICLE BASED VACCINE PLATFORMFIELD OF THE INVENTION[1] The invention is based on fusion proteins comprising immunogenic antigens and partial cell adhesion proteins expressed in secreted extracellular vesicles for use in inducing immune responses in mammals. The invention is applicable as a novel platform technology for the development of vaccines, for methods of vaccination, for example against diseases or the generation of immune responses in mammals. The invention is for example useful as a platform technology for the generation of novel antibodies. The invention provides the cell adhesion protein based fusion proteins, recombinant cells, extracellular vesicles, and methods and uses thereof, for vaccination and vaccine development against for example infectious diseases.DESCRIPTION[2] Zoonotic bacterial and viral infections constitute a practically infinite and growing list of pathogens with the potential to cause human diseases of varying severity, modes of transmission, and epidemiology. Although there is more than a dozen of bacterial zoonotic diseases, each affecting hundreds of thousands of patients annually, viral zoonoses continually emerge and are associated with limited or expanded geographic outbreaks. The severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) virus is the causative agent of the Corona Virus Disease- 19 (COVID-19) pandemic and was first identified in Wuhan, China, in late of 2019. Numerous vaccines were rapidly developed to control the pandemic and protect people from a potentially lethal disease. The vaccines aim to mediate protective antibody responses against the viral spike protein. SARS-CoV-2 spike protein S1 subunit receptor binding domain (RBD) is most important for virus entry via binding to the host cells surface receptor angiotensin-converting enzyme-2 (ACE-2), and thus the initiation of infection. The associated S2 subunit assists with the help of cellular proteases in virus integration and release of the viral genome into the cell. This makes SARS-CoV-2 spike protein, especially RBD, the most important target for scientists in developing therapeutics and vaccines. Long-term efficacy of COVID-19 vaccines (BNT162b2 [Pfizer- BioNTech], mRNA-1273 [Moderna], ChAdOxI nCoV-19 [AZD1222; Oxford-AstraZeneca], and Ad26.COV2.S [Janssen]) against infections, hospitalizations, and mortality differed based on recipient age and health status as well as the SARS-CoV-2 variant. Although mRNA and vectorbased vaccines show good efficacy, they still have some limitations, such as mRNA vaccines poor stability and vector-based vaccines pre-existing antibodies. Thus, it is still timely to develop novel concepts for vaccination.[3] Extracellular vesicles (EVs) represent a novel platform for antigen delivery to be further investigated for vaccine development. This based on EV roles in basic processes of innate and adaptive immunity, including inflammation, antigen presentation, and development and activationof B- and T-cells. EVs as part of an ancient intercellular communication system play an important role in transporting several factors derived from parental cells, protecting luminal payloads, and providing information to elucidate the molecular mechanisms of various diseases and pathological conditions. The similarities between EVs and viruses are numerous, most importantly using the same cellular machinery in EV production versus virus replication, EV substrate loading versus virus assembly, and EV secretion versus virus egress. Furthermore, viruses can increase the production of EVs and recruit them to spread their genetic material or proteins versus the ability of EVs to regulate viral infection by transporting immunomodulatory molecules and viral antigens to initiate antiviral immune responses. In addition, their versatility, efficacy, biocompatibility, and safety advance their usefulness as novel therapeutics, drug delivery carriers, and vaccine platforms. Thus, they are now considered one of the most important entities in the battle against COVID-19 and hence any classical or newly emerged virus. Unlike viral vector- and virus-like particle (VLP)-based vaccine platforms, EV-based vaccine platforms are non- immunogenic and do not face the issue of pre-existing antibodies that can reduce platform immunogenicity as well as the potency of heterologous antigens they presented. Thus, by genetically engineering of EV-producing cells, immunogenic proteins are effectively targeted onto the surface of non-immunogenic EVs.[4] EVs are a group of biologically nanosized bilayered membrane vesicles produced by nearly all cells. They are ubiquitous in bodily fluids, such as blood, saliva, and breast milk. EVs can be classified into two predominant classes, exosomes and microvesicles, based on their biogenesis. Exosomes are 70-150 nm EVs that initially demonstrate formation as intraluminal vesicles inside multivesicular bodies (MVBs) and are released after fusion of MVBs with the plasma membrane. Microvesicles are 100-1000 nm and are formed by the outward budding of the plasma membrane, a process regulated by the translocation of phospholipids. Although most EVs fail to trigger immune responses, their cell-to-cell communication is beneficial, where immune professional antigen presenting cells (APCs); such as dendritic cell (DC-), B lymphocyte (BL-), and macrophage (MP-)EVs carry MHC I and II that are required for antigen presentation as well as necessary co-stimulatory molecules that have the ability to promote T- cell responses. A study showed that DC-EVs pulsing with tumor peptides can be used as an effective non-cellular vaccine to prime cytotoxic T-lymphocytes (CTLs) in vivo.[5] Thus, it is an object of the invention to provide a variable and easy platform for inducing immune responses to target antigens in mammals.BRIEF DESCRIPTION OF THE INVENTION[6] The inventors have solved the problem by selecting cell adhesion molecules, such as the intercellular adhesion molecule 1(ICAM-1) transmembrane protein for anchoring of foreign antigens.[7] Generally, and by way of brief description, the main aspects of the present invention can be described as follows:[8] In a first aspect, the invention pertains to a fusion protein comprising in N- to C-terminal direction:(a) A protein cell trafficking portion (signal peptide);(b) A protein fusion domain comprising a protein sequence derived from a cargo protein, and at least a partial sequence of an extracellular domain (ECD) of a cell adhesion protein;(c) A transmembrane region of the cell adhesion protein;(d) At least a partial intracellular sequence of the cell adhesion protein.[9] In a second aspect, the invention pertains to a nucleic acid, or nucleic acid construct (NAC) encoding the fusion protein of the invention.

[0010] In a third aspect, the invention pertains to a nucleic acid vaccine expression construct comprising a fusion protein expression cassette, wherein the fusion protein expression cassette comprises in operable linkage:(a) A first coding sequence encoding a protein cell trafficking portion (signal peptide);(b) A second coding sequence, comprising an acceptor element for accepting (cloning into) a donor coding sequence of a cargo protein, and a fusion protein coding region of at least a partial sequence of an extracellular domain (ECD) of a cell adhesion protein, such as an ICAM1 protein, or of an ICAM1 variant,(c) A third coding sequence encoding transmembrane region of the cell adhesion protein, such as the ICAM1 protein, or of an ICAM1 variant,(d) A fourth coding sequence encoding at least a partial intracellular sequence of the cell adhesion protein, such as the ICAM1 protein, or of an ICAM1 variant.

[0011] In a fourth aspect, the invention pertains to a recombinant cell comprising the nucleic acids or fusion proteins of the invention.

[0012] In a fifth aspect, the invention pertains to an extracellular vesicle (EV) comprising a fusion protein of the invention, preferably on its surface.

[0013] In a sixth aspect, the invention pertains to a method of inducing an immune reaction against a target protein in an animal, the method comprising administering to the animal(a) a fusion protein of the previous aspects, wherein the cargo protein comprises a protein sequence of the target protein; and / or(b) an expressible nucleic acid encoding the fusion protein of the previous aspects, wherein the cargo protein comprises a protein sequence of the target protein.(c) an extracellular vesicle comprising a fusion protein of the previous aspects, wherein the cargo protein comprises a protein sequence of the target protein.

[0014] In a seventh aspect, the invention pertains to a vaccine composition comprising the fusion protein, a nucleic acid, an EV or a recombinant cell in accordance with any of the aspects of the invention.DETAILED DESCRIPTION OF THE INVENTION

[0015] In the following, the elements of the invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine two or more of the explicitly described embodiments or which combine the one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0016] In a first aspect, the invention pertains to a fusion protein comprising in N- to C-terminal direction:(a) A protein cell trafficking portion (signal peptide);(b) A protein fusion domain comprising a protein sequence derived from a cargo protein, and at least a partial sequence of an extracellular domain (ECD) of a cell adhesion protein;(c) A transmembrane region of the cell adhesion protein;(d) At least a partial intracellular sequence of the cell adhesion protein.

[0017] The invention surprisingly identified a novel technology for the expression and presentation of antigenic sequences on extracellular vesicles. The invention relies on the expression of extracellular fusions domains derived from cell adhesion molecules (cell adhesion proteins) which are used as a scaffold for the presentation of an antigenic sequences that is intended to be presented on EV surfaces. The cell adhesion molecules therefore function as scaffold for antigen presentation. This technology allows for the presentation of any antigenic sequence to a hosts immune system and thereby consitutes an excellent platform for thedevelopment of vaccines, antibodies or general for the targeted antigen specific induction of immune responses in vertebrates. According to the invention, a partial cell adhesion protein comprises: an intracellular domain, or parts thereof, a transmembrane domain and at least a partial extracellular domain of the cell adhesion molecule. In this protein construct an antigenic sequence is fused into the extracellular domain of the cell adhesion molecule and thereby constitutes a fusion protein comprising both extracellular domain sequences of the cell adhesion molecule and the antigenic sequences. Any antigenic sequence against which an immune response is desired can be used in accordance with the invention. The antigenic sequences may be fused at any position in the extracellular part of the cell adhesion molecule, and may be an internal fusion or at the N-terminal end of the fusion protein.

[0018] Most preferably, the cell adhesion molecule of the invention is an ICAM1 protein (such as a human ICAM1) wherein the antigenic sequence is introduced within the immunoglobulin like domains that form part of the extracellular domain of ICAM1. Most preferably the antigenic sequence is introduced inbetween the Spacer 1 (S1) domain and the S5 domain of ICAM1 (see also Figure 1A). The antigenic sequence may be inserted at any location, either as a pure insertion (without replacing any ICAM1 sequences) or replacing one or more extracellular sequences from ICAM1. In a preferred embodiment of the invention insertion of the antigenic sequences is done by replacing all spacer and immunoglobulin-like domains in between. However, depending on the length and / or structure of the antigenic sequence to be inserted into the extracellular domain of ICAM1 , the length of sequences to be replaced may be adjusted to be in a similar range as the length of the antigenic sequence. Inserted sequences and replaced sequences may therefore in preferred embodiments be not significantly different, for example the shorter (in amino acid number or structurally measured distance) sequence may not be less than 50%, preferably not less than 60%, 70%, 80% most preferably not less than 90% of the length of the longer sequences. As mentioned, the antigenic sequence preferably replaces a part of the extracellular domain of human ICAM1 between the N-terminal signal peptide and one of the immunoglobulin-like domains. Preferably, the length of the antigenic sequence fused into the ICAM 1 extracellular region matches the length of replaced sequence in the original ICAM 1 region.

[0019] In alternative embodiments, the cell adhesion molecule is human ICAM1 , and wherein the at least a partial sequence of an extracellular domain (ECD) is when compared to the full length human ICAM1 in that one or more amino acids are removed in the extracellular domain between the signal peptide and the transmembrane domain, preferably wherein the one or more amino acids are 5 or more, 10 ore more or 20 or more amino acids, or are at least one or more of a spacer or other extracellular domain of human ICAM 1.

[0020] The term “cell adhesion protein” or “cell adhesion molecule” are used herein synonymously. The terms pertain to a large family of cell adhesion proteins that comprise an extracellular region for example in the form of a structure that serves as a cell recognition site and that is involved in in the mediation of cell-to-cell interactions, preferably adhesion to form cell bonds, for example in tissues. The term “cell adhesion protein” includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the nativesequence of the adhesion protein, including synthetically produced small-molecule entities and pharmaceutically acceptable derivatives and salts thereof.

[0021] The term "ICAM," or "intercellular adhesion molecule," refers to any of several type I membrane glycoproteins of the immunoglobulin superfamily. ICAMs act as ligands for leukocyte adhesion to target cells, in conjunction with LFA-I. LFA-1 / TCAM interactions mediate adhesion between many cell types. There are three subclasses of ICAM. ICAM-I (CD54), has a molecular mass of 90-115 kDa (see Figure 4(A)) and is expressed on B and T cells, endothelial, epithelial, and dendritic cells as well as fibroblasts, keratinocytes, and chondrocytes. They are inducible in 12-24 hours by cytokines including gamma interferon, interleukin-ip, and tumor necrosis factor-a. Examples of ICAM-I include ICAM-I-HUMAN, 532 amino acids (57.76 kDa). ICAM-2 (CD102), has a molecular mass of about 55-65 kDa and is constitutively expressed on endothelial cells, some lymphocytes, monocytes and dendritic cells. Examples of ICAM-2 include ICA2_HUMAN, 275 amino acids (30.62 kDa). ICAM-3 (CD50) has a molecular mass of 116-140 kDa, and is constitutively expressed on monocytes, granulocytes and lymphocytes. Upon physiological stimulation, ICAM-3 becomes rapidly and transiently phosphorylated on serine residues. Examples of ICAM-3 include ICA3_HUMAN, 547 amino acids (59.32 kDa).

[0022] In particular preferred in context of the invention is ICAM1. ICAM1 is a member of the immunoglobulin superfamily (IgSF) cell surface glycoprotein and adhesion receptor involved in the regulation of leukocytes recruitment from circulation to sites of inflammation. As a transmembrane glycoprotein consists of three main parts: a cytosolic, a transmembrane, and an extracellular part. The latter consists of five domains, D1-D5 and a signal peptide on the N- terminus, where D2-4 contains glycosylation site sessential for protein stability and function (Figure 1 , A). It contributes to homeostatic immune responses by acting as a receptor for p2 integrins macrophage-1 antigen (MAC-1) and lymphocyte function-associated- 1 antigen (LFA-1) essential for their trafficking. This central role in mediating leukocyte extravasation and involvement in a variety of diseases has made ICAM1 an attractive therapeutic target in pathological inflammatory conditions. In context of the present invention the goal was to establish a novel and very flexible EV-based vaccines platform for presenting pathogenic peptides (e.g., SARS-CoV-2 RBD) via a well selected and characterized truncated ICAM-1 anchor as an example. Therefore, the in context of the examples, the chosen variant ICAM-1_AC10_eGFP(anchor) still efficiently delivered to the plasma membrane and re-stored in EVs in a pattern like the full-length (FL) version ICAM-1_FL_eGFP (native).

[0023] In a preferred embodiment of the disclosed invention the cell adhesion protein is a protein involved in the mediation of cell-to-cel I interactions, and preferably is a protein selected from the intracellular cell adhesion molecules (ICAM), and most preferably is ICAM1 or a variant thereof. Most preferably thefusion protein is an ICAM1 such as human ICAM (UniProt Idenfication P05362).

[0024] In preferred embodiments of the invention the invention, the fusion protein of the invention is based on the SEQ ID NO: 1 , or a variant protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, as cell adhesion protein in accordance with the herein described aspects and embodiments of the invention.

[0025] In certain preferred embodiments of the invention, the fusion protein of the invention comprises the anchor sequence of SEQ ID NO: 1 , or a variant protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0026] In certain preferred embodiments of the invention, the fusion protein is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 , or, in some embodiments, wherein the fusion protein does not comprise the eGFP sequence. In the latter case, the fusion protein may comprise a different tag, such as any other detectable sequence, or no sequence tag.

[0027] For clarity of the invention, a fusion protein in accordance with the invention is comprised by a cell adhesion protein as anchor fused to a target antigen sequence and the antigen sequence is a sequence not derived from the respective cell adhesion protein. The term shall not be construed to include full length cell adhesion proteins wherein a part of the inherent full-length sequence is defined as “antigenic”. The present invention pertains to real fusion proteins composed of two sequences that are artificially fused together in accordance with the various aspects and embodiments of the invention. Therefore, preferably the protein sequence derived from the cargo protein does not comprise an ICAM1 protein sequence, preferably does not comprise the sequence of a cell adhesion protein.

[0028] The fusion protein may comprise further additional protein elements such as a detectable signal or tag sequence, such as a purification tag or a fluorescent protein sequence. Also other sequence that could be advantageous for a specific application may be included in the final sequence.

[0029] Preferred antigenic sequences can be derived from any protein for which an immune response in a vertebrate is desired. Either for research purposes, for antibody generation or vaccination / treatment of diseases. Preferred may be antigenic sequences from proteins knownto be involved in infectious diseases or proliferative disorders such as cancer. Viral proteins are preferred, such as a corona virus derived antigenic (spike protein) sequence. However, any protein sequence that can serve as an antigen for inducing immune responses can be used, and the present invention shall not be restricted to any specific category of targets.

[0030] In some preferred embodiments of the invention the antigenic sequence to be introduced into the fusion domain of the invention has a length of at least 5 amino acids (AA), preferably of at least 8, 9 or 10 AA to allow for eliciting an immune response. However, also longer antigenic sequences could be used. As such an antigenic sequence in accordance with the invention may have a length of at 4 to 5000 AA, preferably of 4 to 1000 amino acids, more preferably of 4 to 500 AA, more preferably of 4 to 300 AA. Alternative or additional embodiments of the invention relate to antigenic sequences having a length of 10 to 5000 AA, preferably of 1 to 1000 amino acids, more preferably of 10 to 500 AA, more preferably of 10 to 300 AA. Further alternative or additional embodiments of the invention pertain to antigenic sequences having a length of 20 to 5000 AA, preferably of 20 to 1000 amino acids, more preferably of 2 to 500 AA, more preferably of 20 to 300 AA. Most preferred alternative or additional embodiments pertain to antigenic sequences having a length of 50 to 5000 AA, preferably of 50 to 1000 amino acids, more preferably of 50 to 500 AA, more preferably of 50 to 300 AA.

[0031] In a second aspect, the invention pertains to a nucleic acid, or nucleic acid construct (NAC) encoding the fusion protein of the invention.

[0032] For example a nucleic acid of the invention may encode for an human ICAM protein, but wherein at least part of the extracellular domain encoding nucleic acid sequences have been removed, and / or replaced with an insertion site for inserting non ICAM1 sequences, such as preferably antigenic sequences. In preferred embodiments, the nucleic acid comprises a sequence encoding a protein that is at least 60%, 70%, 80%, 90%, 95%, 96% or 100% identical to the sequence shown in SEQ ID NO 2.

[0033] In some aspect the invention provides a nucleic acid sequence encoding for a deletion mutation of human ICAM1 , such as a protein according to SEQ ID NO: 1 , preferably wherein the delection mutant is a deleted in at least one extracellular region, such as at least 10 amino acids or more of the extracellular region, preferably wherein the deletion is of one or more D and / or S domains as described herein before.

[0034] In a third aspect, the invention pertains to a nucleic acid vaccine expression construct comprising a fusion protein expression cassette, wherein the fusion protein expression cassette comprises in operable linkage:(a) A first coding sequence encoding a protein cell trafficking portion (signal peptide);(b) A second coding sequence, comprising an acceptor element for accepting (cloning into) a donor coding sequence of a cargo protein, and a fusion protein coding region of at least a partial sequence of an extracellular domain (ECD) of a cell adhesion protein, such as an ICAM1 protein, or of an ICAM1 variant,(c) A third coding sequence encoding transmembrane region of the cell adhesion protein, such as the ICAM1 protein, or of an ICAM1 variant,(d) A fourth coding sequence encoding at least a partial intracellular sequence of the cell adhesion protein, such as the ICAM1 protein, or of an ICAM1 variant.

[0035] In a preferred embodiment the first, second, third and fourth coding sequence are in operable linkage such, that they allow for expression of a fusion protein comprising all encoded elements.

[0036] A nucleic acid construct of expression construct according to the invention may comprise (or consist of) a DNA or RNA (mRNA) molecule which includes an open reading frame encoding a fusion protein of the invention, and for example together with upstream and downstream elements (such as 5' and / or 3' UTRs and / or poly-A stretch) that enables expression of the fusion protein, and preferably enhancing stability of the mRNA and / or expression of the fusion protein. The use of mRNA as NACs to introduce into and express polynucleotides in cells is described, for example, in Zangi et al in Nat. Biotechnol. vol. 31 , 898-907 (2013), Sahin et al (2014) Nature Reviews Drug Discovery 13:759 and by Thess et al in Mol. Ther. vol. 23 no.9, 1456-1464 (2015). Particular UTRs that may be comprised in an mRNA NAC of the invention include: 5'UTR of a TOP gene (WO2013 / 143699), and / or a histone stem-loop (WO 2013 / 120629). An mRNA NAC of the invention may further comprise one or more chemical modifications (EP 1 685 844); including a 5'-cap, such as m7G(5')ppp, (5'(A,G(5')ppp(5')A or G(5')ppp(5')G and / or at least one nucleotide that is an analogue of naturally occurring nucleotides, such as phosphorothioates, phosphoroamidates, peptide nucleotides, methyl phosphonates, 7-deaza-guanosine, 5- methylcytosine or inosine.

[0037] NAC according to the invention are preferably DNA based expression constructs. DNA based NAC in context of the invention mean any polynucleotide designed to transcribe an RNA. For example, a construct that contains at least one promoter which is or may be operably linked to a downstream gene, coding region, or polynucleotide sequence encoding for a fusion protein of the invention. An "expression vector" is a polynucleotide comprising a promoter which can be operably linked to a second polynucleotide. Transfection or transformation of the expression construct into a recipient cell allows the cell to express a fusion protein of the invention An expression construct may be a genetically engineered plasmid, virus, recombinant virus, or an artificial chromosome derived from, for example, a bacteriophage, adenovirus, adeno-associatedvirus, retrovirus, lentivirus, poxvirus, or herpesvirus. Such expression vectors can include sequences from bacteria, viruses or phages. Such vectors include chromosomal, episomal and virus-derived vectors, for example, vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, and viruses, vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, cosmids and phagemids. An expression construct can be replicated in a living cell, or it can be made synthetically. For purposes of this application, the terms "expression construct", "expression vector", "vector", and "plasmid" are used interchangeably to demonstrate the application of the invention in a general, illustrative sense, and are not intended to limit the invention to a particular type of expression construct.

[0038] In a fourth aspect, the invention pertains to a host cell, preferably a recombinant cell comprising the nucleic acids or fusion proteins of the invention.

[0039] The term "host cell" refers to a cell line, which is capable to produce a recombinant protein after introducing an expression vector. The term "recombinant cell line" refers to a cell line into which a recombinant expression vector has been introduced for the expression of a fusion protein of the invention. It should be understood that "recombinant cell line" does not only mean the particular subject cell line but also the progeny of such a cell line. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical! to the parent cell, but is still included within the scope of the term "recombinant cell line" as used herein. The host cell as preferably used according to the present invention is a vertebrate cell line, most preferably a vertebrate cell line capable of producing, optionally secreting, extracellular vesicles. Most preferably, the host cell of the invention is a Mesenchymal Stromal Cell (MSC).

[0040] To produce vaccines in context of the invention, a host cell may be preferred which is less immune suppressive as an MSC. Host cells according to the invention in such embodiments may be preferably suspension cells, such as modified HEK cells (for example Expi293).

[0041] In a fifth aspect, the invention pertains to an extracellular vesicle (EV) comprising a fusion protein of the invention, preferably on its surface.

[0042] In a sixth aspect, the invention pertains to a method of inducing an immune reaction against a target protein in an animal, the method comprising administering to the animal;(a) a fusion protein of any one of claims 1 to 6, wherein the cargo protein comprises a protein sequence of the target protein; and / or(b) an expressible nucleic acid encoding the fusion protein of any one of claims 1 to 6, wherein the cargo protein comprises a protein sequence of the target protein.(c) an extracellular vesicle comprising a fusion protein of any one of claims 1 to 6, wherein the cargo protein comprises a protein sequence of the target protein.

[0043] In a seventh aspect, the invention pertains to a vaccine composition comprising the fusion protein, a nucleic acid, an EV or a recombinant cell in accordance with any of the aspects of the invention.

[0044] Immunization with the fusion protein loaded EVs of the invention may be conducted with one or more, preferably two or more steps of immunization of a vertebrate. In certain preferred aspects, the immunization of a vertebrate may comprise a priming and a boosting immunization.

[0045] The terms “of the [present] invention”, “in accordance with the invention”, “according to the invention” and the like, as used herein are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0046] As used herein, the term “comprising” is to be construed as encompassing both “including” and “consisting of”, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention. Where used herein, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±20%, ±15%, ±10%, and for example ±5%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated.

[0047] It is to be understood that application of the teachings of the present invention to a specific problem or environment, and the inclusion of variations of the present invention or additional features thereto (such as further aspects and embodiments), will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein.

[0048] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equallyto all aspects and embodiments which are described.

[0049] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.

[0050] In view of the above, it will be appreciated that the present invention also relates to the following itemised embodiments:Item 1. An Intercellular Adhesion Molecule 1 (ICAM1) fusion protein comprising in N- to C- terminal direction:A protein cell trafficking portion (signal peptide);(a) A protein fusion domain comprising a protein sequence derived from a cargo protein, and at least a partial sequence of an extracellular domain (ECD) of an ICAM1 protein, or of an ICAM1 variant;(b) A transmembrane region of an ICAM1 protein, or of an ICAM1 variant;(c) At least a partial intracellular sequence of an ICAM1 protein, or of an ICAM1 variant.Item 2. The ICAM1 fusion protein of item 1 , wherein the protein sequence derived from the cargo protein does not comprise an ICAM1 protein sequence.Item 3. The ICAM1 fusion protein of item 1 or 2, further comprising a detectable signal or tag sequence, such as a purification tag or a fluorescent protein sequence.Item 4. The ICAM1 fusion protein of item 1 or 2, wherein the ICAM1 is human ICAM (UniProt Idenfication P05362)Item 5. The fusion protein of any one of item 1 to 4, wherein the protein fusion domain comprises the structure according to the SEQ ID NOs: 1 , 2 and 4.Item 6. A nucleic acid construct comprising a nucleic acid sequence encoding the ICAM 1 fusion protein of any one of items 1 to 5.Item 7. A nucleic acid vaccine expression construct, comprising a fusion protein expression cassette, wherein the fusion protein expression cassette comprises in operable linkage:(a) A first coding sequence encoding a protein cell trafficking portion (signal peptide);(b) A second coding sequence, comprising an acceptor element for accepting (cloning into) a donor coding sequence of a cargo protein, and a fusion protein coding region of at least a partial sequence of an extracellular domain (ECD) of an ICAM1 protein, or of an ICAM1 variant,(c) A third coding sequence encoding transmembrane region of an ICAM1 protein, or of an ICAM1 variant,(d) A fourth coding sequence encoding at least a partial intracellular sequence of an ICAM1 protein, or of an ICAM1 variant.Item 8. The nucleic acid vaccine expression construct of item 7, wherein the first, second, third and fourth coding sequence are in operable linkage such, that they allow for expression of a fusion protein comprising all encoded elements.Item 9. A recombinant cell comprising an ICAM1 fusion protein of any one of items 1 to 5, a nucleic acid of item 6 or a nucleic acid vaccine expression construct of items 7 or 8.Item 10. The recombinant cell of item 9, wherein the cell capable of secreting extracellular vesicles (EV).Item 11. An extracellular vesicle (EV) comprising an ICAM1 fusion protein of any one of items 1 to 5.Item 12. A method of inducing an immune reaction against a target protein in an animal, the method comprising administering to the animal(e) an ICAM1 fusion protein of any one of items 1 to 5, wherein the cargo protein comprises a protein sequence of the target protein; and / or(f) an expressible nucleic acid encoding the ICAM1 fusion protein of any one of items1 to 5, wherein the cargo protein comprises a protein sequence of the target protein.(g) an extracellular vesicle comprising an ICAM1 fusion protein of any one of items 1 to 5, wherein the cargo protein comprises a protein sequence of the target protein.Item 13. The method of item 12, wherein the method is for vaccination of an animal, and / or for generating an antibody against the target protein in the animal.BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCES

[0051] The figures show:

[0052] Figure 1 : shows a schematic representation of: A) full-length intercellular adhesion molecule 1 (ICAM-1 signal peptide (L), five extracellular immunoglobulin-like domains (D1-D5), six spacer regions (S1 S5, ST), transmembrane domain (T), and cytoplasmic tail (Cyto)) and its 9 designed variant truncated constructs (C1C11) mainly uniquely designed construct C10, theanchor of choice. The full-length severe acute respiratory syndrome coronavirus type 2 (SARS- CoV-2) spike protein (S: segments of S1 and S2 include NTD, RBD, CTD1 , CTD2, S1 / S2, S2’, FP, FPPR, HR1 , CH CD, HR2, TM, and CT). B) expressed FL: ICAM-1_FL_eGFP (native), C10: ICAM-1_AC10_eGFP(anchor), and RBD: ICAM-1_AC10_RBD_eGFP (vaccine) fusion proteins delivered to the plasma membrane and to the components of the endosomal compartment as shown by eGFP subcellular distribution and cell morphology by phase contrast microscopy.

[0053] Figure 2: shows a flow cytometric analysis of HEK293T cells wild type (WT) and transduced with FL: ICAM1_FL_eGFP (native), C10: ICAM-1_AC10_eGFP (anchor), and RBD: ICAM-1_AC10_RBD_eGFP (vaccine) cells stained with antibodies against CD54 and SARS-CoV- 2 RBD to analyze these proteins expression on the surface of the transduced cells via measuring cells fluorescence intensity.

[0054] Figure 3: shows a quantification of A) eGFP+, B) RBD+, C) CD63+, and D) CD81+ EVs derived from HEK293T cells wild type (WT) and transduced with FL: ICAM-1_FL_eGFP (native), C10: ICAM-1_AC10_eGFP (anchor), and RBD: ICAM-1_AC10_RBD_eGFP (vaccine). The EVs of 1 x 108cells of each transduced HEK293T cell line after one day of incubation were quantified using imaging flow cytometry.

[0055] Figure 4: shows a Western blots of HEK293T-EV preparations (WT: wild type, FL: ICAM- 1_FL_eGFP (native), C10: ICAM-1_AC10_eGFP (anchor), and RBD: ICAM-1_ AC10_RBD_eGFP (vaccine)) according to their reactivity under different SDS-PAGE conditions. RBD, GFP, Syntenin, and Calnexin under reducing, whereas CD81 and CD63 under non-reducing conditions resolved by SDS-PAGE were transferred to PVDF membranes and stained with anti- RBD / anti-GFP / pooled vaccinated mice sera, anti-CD81 / anti-CD63, and anti-Syntenin / anti- Calnexin. For controls, CL-RBD: HEK293T ICAM-1_ AC10_RBD_eGFP (vaccine) cell lysate and RC-RBD: recombinant RBD (319-541 a. a., Prospec Cat # sars-049 & Lot # 1020PCOV2S1) were used. Arrows within each horizontal set of western blots represent the same membrane that restored after treatment with a stripping buffer (Restore TM Western Blot Stripping- Buffer, Thermo Scientific TM, USA, 21059).

[0056] Figure 5: shows a schematic representation of vaccination of mice with MPL / Alum adjuvanted EV-RBD on day 0 and boost-immunized on day 25. Serum was taken as indicated in the schemeto be tested in a neutralization assay.

[0057] Figure 6: shows antibody responses to EV-RBD. Mice were immunized with MPL / Alum adjuvanted EV-RBD at days 0 and 25. Serawas diluted at a 1000 dilution and tested for the presence of RBD-specific Abs by ELISA. As a control, purified serum from naive mice were used.

[0058] Figure 7: shows neutralization of SARS-CoV-2 virus by sera from mice immunized with EV-RBD. BALB / c mice were immunized with EVs-RBD at days 0 and 25. Sera were taken at days25 and 42 and tested for the neutralization of SARS-CoV-2 virus. Therefore, serial dilutions of the 42 days boosted sera (1 :20 - 1 :320) were pre-incubated with 25 TCID50 of SARS-CoV-2 virus for 1h at 37°C and then added to confluent Vero E6 cell monolayers. After 48 h, the cells were stained with crystal violet and analyzed for cytopathic effects using transmitted light microscopy. Scale bar = 200 pm.

[0059] SEQ ID NO 1 : human full length ICAM-1 (CD54) = native

[0060] MAPSSPRPALPALLVLLGALFPGPGNAQTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGI ETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPS WQPVGKNLTLRCQVEGGAPRANLTVVLLRGEKELKREPAVGEPAEVTTTVLVRRDHHGANFSC RTELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPRVLEVDTQGTVVCSLDGLFPVSEAQV HLALGDQRLNPTVTYGNDSFSAKASVSVTAEDEGTQRLTCAVILGNQSQETLQTVTIYSFPAPN VILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSATLEVA GQLIHKNQTRELRVLYGPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPL PIGESVTVTRDLEGTYLCRARSTQGEVTRKVTVNVLSPRYEIVIITVVAAAVIMGTAGLSTYLYNR QRKI KKYRLQQAQKGTPM KPNTQATPP

[0061] SEQ ID NO 2: ICAM-1_AC10 = Anchor

[0062] MAPSSPRPALPALLVLLGALFPGPGNAQTSVSPSKVILPRGTGGQLIHKNQTRELRVLY GPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPIGESVTVTRDLEGTYL CRARSTQGEVTRKVTVNVLSPRYEIVIITVVAAAVIMGTAGLSTYLYNRQRKIKKYRLQQAQKGT PM KPNTQATPP

[0063] SEQ ID NO 3: ICAM-1_AC10_HSV-2_gD = Vaccine I (Vaccine Example 1)

[0064] MAPSSPRPALPALLVLLGALFPGPGNAQTSVSPSKVILPRGTQRLQGKYALADPSLKMA DPNRFRGKNLPVLDRLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRSVLLHAPSEA PQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECPYNKSLGVCPIRTQPRWSYYDSF SAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRARASCKYALPLRIPPAACLTSKA YQQGVTVDSIGMLPRFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPED PEDSALLEDPAGTVSSQIPPNWHIPSIQDVAPHHAPAAPSNPRTRCGTGGQLIHKNQTRELRVL YGPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPIGESVTVTRDLEGT YLCRARSTQGEVTRKVTVNVLSPRYEIVIITVVAAAVIMGTAGLSTYLYNRQRKIKKYRLQQAQK GTPM KPNTQATPP

[0065] SEQ ID NO 4: ICAM-1_AC10_SARS-CoV-2_RBD = Vaccine II (Vaccine Example 2)

[0066] MAPSSPRPALPALLVLLGALFPGPGNAQTSVSPSKVILPRGTQRLQGRVQPTESIVRFP NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTN VYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRK SNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFRTRCGTGGQLIHKNQTRELRVLYGPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPIGESVTVTRDLEGTYLCRARSTQGEVTRKVTVNVLSPRYEIVIITVVAAAVIMGTAGLSTYLYNRQRKIKKYRLQQAQKGTPMKPNTQATPPEXAMPLES

[0067] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0068] EV-based cell-free vaccines as a novel and flexible platform would be a promising practical and economical alternative to most vaccine strategies, and this stems from EVs displaying anchor that could be any bioengineered selective surface protein rather than natural APC-EVs MHC molecules. At first, after selecting ICAM-1 surface protein, it was demonstrated that full-length (FL) version ICAM-1_FL_eGFP (native) (Figure 1 , A) fusion protein is effectively delivered to EVs.

[0069] With the aim of creating an antigen anchor of limited size, the inventors subsequently generated several truncated variants with deletions in their extracellular domain. Variants lacking the N-terminus failed to localize to the plasma membrane and were not recovered in EVs (data not shown). The variant still efficiently delivered to the plasma membrane and restored in EVs in a pattern similar to the native was the truncated version in which almost the whole extracellular domain, but the N-terminus was deleted and decoded as ICAM-1_AC10_eGFP (anchor) (Figure 1 , A). The inventors cloned the coding region of the SARS-CoV-2 RBD into the deletion site of ICAM- 1_AC10_eGFP encoding plasmid to obtain the fusion protein ICAM-1_AC10_RBD_eGFP (vaccine) (Figure 1 , A). The ICAM-1_FL_eGFP (native), ICAM-1_AC10_eGFP (anchor), and ICAM-1_AC10_RBD_eGFP (vaccine) constructs cloned into pCI6-IEG-wo employed to generate lentiviral systems required for integration into the genome of HEK293T cells and thus EVproducing cell lines (Figure 1 , B).

[0070] To detect surface expressed proteins (CD54 and SARS-CoV-2 RBD) on the surface of HEK293T wild type (WT) and transduced ICAM-1_FL_eGFP (native), ICAM- 1_AC10_eGFP (anchor), and ICAM-1_AC10_RBD_eGFP (vaccine) cells, FACS analysis (qualitative) was employed (Figure 2). It has been shown that only the native cell line ex- pressed CD54 and only the vaccine cell line expressed RBD on the surface. In contrast, IFCM analysis (quantitative) was employed (Figure 3) to detect surface expressed proteins (ICAM-1_FL, ICAM-1_AC10, and ICAM-1_AC10_RBD) as well as CD9, CD63, and CD81 EVs most enriched markers on the surface of HEK293T wild type (WT) and transduced ICAM-1_FL_eGFP (native), ICAM- 1_AC10_eGFP (anchor), and ICAM1_AC10_RBD_eGFP (vaccine) EVs. The EV preparations (WT, FL, C10, and RBD) were examined using IFCM for eGFP, RBD, CD63, and CD81 (Figure 3). The FL, C10, and RBDEV preparations display a clear signal for eGFP, which indicates the presence of fusion proteins in the EVs. To verify whether RBD can also be detected at the EVlevel, the preparations were also stained with an anti-RBD antibody. This shows that an EV population is detected only in the expected RBD preparation. CD63 and CD81 were used as further markers to check the preparations, which generally should show the quality of the preparations and can be detected regardless of the genetic modification. Here, all EV preparations show positive signals in similar concentration ranges. Only CD81 is less measured in the RBD preparation. In addition to FACS (Figure 2) and IFCM (Figure 3) analysis, EV RBD production in a culture supernatant of stable HEK293T ICAM-1_AC10_RBD_eGFP (vaccine) cells line was confirmed by a western blot band detected at a position corresponding to an approximate molecular weight over 70 kDa (Figure 4; top set). CD63 and CD81 were also detected, similar to the IFCM analysis, with weaker bands observed for CD63 compared to the bands for CD81 (Figure 4; middle set). EV preparations (Figure 4 bottom set); the presence of Syntenin (EVs positive marker), whereas the absence of Calnexin (EVs negative marker) strongly reveals the purity of EV preparations isolated using PEG ultracentrifugation-based purification strategy and is in agreement with previous studies. Thus, western blots confirmed that fusion of SARS-CoV- 2_RBD as a model encoded antigen to the membrane-associated ICAM-1_AC10 truncated variant success fully tether RBD to the surface of EVs in vitro.

[0071] Prior studies indicated that vaccine-induced nAbs were correlated with protection against SARS-CoV-2 virus infection [55-61], Hence, we investigated whether the EV-RBD vaccine could mediate n-Abs responses in mice. Therefore, we immunized BALB / c mice with EV-RBD formulated with Alum / MPL adjuvants, in which the applied dose of 30 pl was injected into the thigh muscle per vaccination (Figure 5). We used a standard neutralization assay to determine the neutralizing antibody titers in sera harvested on days 25 and 42 upon the first vaccination. The presence of RBD-specific Abs using an ELISA assay. RBD-specific Abs could be detected in serum samples derived from mice immunized with EV-RBD vaccine. The highest amounts of RBD-specific Abs could be observed in mice being two times vaccinated with EV-RBD vaccine (Figure 6).

[0072] Next, the three mice sera from blood collected at day 42, i.e. , 17 days after the boost dose were adjusted to serial dilutions and tested for the neutralization of the SARS-CoV-2 virus in Vero cell cultures. The cell cultures were infected with 25 TCID50 SARS-CoV-2 virus for 1 h at 37°C to infect single cells within the cell layer. The inoculation medium was then removed, and the cultures were inoculated with medium containing serial dilutions of the sera (1 :20-1 :320) from the immunized mice. Medium 2% DM EM only and with SARS-CoV-2 virus were used as negative and positive controls respectively. 48 h after infection, the cells were analyzed for plaque formation, which reflects the infection of cells (Figure 7).

[0073] Naive mice sera had no impact on the neutralization, since plaque formation was observed and the number of infected cells was similar to the 2% DMEM medium positive control.In contrast, ranking the three mice boosted sera effectiveness in neutralizing SARS-CoV-2 virus at lower dilutions shown in (Figure 7) was as follows; 112 (1 :160) > II3 (1:40) > 11 (1:20). The plaque size was slightly reduced and the number of SARS-CoV-2 infected cells per slide was lower when compared to the 2% DMEM medium positive or naive mice sera controls. Notably, EV-RBD induced Abs responses that completely neutralized SARS-CoV-2 virus. Plaque reduction was observed only when the SARS-CoV-2 infected cell cultures were treated with sera dilutions derived from EV-RBD immunized mice 17 days after the boost dose vaccination. Thus, sera contained RBD-specific Abs harvested after the boost dose had the only neutralization capability than sera derived after the prime dose vaccination (data not shown). Taken together, these data demonstrate that EV-RBD can induce SARS-CoV-2 virus-Abs. Furthermore, two immunizations are needed to achieve a sufficient n-Abs concentration required for complete SARS-CoV-2 virus neutralization in cell culture.

Claims

CLAIMS1. An Intercellular Adhesion Molecule 1 (ICAM1) fusion protein comprising in N- to C- terminal direction:(a) A protein cell trafficking portion (signal peptide);(b) A protein fusion domain comprising a protein sequence derived from a cargo protein, and at least a partial sequence of an extracellular domain (ECD) of an ICAM1 protein, or of an ICAM1 variant;(c) A transmembrane region of an ICAM1 protein, or of an ICAM1 variant;(d) At least a partial intracellular sequence of an ICAM1 protein, or of an ICAM1 variant.

2. The ICAM1 fusion protein of claim 1 , wherein the protein sequence derived from the cargo protein does not comprise an ICAM1 protein sequence.

3. The ICAM1 fusion protein of claim 1 or 2, further comprising a detectable signal or tag sequence, such as a purification tag or a fluorescent protein sequence.

4. The ICAM1 fusion protein of claim 1 or 2, wherein the ICAM1 is human ICAM (UniProt Idenfication P05362)5. The fusion protein of any one of claims 1 to 4, wherein the protein fusion domain comprises the structure according to the SEQ ID NOs: 1, 2 and 4.

6. A nucleic acid construct comprising a nucleic acid sequence encoding the ICAM1 fusion protein of any one of claims 1 to 5.

7. A nucleic acid vaccine expression construct, comprising a fusion protein expression cassette, wherein the fusion protein expression cassette comprises in operable linkage:(a) A first coding sequence encoding a protein cell trafficking portion (signal peptide);(b) A second coding sequence, comprising an acceptor element for accepting (cloning into) a donor coding sequence of a cargo protein, and a fusion protein coding region of at least a partial sequence of an extracellular domain (ECD) of an ICAM1 protein, or of an ICAM1 variant,(c) A third coding sequence encoding transmembrane region of an ICAM1 protein, or of an ICAM1 variant,(d) A fourth coding sequence encoding at least a partial intracellular sequence of an ICAM1 protein, or of an ICAM1 variant.

8. The nucleic acid vaccine expression construct of claim 7, wherein the first, second, third and fourth coding sequence are in operable linkage such, that they allow for expression of a fusion protein comprising all encoded elements.

9. A recombinant cell comprising an ICAM1 fusion protein of any one of claims 1 to 5, a nucleic acid of claim 6 or a nucleic acid vaccine expression construct of claim 7 or 8.

10. The recombinant cell of claim 9, wherein the cell capable of secreting extracellular vesicles (EV).

11. An extracellular vesicle (EV) comprising an ICAM1 fusion protein of any one of claims 1 to 5.

12. A method of inducing an immune reaction against a target protein in an animal, the method comprising administering to the animal(a) an ICAM1 fusion protein of any one of claims 1 to 5, wherein the cargo protein comprises a protein sequence of the target protein; and / or(b) an expressible nucleic acid encoding the ICAM1 fusion protein of any one of claims 1 to 5, wherein the cargo protein comprises a protein sequence of the target protein.(c) an extracellular vesicle comprising an ICAM1 fusion protein of any one of claims 1 to 5, wherein the cargo protein comprises a protein sequence of the target protein.

13. The method of claim 12, wherein the method is for vaccination of an animal, and / or for generating an antibody against the target protein in the animal.

14. A vaccine composition, comprising an ICAM1 fusion protein of any one of claims 1 to 5, a nucleic of claim 6, a recombinant cell of claim 9 or 10, or an EV of claim 11.

15. The vaccine composition of claim 14, wherein the cargo protein or target protein comprises an RBD protein sequence.

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