Immunogenic constructs for protein-based adjuvantation

Abstract

The present invention relates to an immunogenic construct with the capability to activate the mammalian immune system. In particular, the present invention relates to an immunogenic construct comprising bacterial proteins or fragments thereof that can both target and activate immune cells through interaction with pattern recognition receptors (PRR) expressed on the immune cells.

Description

[0001] IMMUNOGENIC CONSTRUCTS FOR PROTEIN-BASED ADJUVANTATION

[0002] Technical field of the invention

[0003] The present invention relates to an immunogenic construct with the capability to activate the mammalian immune system. In particular, the present invention relates to an immunogenic construct comprising bacterial proteins or fragments thereof that can both target and activate immune cells through interaction with pattern recognition receptors (PRR) expressed on the immune cells.

[0004] Background of the invention

[0005] Vaccines are used to safely induce an immune response that leads to protection against infectious or carcinogenic disease. An essential component of vaccines is one or more antigenic components, such as proteins, derived from either the pathogen (or tumour) or produced synthetically to represent components of the pathogen or tumour. Vaccines are generally classified as live or non-live vaccines to distinguish those vaccines that contain live attenuated replicating strains of a pathogenic organism from those that contain components of a pathogen or killed / inactive whole organisms. Several other vaccine platforms have been developed over the past decades, including viral vectors, nucleotide-based RNA and DNA vaccines, and virus-like particles.

[0006] Most non-live vaccines contain an adjuvant, which is an agent used to initiate and enhance the immune response against the antigen. There is a limited number of adjuvants approved for use in licensed vaccines today, including aluminium salts (generally referred to as "alum" adjuvants) used as a vaccine adjuvant for more than 80 years and liposome-based adjuvants, oil-in-water emulsions, and monophosphoryl lipid A (MPL) used as adjuvants in the past few decades. Although aluminium salt adjuvants have proven to be efficient in inducing a robust antibody (Th2) response towards co-administered antigen(s), they are largely ineffective at stimulating a cellular (Thl) immune response, which is important for protection against viral disease and carcinogenic disease, that is, for tumour surveillance. Further, aluminium salts have the potential to cause rare severe local and systematic side effects including sterile abscesses, eosinophilia, and macrophagic myofasciitis.

[0007] The main obstacle to the development of improved human adjuvants is the problem of local and systematic toxicity and adverse reactions, including inflammation and granuloma formation at the site of injection, pyrogenicity, nausea, adjuvant arthritis, uveitis, eosinophilia, allergy, anaphylaxis, or immunotoxicity. Ideally, adjuvant formulations should be suited for use with a wide range of potential vaccine antigens to increase vaccine potency without inducing increased local or systemic toxicity. Further, they should ideally be safe and reactive for use in low-responder populations including children, the elderly and immunocompromised individuals. Compounds that can successfully enhance adaptive immune responses and at the same time are well tolerated, safe, and non-toxic to the host remain highly elusive. There is increasing evidence that the immune response and protection can be further enhanced by novel adjuvants targeting danger signals in the innate immune system, such as pattern recognition receptors (PRR), expressed by immune cells of the innate immune system. Out of the hundreds of compounds known to be innate immune activators and possess vaccine adjuvant potential, less than a handful are approved for use in humans, and just two compounds, aluminium salts and MPL, being approved by the FDA for human vaccine use in the US.

[0008] Another major obstacle is to find a potent adjuvant that can induce a long-term immune response, including generating a CD8+T cell memory response. Current vaccines today induce a Th2-type immune response, leading to B-cell activation and production of antibodies against the target antigen. New adjuvant strategies capable of inducing Thl- and Thl7 immune response, leading to the generation of an antigenreactive CD8+memory T cell population are needed for use in effective viral and cancer vaccines.

[0009] Thus, there is an unmet need for safe adjuvants that induce improved immune responses for a broad range of vaccine antigens.

[0010] Hence, it would be advantageous to provide a potent immunogenic construct capable of adjuvating an improved immune response independently of the nature of the antigen. In particular, it would be advantageous to provide an immunogenic construct capable of inducing long-term immune responses.

[0011] Summary of the invention

[0012] The present invention relates to an immunogenic construct comprising a dual-purpose targeting moiety and an antigenic moiety. In particular, the dual-purpose targeting moiety is capable of (i) delivering the antigen to immune cells and (ii) inducing an adjuvating effect. The targeting moiety comprises bacterial proteins or fragments thereof that can both target and activate immune cells through interaction with pattern recognition receptors (PRR) expressed on the immune cells. Examples of such bacterial proteins are prokaryotic protein elongation factor thermo unstable (EF-Tu), L-lactate dehydrogenase (LDH), Protein F (PF) or single domains, or shorter fragments thereof. These bacterial proteins are capable of interacting directly with immune cells and therefore facilitate an enhanced immune response to any vaccine antigen guided by the immunogenic construct vehicle.

[0013] Thus, an object of the present invention relates to the provision of a platform technology for improving acute and prolonged immune responses to vaccine antigens.

[0014] In particular, it is an object of the present invention to provide immunogenic constructs which may act as active antigenic ingredients in viral, bacterial and / or cancer vaccines.

[0015] Thus, an aspect of the present invention relates to an immunogenic construct comprising a targeting moiety and an antigenic moiety.

[0016] An embodiment of the present invention relates to an immunogenic construct, wherein the targeting moiety comprises a protein or polypeptide selected from the group consisting of elongation factor thermo unstable (EF-Tu), L-lactate dehydrogenase (LDH), Protein F (PF), or single domains, or shorter fragments thereof.

[0017] Another aspect of the present invention relates to a vaccine or immunogenic composition comprising an immunogenic construct as described herein.

[0018] A further aspect of the present invention relates to an immunogenic construct or a vaccine or immunogenic composition as described herein for use as a medicament.

[0019] Yet another aspect of the present invention relates to an immunogenic construct or a vaccine or immunogenic composition as described herein for use in vaccination or immunization of a subject against viral and / or bacterial infections and / or cancer.

[0020] An even further aspect of the present invention relates to use of a targeting moiety for targeting an antigen to an immune cell, wherein said targeting moiety comprises a protein or polypeptide selected from the group consisting of elongation factor thermo unstable (EF-Tu), L-lactate dehydrogenase (LDH), Protein F (PF), or single domains, or shorter fragments thereof.

[0021] A still further aspect of the present invention relates to a kit comprising: i) an immunogenic construct or a vaccine or immunogenic composition as described herein, and ii) optionally, instructions for use. Yet another aspect of the present invention relates to a nucleic acid comprising a sequence encoding an immunogenic construct as described herein.

[0022] A further aspect relates to a recombinant expression vector comprising a nucleic acid as described herein operatively linked to one or more control sequences suitable for directing the production of the immunogenic construct in a suitable host.

[0023] A still further aspect relates to a recombinant host cell comprising a recombinant expression vector as described herein.

[0024] An even further aspect of the present invention relates to a method for provision of an immunogenic construct as described herein, said method comprising the steps of: i) provision of one or more nucleic acids at least encoding: a) the targeting moiety, and b) the antigenic moiety; and ii) expression of said one or more nucleic acids, thereby providing the immunogenic construct as described herein.

[0025] A further aspect relates to an immunogenic construct as described herein obtained from the method as described herein.

[0026] Brief description of the figures

[0027] Figure 1 shows five graphs labeled A-E showing that incubation of human monocyte- derived dendritic cells (MoDCs) with recombinant EF-Tu or EF-Tu domain 1 leads to maturation, IL-23 secretion, and upregulation of co-stimulatory surface ligands important for T cell activation. Figure A shows that human MoDCs incubated with recombinant EF-Tu, EF-Tu domain 1 mature and lose their macropinocytotic ability. Stimulated MoDCs were incubated with Alexa-488 labeled ovalbumin (OVA) and uptake was measured by flow cytometry. Viable CDllc+ cells were included in the analysis. Figure B shows that human MoDCs secrete IL-23 upon incubation with recombinant EF-Tu and EF-Tu domain 1. Cytokine levels in cell culture supernatants were measured by ELISA. Figure C-E show that CD40, CD86, and HLA-DR are upregulated upon stimulation with recombinant EF-Tu and EF-Tu domain 1.

[0028] Figure 2 shows a bar chart showing that EF-Tu orthologs from non-typeable Haemophilus influenzae, Escherichia coli, Streptococcus pneumoniae, and Listeria monocytogenes all induce IL-23 secretion in human MoDCs. Cytokine levels in cell culture supernatants were measured by ELISA.

[0029] Figure 3 shows four figures labeled A-D showing that incubation with recombinant EF- Tu stimulates murine bone marrow-derived dendritic cells (BMDCs). This is evidenced by IFN-y secretion (Panel A) and upregulation of CD40 (Panel B), CD86 (Panel C), and MHC-II (Panel D). Cells were incubated with indicated stimulants and thereafter, IFN- y in cell culture supernatants was assessed by ELISA (A) and cells analysed by flow cytometry (B-D).

[0030] Figure 4 shows three figures labeled A-C showing that cross-priming of CD8+T-cells by murine BMDCs is enhanced by pulsing with OVA adjuvanted by EF-Tu. Panel A shows representative flow cytometry profiles of CFSE-labeled CD8+T cells from OT-I mice harboring transgenic T cell receptors specific for OVA residues 257-264 (H2-Kbrestricted) after co-culture (1 :4) with BMDCs pulsed with EF-Tu (5 pg / ml) + OVA (50pg / ml), OVA (50pg / ml) or medium only. CFSELOW (box gates) indicate primed, proliferating CD8+T cells. In panel B, the percentage CFSELOW CD8+T cells after coculture with BMDCs pulsed with indicated stimuli, and the co-culture ratio is shown. Figure 4C demonstrates that murine BMDCs pulsed with OVA together with EF-Tu cross-present more OVA257'264(H2-Kbrestricted peptide) on the cell surface compared with OVA only (control).

[0031] Figure 5 shows MHC-I antigen presentation. Murine bone marrow-derived dendritic cells were exposed to EF-Tu-OVA recombinant overlapping peptide (ROP) fusion protein containing two repeats of the ovalbumin SIINFEKL epitope preferentially presented in MHC-I of mouse strain C57BL / 6J or indicated controls for 24h. Surface MHC-I complexes with bound SIINFEKL peptide were detected by flow cytometry. Viable, CDllc+ cells were analyzed. For the SIINFEKL control, peptide was added 30 min prior to staining. Data represent 4 independent experiments with cells from 4 different mice (*** = p<0.001,* = p<0.05).

[0032] Figure 6 shows that an increased amount of circulating CD8+ T cells is achieved upon immunization of C57BL / 6J mice with recombinant EF-Tu. Representative flow cytometry profiles of circulating CD8+ T cells 7 days after immunization with recombinant EF-Tu (20 pg) + OVA (100 pg; n = 2), curdlan (100 pg) + OVA (n = 2) or OVA only (n = 2) is shown. Figure 7 demonstrates that immunization with EF-Tu domain 1 (EF-Tu_Dl; 20 pg; n = 8) with model tumour antigen OVA (100 pg) offers significant protection upon tumour challenge compared with curdlan + OVA (100 pg + 100 pg; n = 8). Mice were immunized twice and challenged with 105B16F10 murine melanoma cell line expressing full-length OVA (B16.OVA) s.c in the left flank. Plot shows the survival of tumour-bearing C57BL / 6J mice.

[0033] Figure 8 shows three figures labeled A-C showing that fusion proteins consisting of EF- Tu domain 1 and viral antigens from SARS-CoV-2 stimulate human monocyte-derived dendritic cells (MoDCs), resulting in increased priming of CD8+ T cells. Figure 8A is a drawing showing two fusion proteins of EF-Tu domain 1 (denoted EF-Tu_Dl) with 216 aa sequence from SARS-CoV-2 spike protein (denoted DIS) or 181 aa nucleocapsid protein (denoted DIN). Both contain a (GGGS)2 linker. Figure 8B shows the fraction of CD8+IFN-y+upon co-culture with MoDCs pulsed with fusion protein DIN, DIS, truncated SARS-CoV-2 N protein (denoted N), or truncated SARS-CoV-2 S protein (denoted S). Figure 8C shows representative flow cytometry profiles of human MoDCs incubated with fusion protein DIN or truncated SARS-CoV-2 N protein. Incubation with DIN stimulates more upregulation of T cell-stimulating membrane proteins CD80 and CD86, which leads to increased priming of CD8+T cells.

[0034] Figure 9 shows that EF-Tu orthologs from S. pneumoniae, E. coli, L. monocytogenes, and Haemophilus influenzae bind to Dectin-1. The binding was tested using ELISA. Polysorp plates were coated with 25nM fc-hDectin-la and incubated with 25nM recombinant protein. Detection was performed with o-HIS polyclonal antibodies (pAb).

[0035] Figure 10 shows two figures labeled A-B showing that EF-Tu can bind and activate Dectin-1 expressed on A549 cells. Figure 10A shows EF-Tu binding to the surface of A549 cells transfected with Dectin-1 or empty vector (vector) as measured by flow cytometry with rabbit o-EF-Tu pAb. Figure 10B shows IL-8 in cell culture supernatant upon incubation of transfected A549 cells with EF-Tu or LPS.

[0036] Figure 11 shows that Dectin-1 is important for the effect of EF-Tu on dendritic cells. Murine BMDCs derived from wild-type C57BL / 6J mice and Dectin-1 KO C57BL / 6J Clec7a- / -) mice were incubated with EF-Tu (5 pg / ml). IFN-y secretion was assessed by ELISA.

[0037] Figure 12 shows that EF-Tu induces Dectin-l-dependent downstream signaling when coated on a bead surface. Human embryonic kidney 293 reporter cells (HEK) that express NF-KB-inducible SEAP (secreted embryonic alkaline phosphatase) with Dectin- 1 or without (Control) were incubated for 24h with nickel beads coated with HIS-tagged EF-Tu, LDH, or uncoated (Empty beads) at indicated volumes (Ipl beads contain 0.8pg of protein). NF-KB activation was assessed by measuring the relative SEAP concentration in the conditioned media.

[0038] Figure 13 shows (A) NF-KB activation in HEK reporter cells expressing Dectin-1 and HEK control, respectively, after 24h incubation with protein-coated LNPs carrying 20ng eGFP mRNA, and (B) eGFP expression after 24h incubation with protein-coated LNPs carrying 20ng eGFP mRNA as measured by fluorescence intensity in both cell lines, respectively. LNPs were coated with EF-Tu, LDH, PF, or ovalbumin (OVA) at a protein- to-lipid ratio (w / w) of 1 :24.

[0039] Figure 14 shows dose-dependent binding of fc-h Dectin-1 to immobilized recombinant proteins by ELISA. Polysorb plates were coated with increasing concentrations (0-1 pg / mL) of recombinant EF-Tu (A), Protein F (B), or LDH (C), then blocked and incubated with 0.1 pg / mL fc-hDectin-1. Bound Dectin-1 was detected using an HRP- conjugated anti-human Fc antibody with TMB substrate, and absorbance was read at 450 nm. Data are presented as mean ± SEM of three independent experiments.

[0040] Figure 15 shows that EF-Tu domain 1 induces Dectin-l-dependent downstream signaling when coated on a bead surface comparable to full-length EF-Tu. Human embryonic kidney 293 reporter cells (HEK) that express NF-KB-inducible SEAP (secreted embryonic alkaline phosphatase) with Dectin-1 or without (Control) were incubated for 24h with 4 pl nickel beads coated with HIS-tagged EF-Tu domain 1, EF- Tu, or uncoated (Empty beads). 4 pl beads contain roughly 3.2 pg of protein. NF-KB activation was assessed by measuring the SEAP concentration in the conditioned media. Data represent mean ± SEM from at least 3 independent experiments. Statistical comparisons between cell types for each stimulus were performed using unpaired two-tailed t-tests. Significance levels: *** p < 0.001, ** p < 0.01.

[0041] Detailed description of the invention

[0042] Definitions

[0043] Prior to outlining the present invention in more detail, a set of terms and conventions is first defined:

[0044] Immunogenic construct In the present context, the term "immunogenic construct" refers to a construct comprising a targeting moiety and an antigenic moiety. The targeting moiety is capable of directing the immunogenic construct to immune cells and providing an adjuvating effect. The antigenic moiety comprises a vaccine antigen of native or synthetic origin. The targeting moiety may be covalently or non-covalently attached to the antigenic moiety. Ways of attachment include, but are not limited to, covalent immobilization, through a linker moiety or by coating of the targeting moiety onto the antigenic moiety.

[0045] Fusion protein

[0046] In the present context, the term "fusion protein" refers to an immunogenic construct wherein the targeting moiety and an antigenic moiety are expressed as a single protein. The targeting moiety and antigenic moiety may be connected through a linker moiety.

[0047] EF-Tu

[0048] In the present context, the term "EF-Tu" refers to Elongation factor thermo unstable (EF-Tu) which is a bacterial elongation factor that is highly conserved across various bacterial species and can activate the mammalian immune system, e.g. through pattern recognition receptors (PRRs).

[0049] LDH

[0050] In the present context, the term "LDH" refers to L-lactate dehydrogenase. LDH can interact with the human immune system through binding to PRRs, such as Dectin-1 or other pattern recognition receptors (PRR).

[0051] L-lactate dehydrogenase may also be referred to as Lactate dehydrogenase and the terms may be used interchangeably herein.

[0052] PF

[0053] In the present context, the term "PF" refers to Protein F which is a bacterial metalbinding receptor belonging to the adenosine triphosphate-binding cassette (ABC) transporter family. PF can interact with the human immune system through binding to PRRs, such as Dectin-1.

[0054] Targeting moiety

[0055] In the present context, the term "targeting moiety" refers to an amino acid sequence, protein, or peptide capable of interacting with immune cells and inducing an adjuvating effect. Including a targeting moiety in the immunogenic construct entails increased affinity for immune cells over other cell types or non-relevant targets.

[0056] Affinity (ko)

[0057] In the present context, the term "affinity" refers to the binding affinity or strength of interaction between the targeting moiety and its target, including, but not limited to, receptors located on immune cells. The binding affinity may be described by the dissociation constant (ko) in molar units as conventionally defined.

[0058] Accordingly, the terms "binding affinity" and "dissociation constant" are used interchangeably herein.

[0059] The targeting moiety may be understood to have an affinity for a target if the dissociation constant ko is equal to or less than about 1.0 x 10'5M.

[0060] Antigenic moiety

[0061] In the present context, the term "antigenic moiety" refers to a part of the immunogenic construct that act as an antigen, e.g., when presented to immune cells. Thus, the antigenic moiety may comprise one or more antigens, such as antigenic proteins, peptides, polypeptides or nucleic acids. The antigenic moiety may also comprise a vehicle for protecting and / or delivering cargo, such as nucleic acids or proteins, to immune cells. The vehicle may be a particle, such as a lipid nanoparticle (LNP). By way of example, the antigenic moiety can be a LNP comprising DNA or mRNA as already known from mRNA vaccines.

[0062] Antigen

[0063] In the present context, the term "antigen" refers to any molecule or molecular structure that is capable of activating the immune system and triggering an immune response. Antigens include, but are not limited to, proteins, peptides, nucleotides, ribonucleotides, messenger ribonucleic acid (mRNA), and polysaccharides. Antigens may also be fragments of larger molecular structures, such as specific epitopes of high immunogenicity.

[0064] The origin of the antigen may be exogenous or endogenous. The latter may for example be the situation in case of viral infection. Antigens also include neo-epitope antigens, i.e. antigens that are not present in the normal human genome. Particle

[0065] In the present context, the term "particle" refers to a vehicle for protecting and / or delivering cargo, such as nucleic acids or proteins, to immune cells. For example, the vehicle may be a lipid-based particle, a polymer-based particle, a peptide-based particle, a bead-based particle and a virus-like particle.

[0066] Lipid nanoparticle (LNP)

[0067] In the present context, the term "lipid nanoparticle (LNP)" refers to a vehicle comprising primarily lipids, typically a substantially spherical nanosized particle. The LNP may comprise a single outer lipid layer and lipids in the core of the particle. LNPs typically have an average diameter of 10-1000 nm. Nucleic acids may be interacting with the lipids in the core to trap them inside the LNP. The LNP may be used to deliver nucleic acid material to a host cell within a target subject. The nucleic acid material may be complexed and / or encapsulated within the lipid formulation.

[0068] The term lipid is used here in a broad sense and includes, but is not limited to, phospholipids, triglycerides, diglycerides, monoglycerides, fatty acids, steroids, and waxes.

[0069] Electrostatic interaction

[0070] In the present context, the term "electrostatic interaction" refers to an attractive or repulsive force between charged moieties. Accordingly, the electrostatic interaction may be an attractive force between a charged moiety and an oppositely charged surface. Electrostatic interactions may be used as a method of coating proteins, peptides, polypeptides or nucleic acid onto the surface of particles, such as a LNPs. In particular, electrostatic interaction may be used to coat the targeting moiety onto the surface of particles, such as LNPs.

[0071] Neo-epitope

[0072] In the present context, the term "neo-epitope" refers to a class of antigenic epitopes that arise from tumour-specific mutations within a tumour. Neo-epitopes are unique epitopes exclusively expressed in tumours and not in healthy tissue and is therefore recognized as new and foreign to the immune system. They bind to MHC molecules and are presented to immune cells and can initiate an immune response towards the tumour.

[0073] The antigenic epitope may also arise from a specific mutation within a microbe. Immune cell

[0074] In the present context, the term "immune cell" refers to a cell that is part of the immune system and helps the body fight infections and other diseases. These include, but are not limited to, lymphocytes and monocytes.

[0075] Antigen-presenting cells (APCs)

[0076] In the present context, the term "antigen-presenting cell (APCs)" refers to any cell capable of displaying antigens on their surface. Immune cells typically display antigens as protein fragments (or antigenic peptides) bound by MHC molecules, thereby activating cells of the immune system, such as T helper cells and cytotoxic T cells.

[0077] Antigen-presenting cells include, but are not limited to, B cells, dendritic cells and macrophages.

[0078] Antigenic peptide

[0079] In the present context, the term "antigenic peptide" refers to an amino acid sequence or protein fragment derivate of pathogenic or cancerous origin that binds to MHC molecules and is presented to immune cells and initiates an immune response towards the pathogen or cancer.

[0080] The terms "antigenic" and "immunogenic" are used interchangeably herein.

[0081] Pattern Recognition Receptor (PRR)

[0082] In the present context, the term "pattern recognition receptors (PRR)" refers to a group of sensor molecules capable of detecting molecules typical for microbes and pathogens. PRRs are typically expressed by cells of the innate immune system and are capable of recognizing pathogen-associated molecules patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs), which relate to microbe-specific molecules (e.g. nucleic acids, carbohydrates, or peptides) and molecules released by damaged or dead cells, respectively.

[0083] PRRs include, but are not limited to, toll-like receptors (TLRs) and C-type lectin receptors (CLRs). An example of a PRR is Dectin-1.

[0084] Fragment

[0085] In the present context, the term "fragment" when used in reference to polypeptides refers to polypeptides that are shorter than the full-length polypeptide by virtue of truncation at either the N-terminus or C-terminus of the protein or both, and / or by deletion of an internal portion or region of the protein. Fragments of a polypeptide can be generated by methods known in the art.

[0086] It is to be understood that fragments maintain immunogenicity and the ability to function both as a delivery module and to provide an adjuvating effect.

[0087] Primary cancer

[0088] In the present context, the term "primary cancer" refers to any cancer of the original or primary site in the body at which a first tumour is located, i.e., where the cancer originates from. Primary cancers relate to a non-metastatic stage of the disease, i.e. it is to be distinguished from metastatic cancer.

[0089] High mutational burden tumour

[0090] In the present context, the term "high mutational burden tumour" refers to a tumour with a large number of somatic gene mutations that occur in the genome of tumour cells. Tumour mutational burden (TMB) may be used as a type of biomarker quantified by the number of non-inherited mutations per million bases (Mb) of investigated genomic sequence. A high mutational burden tumour may be defined to comprise at least 10 mutations per Mb.

[0091] The TMB may be determined by e.g. next generation sequencing or similar technologies.

[0092] Linker moiety

[0093] In the present context, the term "linker moiety" refers to a nucleic acid or amino acid sequence or a protein that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds. Thus, the linker moiety may be a peptide linker that connects the targeting moiety with the antigenic moiety.

[0094] The linker moiety may also be a crosslinking reagent, which chemically crosslinks the antigenic moiety to the targeting moiety. Crosslinking reagents contain two or more reactive ends capable of chemically attaching to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules, herein the antigenic moiety and targeting moiety.

[0095] Expression vector

[0096] In the present context, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.

[0097] Pharmaceutically acceptable

[0098] In the present context, the term "pharmaceutically acceptable" refers to molecular entities and compositions that are suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0099] Subject

[0100] In the present context, the term "subject" refers to a human or non-human species including primates, livestock animals e.g. sheep, cows, pigs, horses, donkeys, goats, laboratory test animals e.g. mice, rats, rabbits, guinea pigs, hamsters, companion animals e.g. dogs, cats, avian species e.g. poultry birds, aviary birds, reptiles and amphibians.

[0101] Sequence identity

[0102] In the present context, the term "sequence identity" is here defined as the sequence identity between proteins at the amino acid level. The protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned.

[0103] To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g. gaps may be introduced in the sequence of a first amino acid sequence for optimal alignment with a second amino acid sequence). The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions / total # of positions (e.g., overlapping positions) x 100).

[0104] In one embodiment, the two sequences are the same length. In another embodiment, the two sequences are of different lengths and gaps are seen as different positions. One may manually align the sequences and count the number of identical amino acids. Alternatively, the alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm. Such an algorithm is incorporated into the XBLAST program of (Altschul et al. 1990). BLAST protein searches may be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecule of the invention.

[0105] To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized. Alternatively, PSI-Blast may be used to perform an iterated search, which detects distant relationships between molecules. When utilising the XBLAST and Gapped BLAST programs, the default parameters of the respective programs may be used. See http: / / www.ncbi.nlm.nih.gov. Alternatively, sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov / cgi-bin / BLAST). Generally, the default settings with respect to e.g. "scoring matrix" and "gap penalty" may be used for alignment.

[0106] The percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.

[0107] About

[0108] Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, weights, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ±10% about the number 10, which is anything between 9% and 11%).

[0109] Immunogenic constructs

[0110] In order to mount an effective immune response towards vaccine antigens derived from pathogens such as bacteria, viruses or fungi, it is advantageous to target PRRs expressed by various immune cells, including APCs, recognizing molecules derived from pathogens to quickly mount an unspecific inflammatory response. PRRs expressed on APCs are also crucial for initiating an adaptive immune response, since activation of APCs through PRR interaction initiates lymph node migration and subsequent priming of T- and B-lymphocytes, resulting in cell-mediated and humoral immunity, leading to a long-term memory response.

[0111] A PRR target to initiate such an immune response towards vaccine antigens is Dectin- 1 CLEC7A), expressed by immune cells of the innate immune system, including APCs. It is a type II transmembrane receptor that recognizes p-glucan and is important in anti-fungal immunity. Dectin-1 activation ultimately leads to the induction of chemokine and cytokine synthesis, ligand phagocytosis, respiratory burst, and arachidonic acid metabolization, depending on the cell type and signal coupling with toll-like receptors (TLRs). In dendritic cells (DCs), activation via Dectin-1 causes maturation, increased antigen presentation through MHC class II complex, lymph node migration, and potent cross-priming of CD8+T cells leading to the presentation of antigen through MHC class I complex. In contrast, the stimulatory effect of Dectin-1 in macrophages is towards phagocytosis and respiratory burst. The type of response is also affected by signal coupling with TLRs. Dectin-1 signalling alone prompts an immune response biased towards Thl and Thl7 type of immune cells together with induction of cytotoxic CD8+T lymphocytes.

[0112] In a tumour context, Dectin-1 stimulation can repolarize tumour-associated macrophages (TAM) into a classical immunostimulatory phenotype, which has been shown to delay tumour progression in mice. The Dectin-1 expression on DCs and macrophages is also vital for stimulating tumouricidal activity in NK cells in the tumour microenvironment; hence this is an attractive immunotherapeutic target.

[0113] Accordingly, PRRs appear to be relevant targets for devising competent vaccines that elicit strong immune responses. However, ligands for PRRs, such as Dectin-1, and their role in inducing long-term immune responses are still somewhat unexplored. Herein is disclosed an immunogenic construct with bacterial proteins having affinity for binding PRRs, such as Dectin-1. In particular, it is disclosed herein that elongation factor thermo unstable (EF-Tu), L-lactate dehydrogenase (LDH) and Protein F (PF) may be utilized as a targeting moiety for interacting with immune cells and thus be part of the immunogenic constructs capable of eliciting improved immune responses. Without being bound by theory, it is contemplated that EF-Tu, LDH and PF can activate the mammalian immune system through interactions with PRRs, such as Dectin-1, expressed on immune cells thereby initiating an adaptive immune response.

[0114] By fusing an antigenic moiety to bacterial proteins capable of targeting antigen- presenting cells and activating the human immune system through binding to PRRs, such immunogenic constructs may be applied in vaccine formulations to initiate a robust Thl type immune response against pathogenic antigens, such as those antigens characteristic for viral and carcinogenic disease. Beyond an adjuvating effect, the bacterial proteins, such as EF-Tu, LDH and PF, furthermore, serves the purpose as a delivery system for directing the antigen to the immune cells due to their affinity for antigen-presenting cells. This dual effect of the immunogenic construct is advantageous in modern vaccine strategies, as it solves two of the main challenges in vaccination, namely effective adjuvantation and targeted delivery to immune cells to initiate an adaptive cellular and humoral immune response.

[0115] Hence, an aspect of the present invention relates to an immunogenic construct comprising a targeting moiety and an antigenic moiety.

[0116] To effectively activate the immune system, it is beneficial to target surface receptors expressed on antigen-presenting cells. Especially in a vaccination strategy, it is advantageous to select epitopes, for adjuvating purposes, that have binding affinity for e.g. PPRs in order to raise an adaptive immune response leading to long-term memory and immunity.

[0117] Therefore, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety has affinity for antigen- presenting cells.

[0118] It is to be understood that in the present context the expression "has affinity for" means that the targeting moiety is able to bind a target, such as a receptor.

[0119] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety has affinity for a pattern recognition receptor (PRR).

[0120] The immunogenic construct described herein may especially have affinity for receptors from the CLEC family or C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily.

[0121] It is demonstrated herein that the bacterial proteins EF-Tu, LDH and PF have high affinity for Dectin-1, a PRR positioned on the cell surface of antigen-presenting cells. Immunogenic constructs comprising these bacterial proteins are therefore capable of specifically targeting immune cells leading to a strong specific immune response to the antigenic moiety of the immunogenic construct. Accordingly, the immunogenic constructs described herein facilitate enhanced contact of the antigenic moiety with antigen-presenting cells and ensure the propagation of a strong immune response through the adjuvating effect of bacterial proteins, such as EF-Tu, LDH and PF. Furthermore, activation of antigen-presenting cells through Dectin-1 causes increased antigen presentation through MHC class I and II complexes and potent cross-priming of CD8+T cells leading to activation of an adaptive immune response.

[0122] Therefore, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety has affinity for Dectin-1.

[0123] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety has a binding affinity (kD) for Dectin- 1 in the range of about 1.0 x 10'9M to about 1.0 x 10'5M, such as about 5.0 x 10'8M to about 1.0 x 10'6M.

[0124] Dectin-1 exists in two isoforms - isoform a (SEQ ID NO:7) and isoform b (SEQ ID NO:8). The immunogenic construct may target either of the isoforms of Dectin-1.

[0125] Therefore, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein said Dectin-1 comprises an amino acid sequence selected from SEQ ID NO:7 or SEQ ID NO:8, or an amino acid sequence having at least 90% sequence identity to the full-length sequence of SEQ ID NO:7 or SEQ ID NO:8.

[0126] EF-Tu, LDH and PF are highly conserved across various bacterial species and the origin of the targeting moiety is therefore not limited to any specific bacterial species. Notably, EF-Tu shares at least 90% sequence identity across a broad selection of bacterial species, with the overall structure of three similar domains being conserved. It will be appreciated that e.g. EF-Tu and domains and fragments thereof derived from Haemophilus influenzae has been identified as favourable targeting moieties for the preparation of advantageous constructs of high immunogenicity.

[0127] Therefore, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acid sequence originating from a bacterium.

[0128] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acid sequence originating from a bacterium genus selected from the group consisting of Aggregatibacter, Avibacterium, Enterobacter, Escherichia, Haemophilus, Gallibacterium, Brenneria, Caldithrix, Lactococcus, Listeria, Pasteurella, Porphyromonas, Rodentibacter, Salmonella, Shigella, Staphylococcus, and Streptococcus.

[0129] A still further embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acid sequence originating from a bacterium species selected from the group consisting of Haemophilus influenzae, Escherichia coli, Streptococcus pneumoniae, Listeria monocytogenes, Aggregatibacter aphrophilus (Haemophilus aphrophilus), Aggregatibacter segnis, Avibacterium paragallinarum (Haemophilus gallinarum), Brenneria alni, Calditrichaeota bacterium, Enterobacter hormaechei, Escherichia albertii, Escherichia fergusonii, Gallibacterium anatis, Haemophilus aegyptius, Haemophilus haemolyticus, Haemophilus parainfluenzae, Haemophilus pittmaniae, Lactococcus chungangensis, Lactococcus hodotermopsidis, Lactococcus insecticola, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus reticulitermitis, Listeria booriae, Listeria cornellensis, Listeria fleischmannii, Listeria grandensis, Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria riparia, Listeria rocourtiae, Listeria seeligeri, Listeria weihenstephanensis, Listeriaceae bacterium, Pasteurella multocida, Porphyromonadaceae bacterium, Rodentibacter pneumotropicus, Salmonella enterica, Salmonella houtenae, Salmonella infantis, Salmonella typhimurium, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Streptococcus acidominimus, Streptococcus agalactiae, Streptococcus alactolyticus, Streptococcus anginosus, Streptococcus australis, Streptococcus azizii, Streptococcus bovimastitidis, Streptococcus canis, Streptococcus chenjunshii, Streptococcus chosunense, Streptococcus constellatus, Streptococcus criceti, Streptococcus cristatus, Streptococcus cuniculi, Streptococcus downei (Streptococcus sobrinus), Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equi subsp. equi, Streptococcus equi subsp. ruminatorum, Streptococcus equi subsp. Zooepidemicus, Streptococcus equinus (Streptococcus bovis), Streptococcus ferus, Streptococcus gallolyticus, Streptococcus gordonii, Streptococcus gwangjuense, Streptococcus halitosis, Streptococcus henryi, Streptococcus hyointestinalis, Streptococcus ictaluri, Streptococcus infantarius, Streptococcus infantis, Streptococcus iniae (Streptococcus shiloi), Streptococcus intermedius, Streptococcus lactarius, Streptococcus lutetiensis, Streptococcus macacae, Streptococcus macedonicus, Streptococcus massiliensis, Streptococcus merionis, Streptococcus milleri, Streptococcus minor, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus ovis, Streptococcus pantholopis, Streptococcus parasanguinis, Streptococcus parasuis, Streptococcus parauberis, Streptococcus pasteurianus, Streptococcus penaeicida, Streptococcus periodonticum, Streptococcus peroris, Streptococcus pharyngis, Streptococcus phocae, Streptococcus pluranimalium, Streptococcus porcinus, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus rubneri, Streptococcus ruminantium, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sinensis, Streptococcus sobrinus, Streptococcus suis, Streptococcus thermophilus, Streptococcus troglodytae, Streptococcus uberis, Streptococcus urinalis, Streptococcus varani, Streptococcus vestibularis, Streptococcus viridans and Streptococcus xiaochunlingii.

[0130] An even further embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acid sequence originating from Haemophilus influenzae.

[0131] Yet another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acid sequence originating from non-typeable Haemophilus influenzae (NTHi).

[0132] Beyond being found in bacteria, EF-Tu also exists in mammalian mitochondria. Accordingly, mammalian derived EF-Tu may be used as targeting moiety in the immunogenic construct instead of EF-Tu derived from bacteria. For targeting moieties based on mammalian EF-Tu it is preferred that the EF-Tu is not derived from human eukaryotic cells to reduce the risk of unwanted off-target toxicity through an immune response towards self-mitochondrial EF-Tu.

[0133] Thus, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acids sequence originating from a mammal.

[0134] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acids sequence originating from a non-human mammal.

[0135] The dual-purpose targeting moiety is capable of both delivering the antigen to the immune cells and act as an adjuvant by activating the immune system to promote long-lasting memory through cross-presentation of the antigen to T cells. The targeting moiety comprises bacterial proteins, such as EF-Tu, LDH, PF, or single domains, or shorter fragments (peptides) thereof that can both target and activate immune cells through interaction with PRR expressed on the immune cells. These bacterial proteins are capable of interacting directly with immune cells and therefore facilitate an enhanced immune response to any vaccine antigen guided by the immunogenic construct.

[0136] EF-Tu is an essential bacterial elongation factor that is highly conserved across various bacterial species and functions in polypeptide elongation by transporting aminoacyl transfer RNAs to the ribosomal A-site. Antibodies raised against recombinant EF-Tu from non-typeable Haemophilus influenzae (NTHi) recognize surface-exposed EF-Tu on a wide range of bacterial species, highlighting the sequence and structural similarity between different species. The protein is monomeric and consists of three structural domains, referred to as domains I, II and III. Domain I is located N-terminal and is a GTP-binding domain. Domains II and III are oligonucleotide-binding domains.

[0137] LDH is a metabolic enzyme that catalyses the conversion of L-lactase to pyruvate and back and has a crucial function in the respiratory chain. LDH is a conserved surface- exposed protein in various bacterial species, including NTHi where it binds to laminin. Deletion of the LDH gene in Streptococcus pyogenes has been shown to lead to a hypovirulent phenotype.

[0138] PF is an immunogenic, conserved surface-exposed protein, including NTHi and is an important virulence factor interacting with laminin. PF is a bacterial metal-binding receptor belonging to the adenosine triphosphate-binding cassette (ABC) transporter family. EF-Tu, LDH and PF can interact with the human immune system through binding to PRRs, such as Dectin-1.

[0139] Accordingly, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises a protein or polypeptide selected from the group consisting of elongation factor thermo unstable (EF-Tu), L-lactate dehydrogenase (LDH), Protein F (PF), or single domains, or shorter fragments thereof.

[0140] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acid sequence selected from the group consisting of: a) SEQ ID NO: 1 (EF-Tu domain I), SEQ ID NO:2 (EF-Tu domain II), SEQ ID NO:3 (EF-Tu domain III), and combinations thereof, b) SEQ ID NO:4 (LDH), c) SEQ ID NO:5 (PF), and d) an amino acid sequence having at least 90% sequence identity to the full- length sequence of any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, and combinations thereof, or SEQ ID NO:4, or SEQ ID NO: 5.

[0141] A further embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acid sequence selected from the group consisting of: a) SEQ ID NO: 1 (EF-Tu domain I), b) SEQ ID NO:4 (LDH), c) SEQ ID NO:5 (PF), and d) an amino acid sequence having at least 90% sequence identity to the full- length sequence of any one of SEQ ID NO: 1, SEQ ID NO:4, or SEQ ID NO:5.

[0142] Immunogenic constructs comprising EF-Tu domain I has been identified to yield great interactions with antigen-presenting cells. Without being bound by theory, it is considered that this domain comprises epitopes important for recognition and interaction with pattern recognition receptors (PRRs) located on the surface of antigen- presenting cells. Therefore, EF-Tu domain I serves as a relevant protein for activating the immune system.

[0143] Thus, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acid sequence selected from SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity to the full-length sequence of SEQ ID NO: 1.

[0144] Variants of the immunogenic construct may comprise additional domains to broaden the recognition of EF-Tu by antigen-presenting cells. Depending on the application and antigen of the immunogenic construct certain variants of the targeting moiety may be preferred to achieve an optimal immune response. Therefore, the targeting moiety could also include EF-Tu domain II and / or domain III or the full-length protein.

[0145] Thus, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises: a) an amino acid sequence selected from SEQ ID NO:2 and / or SEQ ID NO:3, or b) an amino acid sequence having at least 90% sequence identity to the full- length sequence of SEQ ID NO:2 and / or SEQ ID NO:3.

[0146] Another embodiment of the present invention relates to the immunogenic construct as described here, wherein the targeting moiety comprises SEQ ID NO: 1 and SEQ ID NO:2.

[0147] A further embodiment of the present invention relates to the immunogenic construct as described here, wherein the targeting moiety comprises SEQ ID NO: 1 and SEQ ID NO:3.

[0148] A still further embodiment of the present invention relates to the immunogenic construct as described here, wherein the targeting moiety comprises SEQ ID NO:2 and SEQ ID NO:3.

[0149] Yet another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acid sequence selected from SEQ ID NO:6 (full-length EF-Tu) or an amino acid sequence having at least 90% sequence identity to the full-length sequence of SEQ ID NO:6.

[0150] Immunogenic constructs comprising a targeting moiety based on the bacterial proteins LDH or PF have also been demonstrated to yield favourable delivery and adjuvating properties. Their utility for inclusion as component in a vaccine is therefore contemplated.

[0151] Thus, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acid sequence selected from SEQ ID NO:4 (full-length LDH) or an amino acid sequence having at least 90% sequence identity to the full-length sequence of SEQ ID NO:4.

[0152] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acid sequence selected from SEQ ID NO: 5 (full-length PF) or an amino acid sequence having at least 90% sequence identity to the full-length sequence of SEQ ID NO: 5.

[0153] It is to be understood that the amino acid sequence(s) of the targeting moiety may have a higher sequence identity with the sequences to which they refer (SEQ ID NOs: 1- 6). Thus, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.8%, such as at least 99.9% sequence identity to the full-length sequence of any one of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, and combinations thereof, or SEQ ID NO:4, or SEQ ID NO:5.

[0154] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety comprises an amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.5%, such as at least 99.8%, such as at least 99.9% sequence identity to the full-length sequence of SEQ ID NO:6.

[0155] Parts of the domains of EF-Tu, LDH and PF may encompass particularly favourable immunogenic epitopes. Thus, the targeting moiety may comprise fragments of the bacterial proteins, such as domains I, II and / or III of EF-Tu. Additionally, without being bound by theory, it is contemplated that reduction of the size of the targeting moiety may reduce any steric hindrance that could potentially negatively impact the interaction with the immune cell. Moreover, any unwanted interaction between the targeting moiety and the antigenic moiety may be mitigated by careful selection of domain fragments. Depending on the choice of antigenic moiety, it may consequently be favoured to design immunogenic constructs comprising fragments of the EF-Tu, LDH and / or PF domains.

[0156] Therefore, an embodiment of the present invention relates to an immunogenic construct as described herein, wherein the targeting moiety comprises fragments of an amino acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0157] The antigenic moiety of the immunogenic construct is a determinant characteristic for a targeted pathogen, i.e. it should be specific for the pathogen in order to give rise to a specific immune response. In general, the antigenic moiety comprises antigens which may ideally be selected from proteins, peptides or polypeptides unique to the pathogen. They may for instance be located on the surface of the pathogen, such as in the case of a viral surface protein. The antigen(s) may be selected to target a specific function of the disease, e.g. a protein that is involved in a crucial mechanism controlling the survival of the pathogen in the host or spread / metastases of cancerous disease to other tissue. This may be a beneficial strategy to effectively eliminating the pathogen, e.g. by generation of neutralizing antibodies targeting key functions of the pathogen.

[0158] The antigenic moiety of the immunogenic construct may comprise only a single antigen or several separate antigens. By collecting several highly immunogenic epitopes in a single immunogenic construct, the immune response may be enhanced. The individual immunogenic epitopes of the antigen can be connected by linker moieties, such as linker sequences or linker peptides.

[0159] Therefore, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein said antigenic moiety comprises one or more antigens.

[0160] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein said one or more antigens are selected from the group consisting of proteins, peptides, polypeptides, and nucleic acids.

[0161] A further embodiment of the present invention relates to the immunogenic construct as described herein, wherein said one or more antigens are connected by linker moieties.

[0162] Despite the fact that all antigens (in the term's common sense) are recognized by specific lymphocytes or by antibodies, not every antigen can evoke an immune response. For the purposes herein, it is to be understood that the antigen is preferably immunogenic and capable of initiating an adaptive immune response to induce longterm memory. However, for some variants of the immunogenic construct, such as when the antigenic moiety comprises mRNA, it may be preferred that the antigenic moiety is not immunogenic prior to translation of the mRNA.

[0163] The antigen of the immunogenic construct is not limited to any specific origin. The technology is based on the targeting moiety and its dual role as targeting and adjuvantation entity, and may therefore in principle as concept be used to prepare immunogenic constructs from any antigen. For example, the antigen may be derived from either a virus or cancer cells to allow for high flexibility and applicability of the immunogenic construct in various vaccination strategies, including therapeutic and / or prophylactic vaccines. Thus, the antigen may originate from any organism against which it is desired to provide a vaccine. This could be a microorganism, such as a virus or bacterium. Alternatively, the antigen could be human, for example if the antigen is a cancer-specific antigen.

[0164] Thus, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein said one or more antigens originate from a virus, a bacterium or a mammal.

[0165] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein said mammal is a human.

[0166] A further embodiment of the present invention relates to the immunogenic construct as described herein, wherein said one or more antigens are cancer-specific antigens or virus-specific antigens.

[0167] A still further embodiment of the present invention relates to the immunogenic construct as described herein, wherein said one or more antigens are bacteriumspecific antigens.

[0168] It will be appreciated that the type of cancers from which the antigen may originate include solid and liquid cancers. Preferably, the antigen is derived from a primary cancer.

[0169] Therefore, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein said cancer-specific antigens originate from a cancer selected from the group consisting of ovarian carcinomas, female genital tract malignancy, breast carcinomas, prostatic adenocarcinoma, pancreatic adenocarcinoma, lung cancer (e.g. squamous cell or bronchial cell carcinoma), hepatocellular carcinoma, colorectal adenocarcinoma, gastric adenocarcinoma, cholangiocarcinoma, bladder cancer, tumours of the central nervous system, and malignancies related to the lymphatic system or bone marrow including leukemia, multiple myeloma or lymphoma.

[0170] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein said cancer-specific antigens are from a primary cancer. Several cancer types give rise to tumour-associated antigens, which are self-antigens overexpressed in tumour cells. These tumour-associated antigens are presented in MHC class I molecules on the surface of the tumour and can be exploited as targets for the immune system to eliminate the tumour. Accordingly, immunogenic construct comprising a tumour-associated antigen, such as an antigen expressed on a solid tumour, are thought to be beneficial.

[0171] Thus, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein said cancer-specific antigens are tumour-specific antigens, preferably solid tumour-specific antigens.

[0172] To overcome the common obstacle with tolerance mechanisms and risk of developing self-antigen-reactive T-cells, when using tumour-associated antigens in cancer vaccines, neo-epitopes is a relevant target to consider for solving this issue. Neoepitopes are unique epitopes that evolve from tumour-specific mutations, which makes them an exclusively tumour-specific target not expressed on healthy tissue and are recognized as foreign to the immune system. Without being bound by theory, patients with high mutational burden tumours have more predicted neo-epitopes and consequently more tumour-reactive T cells and therefore respond better to cancer immunotherapy treatment, compared to patient with low mutational burden tumours.

[0173] Therefore, the implementation of neo-epitopes in the immunogenic construct described herein is relevant for increasing the therapeutic outcome of cancer vaccines and leading to efficient anti-tumour immunity. In addition, neo-epitopes present limited risk of activation of low-avidity T-cells and autoimmune reactions because expression of these neo-epitopes is limited to tumours exclusively and not healthy tissue.

[0174] Therefore, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the tumour-specific antigens are neo-epitope antigens, preferably from a high mutational burden tumour or metastases.

[0175] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the tumour-specific antigens are neo-epitope antigens from a low mutational burden tumour.

[0176] As previously described, the immunogenic constructs described herein is in principle a platform technology that may be used to immunize against a broad range of diseases. Accordingly, the antigen comprised in the immunogenic construct can originate from a broad selection of pathogenic viruses.

[0177] Therefore, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein said virus-specific antigens originate from a virus selected from the group consisting of Adenovirus, SARS-CoV-1 virus, SARS-CoV- 2 virus, Corona virus, Norovirus, Papillomavirus, Polyomavirus, Herpes simplex virus (HSV), Alpha herpesvirinae human herpesvirus 1, 2, 3, Human gamma herpesvirus 4, 8 (Kaposi sarcoma), Betaherpesvirinae 5, 6, 7, Varicella zoster virus (VZV), Epstein- Barr virus (EBV), Cytomegalovirus (CMV), Picornavirus, Enterovirus, Rhinovirus, Hepatovirus, Cardiovirus, Aphthovirus, Coxsackie virus, Echovirus, Paramyxovirus, Measles virus, Parainfluenza virus, Mumps virus, Respiratory syncytial virus (RSV), Metapneumovirus, Nipah virus, Hendra viruses, Orthomyxoviruses, Influenza virus, Rhabdovirus, Filovirus, Marburg virus, Ebola virus, Bornavirus, Rabies virus, Reovirus, Rotavirus, Coltivirus, Orbivirus, Norwalk virus, Calicivirus, Rubella virus, Togavirus, Flavivirus, Arbovirus, Bunyavirus, Arena virus, Poxvirus, Parvovirus, Retrovirus, Human immunodeficiency virus (HIV), Human-T cell leukemia virus, and Hepatitis A, B, C, D, G, and E viruses, and combinations thereof.

[0178] Immunogenic constructs comprising antigens derived from SARS-CoV-2 virus have shown promising results and are thus preferred. The spike (S) protein and nucleocapsid (N) protein of SARS-CoV-2 virus have already garnered must interest in a variety of vaccines for battling the COVID-19 pandemic. These proteins also present themselves as suitable antigens for the immunogenic construct described herein. Thus, SARS-CoV- 2 virus antigens include, but are not limited to, the spike (S) protein and nucleocapsid (N) protein.

[0179] Thus, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the virus-specific antigens originate from SARS-CoV-2 virus.

[0180] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the SARS-CoV-2 antigen is selected from the spike (S) protein, the nucleocapsid (N) protein, or fragments thereof.

[0181] A further embodiment of the present invention relates to the immunogenic construct as described herein, wherein said spike (S) protein comprises an amino acid sequence represented by SEQ ID N0:9 (full length spike (S) protein), or an amino acid sequence having at least 90% sequence identity to the full-length sequence of SEQ ID NO:9.

[0182] Yet another embodiment of the present invention relates to the immunogenic construct as described herein, wherein said nucleocapsid (N) protein comprises an amino acid sequence represented by SEQ ID NO: 10 (full length nucleocapsid (N) protein), or an amino acid sequence having at least 90% sequence identity to the full-length sequence of SEQ ID NO: 10.

[0183] Truncated versions of SARS-CoV-2 spike (S) protein and nucleocapsid (N) protein can be implemented as antigen in the immunogenic construct, providing a short version of the full-length amino acid sequence while still being sufficiently immunogenic to effectively activate the immune system.

[0184] Thus, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein said fragment of the spike (S) protein comprises an amino acid sequence represented by SEQ ID NO: 11 and said fragment of the nucleocapsid (N) protein comprises an amino acid sequence represented by SEQ ID NO: 12.

[0185] The antigen(s) may be implemented in the immunogenic construct in various ways. In one variant, the antigenic moiety comprising the antigen(s) is part of the same protein as the targeting moiety, i.e. be designed as a fusion protein. Such fusion proteins can be produced through conventional recombinant expression.

[0186] However, the antigenic moiety is not limited to consisting of a single or multiple antigens. It may also comprise additional components that may help the immunogenic construct in inducing a strong immune response which is relevant for vaccine purposes. In particular, the antigenic moiety may comprise a vehicle that can protect the antigen(s) from degradation or enhance the effect of the antigen(s) when presented to immune cells. One of the purposes of formulating the antigen(s), such as nucleic acid material, in a delivery vehicle is to protect the foreign antigen(s) from opsonization or reticuloendothelial clearance when introduced in a recipient. Protection may be achieved by physically shielding the antigen(s) from harmful substances and / or the immune system of the host. Lipid formulations can entrap, complex, or encapsulate the antigen(s) in the interior of the lipid particles and thereby protect the material from hostile environments. A non-limiting example of a suitable vehicle could be a lipid- based particle, such as a lipid nanoparticle (LNP) that carries nucleic acids. This type of antigenic particle was the basis of the very successful Covid-19 mRNA vaccines. For delivery of mRNA a protective vehicle is crucial because the mRNA is susceptible to degradation by ribonucleases in the extracellular environment.

[0187] An embodiment of the present invention relates to the immunogenic constructs as described herein, wherein the antigenic moiety comprises a particle.

[0188] A further embodiment of the present invention relates to the immunogenic constructs as described herein, wherein the particle is selected from the group consisting of a lipid-based particle, a polymer-based particle, a peptide-based particle, a bead-based particle and a virus-like particle.

[0189] A still further embodiment of the present invention relates to the immunogenic constructs as described herein, wherein the particle is a lipid-based particle.

[0190] A preferred embodiment of the present invention relates to the immunogenic constructs as described herein, wherein the particle is a lipid nanoparticle (LNP).

[0191] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the particle comprises one or more nucleic acids.

[0192] A further preferred embodiment of the present invention relates to the immunogenic construct as described herein, wherein said one or more nucleic acids are encapsulated or trapped in the particle.

[0193] The nucleic acid is not limited to any specific type of nucleic acid material but may be designed for a variety of applications. Notably, nucleic acid-based medicines differentiate themselves from conventional treatments in that they can target the genetic design and underlying cause for disease rather than induce transient therapeutic effects. Thus, nucleic acid-based medicines have the potential to provide long-lasting and / or curative effects, such as immunization of a subject, replenishment of a deficient gene / protein or silencing of undesirable genes. This may be achieved through different modes of action and include gene inhibition, gene addition, gene replacement or gene editing.

[0194] Therefore, an embodiment of the present invention relates to the immunogenic constructs as described herein, wherein said one or more nucleic acids are selected from ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and analogues thereof. Another embodiment of the present invention relates to the immunogenic constructs as described herein, wherein said one or more nucleic acids are selected from the group consisting of messenger RIMA (mRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), single stranded DNA (ssDNA), and double stranded DNA (dsDNA).

[0195] A preferred embodiment of the present invention relates to the immunogenic constructs as described herein, wherein said one or more nucleic acids are mRNA.

[0196] The targeting moiety is attached to the outer surface of the particle so that it can interact with its target. The attachment may be covalent through a linker or the like. However, the targeting moiety is preferably coated onto the particle. Coating is an easy process for preparing the final immunogenic construct wherein the targeting moiety is simply added to the particle, typically in solution or alternatively by spray-coating a preparation of particles. The amount of targeting moiety added to the particle can be adjusted to vary the occupation of targeting moieties on the particle surface. Preferably, the targeting moiety is added in an amount to saturate the particle surface and create a homogenous layer of targeting moieties, such as proteins or fragments thereof. Without being bound by theory, it is contemplated that creation of a homogenous layer of targeting moiety enhance the interaction and / or adjuvating effect of the immunogenic construct with the target immune cell.

[0197] Accordingly, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety is attached to the outer surface of the particle.

[0198] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety is coated or covalently bound to the surface of the particle.

[0199] A preferred embodiment of the present invention relates to the immunogenic construct as described herein, wherein the targeting moiety is coated onto the surface of the particle.

[0200] Electrostatic interaction is forces between nearby electric charged moieties and is either an attractive or repulsive interaction between the moieties. Electrostatic interaction may be used as a method of coating proteins, peptides, polypeptides or nucleic acid on to a lipid nanoparticle surface in the present invention. Thus, an embodiment of the present invention relates to the immunogenic constructs as described herein, wherein the targeting moiety is coated onto the surface of the particle by electrostatic interactions.

[0201] Alternatively, the targeting moiety may be covalently immobilized on the outer surface of the particle. This may be accomplished by known techniques in the art, such as by thiol-maleimide chemistry. The thiol moiety may be positioned on a terminal cysteine of the targeting moiety and the maleimide moiety can be provided on the surface of the particle through functionalization of components of the particle. In the case of a LNP, the maleimide-moiety may be providing by functionalization of lipidated polyethylene glycol (PEG) incorporated in the LNP.

[0202] Thus, an embodiment of the present invention relates to the immunogenic constructs as described herein, wherein the targeting moiety is attached to the outer surface of the particle via a thiol-maleimide covalent coupling.

[0203] Another embodiment of the present invention relates to the immunogenic constructs as described herein, wherein a terminal cysteine of the targeting moiety is covalently coupled to a maleimide moiety on the outer surface of the particle.

[0204] An alternative variant of the immunogenic construct pertains to a fusion protein. Fusion proteins comprising a targeting moiety and an antigenic moiety has been demonstrated herein to yield enhanced immune responses that are greatly beneficial for vaccine development. While fusion proteins may be devised with antigenic moiety and targeting moiety positioned in the immediate vicinity of each other, for many practical purposes it is preferred to connect the antigenic moiety and targeting moiety by a linker moiety. The linker moiety may be attached to the C-terminal part of the targeting moiety and the N-terminal part of the antigen sequence of the antigenic moiety, although it is also possible to interchange the position of the antigenic moiety and targeting moiety.

[0205] Thus, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the immunogenic construct is a fusion protein.

[0206] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein no linker moiety is included.

[0207] A further embodiment of the present invention relates to the immunogenic construct as described herein further comprising a linker moiety. The linker moiety may be a nucleotide- or peptide linker, or a crosslinking reagent for chemically crosslinking the antigenic moiety to the targeting moiety. Such chemical crosslinking may be achieved through binding to functional groups present on the antigen(s) of the antigenic moiety and the targeting moiety. Accordingly, linker moieties include crosslinking reagents containing two or more reactive ends capable of chemically attaching to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules, herein the antigen(s) of the antigenic moiety and the targeting moiety.

[0208] Thus, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the linker moiety is a crosslinking reagent.

[0209] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the crosslinking reagent is selected from the group of NHS esters, imidoesters, NHS-haloacetyls and NHS-maleimides.

[0210] It may be advantageous to use Succinimidyl S-acetylthioacetate (SATA) as the crosslinking reagent as described in (Lin Y, Cheng Q, Wei T. Biophys Rep. 2023).

[0211] Therefore, one embodiment of the present invention relates to the immunogenic construct as described herein, wherein the proteins EF-Tu, LDH or PF are covalently attached to the surface of lipid nanoparticles using SATA as a crosslinking reagent.

[0212] The process of covalently attaching the proteins EF-Tu, LDH or PF to the surface of lipid nanoparticles using SATA is initiated with the modification of the protein to introduce protected thiol groups. Specifically, SATA, which contains an N-hydroxysuccinimide (NHS) ester moiety and an S-acetylated thiol group, is reacted with primary amine groups present on lysine residues of the protein. The NHS ester selectively reacts with these amine functionalities under mild aqueous conditions, forming stable amide linkages and yielding S-acetylthioacetylated proteins.

[0213] Following the conjugation of SATA to the protein, the acetyl-protecting groups are removed to expose free thiol groups. This deacetylation is achieved by treatment with hydroxylamine hydrochloride, which cleaves the S-acetyl moiety, thereby generating reactive thiol (-SH) groups on the protein without compromising protein structure or function. Subsequently, the thiolated protein is conjugated to lipid nanoparticles that incorporate maleimide-functionalized lipids, such as DSPE-PEG-maleimide. The free thiol groups on the protein undergo a chemoselective Michael addition reaction with the maleimide groups on the lipid molecules, forming stable thioether bonds. This reaction results in the covalent anchoring of the protein to the lipid nanoparticle surface via a polyethylene glycol (PEG) linker, which serves to enhance steric accessibility and reduce non-specific interactions.

[0214] The resultant protein-functionalized lipid nanoparticles retain the biological activity of the conjugated protein while enabling targeted delivery, extended circulation time, or enhanced cellular uptake, depending on the intended application.

[0215] The described method provides a robust and versatile platform for the generation of protein-decorated lipid-based nanocarriers suitable for therapeutic, diagnostic, or vaccine delivery purposes.

[0216] Yet another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the linker moiety is positioned in between the targeting moiety and the antigenic moiety.

[0217] A further embodiment of the present invention relates to the immunogenic construct as described herein, wherein the linker is attached to the C-terminal of the targeting moiety and the N-terminal of the antigen(s) of the antigenic moiety.

[0218] It will be appreciated that the linker moiety in the fusion protein can be a peptide linker of various lengths and amino acid content depending on the design of the fusion protein, including considerations as to the size of the fusion protein and nature of the antigen(s) of the antigenic moiety, and targeting moiety and any potential interactions therewith or in between.

[0219] Thus, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the linker moiety is a peptide linker.

[0220] Glycine and serine are preferred amino acids for the linker moiety as these residues contain no side chains and as such will not play any role in protein secondary structure formations but keep the linker flexible. Moreover, serine residues improve solubility and reduce aggregation issues due to its polar nature. Other linkers of interest include, but are not limited to, EAAAK, GPGPG and KK. Therefore, an embodiment of the present invention relates to the immunogenic construct as described herein, wherein the peptide linker is selected from the group consisting of (GGGGS)n, (GGGS)n, SGGGSGGGS and AAY, wherein n is an integer selected from 1, 2 or 3.

[0221] Another embodiment of the present invention relates to the immunogenic construct as described herein, wherein the peptide linker is a (GGGGS)2 linker.

[0222] The targeting moiety of the immunogenic construct entails a dual effect by firstly acting to deliver a specific antigen to immune cells, e.g. to facilitate a specific immune response upon MHO presentation of antigenic peptide. Secondly, the targeting moiety itself induces an adjuvant effect upon interacting with PRRs, such as Dectin-1. Compounds that can successfully enhance adaptive immune responses and at the same time are well tolerated, safe and non-toxic to the host remain highly elusive. The immune response and protection can be further enhanced by adjuvants targeting PRRs, expressed by immune cells of the innate immune system. Therefore, the dual effect of the targeting moiety makes the immunogenic construct advantageous to use in vaccination strategies.

[0223] Accordingly, an aspect of the present invention relates to a vaccine or immunogenic composition comprising an immunogenic construct as described herein.

[0224] It is contemplated that the efficiency of the vaccine or immunogenic composition may be further supported by adding additional traditional adjuvants to the vaccine or immunogenic composition.

[0225] Therefore, another embodiment of the present invention relates to the vaccine or immunogenic composition as described herein, wherein the vaccine or immunogenic composition further comprises one or more adjuvants.

[0226] A further embodiment of the present invention relates to the vaccine or immunogenic composition as described herein, wherein said one or more adjuvants are selected from the group consisting of Freund 's Adjuvants, 3 De-O-acylated monophosphoryl lipid A (3D-MPL), QS21, unmethylated CpG sequences, non-toxic oil-in-water emulsions and aluminium salts.

[0227] The vaccine or immunogenic composition may function with conventional pharmaceutically acceptable carriers. Thus, the immunogenic construct may be provided in any pharmaceutically acceptable carrier and is not limited to any particular one.

[0228] Thus, an even further embodiment of the present invention relates to the vaccine or immunogenic composition as described herein, wherein the vaccine or immunogenic composition further comprises a pharmaceutically acceptable carrier.

[0229] The immunogenic construct, vaccine or immunological composition described herein, can be used as a medicament, e.g. in the treatment or prevention of infection or diseases, such as viral or bacterial infection or cancer. The treatment may be either therapeutic or prophylactic, such as in the case of therapeutic vaccines and prophylactic vaccines.

[0230] Therefore, an aspect of the present invention relates to the immunogenic construct as described herein or the vaccine or immunogenic composition as described herein for use as a medicament.

[0231] The immunogenic construct, vaccine or immunological composition may be used for vaccination, to immunize a subject. Immunization may be against any pathogen for which a suitable antigen can be identified for inclusion in the antigenic moiety. Pathogens may e.g. originate from virus or bacterium. The vaccine may also be utilised for immunizing a subject affected by viral infection or primary cancer as a treatment or inhibition of the disease, or to immunize a healthy subject to prevent the occurrence of viral infection or primary cancer, or to immunize a subject in risk of developing viral infections or primary cancer.

[0232] Accordingly, an embodiment of the present invention relates to the immunogenic construct as described herein or the vaccine or immunogenic composition as described herein for use in vaccination or immunization of a subject against a pathogen.

[0233] Another embodiment of the present invention relates to the immunogenic construct as described herein or the vaccine or immunogenic composition as described herein for use in vaccination or immunization, wherein the pathogen is selected from a virus or a bacterium.

[0234] A further embodiment of the present invention relates to the immunogenic construct as described herein or the vaccine or immunogenic composition as described herein for use in vaccination or immunization of a subject against viral and / or bacterial infections and / or cancer.

[0235] A still further embodiment of the present invention relates to the immunogenic construct, vaccine or immunogenic composition for use as described herein, wherein said immunogenic construct, vaccine or immunogenic composition is administered for prevention, inhibition or treatment of a viral or bacterial infection or cancer.

[0236] Diseases of particular interest are viral infections caused by SARS-CoV-2 as well as non-metastatic cancers. Humans suffering from any such diseases may benefit from treatment or immunization with the immunogenic constructs, vaccines and immunological compositions described herein.

[0237] Therefore, an embodiment of the present invention relates to the immunogenic construct, vaccine or immunogenic composition for use as described herein, wherein the viral infection is caused by SARS-CoV-2.

[0238] Another embodiment of the present invention relates to the immunogenic construct, vaccine or immunogenic composition for use as described herein, wherein the cancer is a primary cancer (non-metastatic cancer).

[0239] Still, a further embodiment of the present invention relates to the immunogenic construct, vaccine or immunogenic composition for use as described herein, wherein the subject is a mammal.

[0240] An even further embodiment of the present invention relates to the immunogenic construct, vaccine or immunogenic composition for use as described herein, wherein the mammal is a human.

[0241] Various administration routes can be used in vaccination with the immunogenic construct, vaccine or immunological composition described herein. The route of administration may be selected to accommodate most appropriately the specific type of vaccine and antigen to increase the likelihood of acquiring long-term immunity as well as take into account any personal or local preferences. Considerations may for instance be given to whether the administration is for children, adolescents or adults, or if the vaccine is to be distributed in areas wherein powder form is preferred. Accordingly, an embodiment of the present invention relates to the immunogenic construct, vaccine or immunogenic composition for use as described herein, wherein the route of administration is selected from the group consisting of parenteral, intravenous, subcutaneous, intramuscular, intradermal, intranasal and oral.

[0242] The immunogenic construct, vaccine or immunological composition described herein is provided in an amount effective to treat, inhibit or prevent disease, such as viral or bacterial infection or cancer. This can be achieved either by vaccination using a single dose or administration of multiple booster doses on top of the initial vaccine dose.

[0243] Thus, another embodiment of the present invention relates to the immunogenic construct, vaccine or immunogenic composition for use as described herein, wherein said immunogenic construct, vaccine or immunogenic composition is administered in a single dose or in multiple doses separated by a period of time.

[0244] As laid out herein, the immunogenic construct, vaccine or immunogenic composition of the present invention may be used in a method of treating a subject in need thereof, e.g. a subject that suffers from viral or bacterial infection or cancer.

[0245] Thus, an aspect of the present invention relates to a method of treatment comprising administration of an immunogenic construct as described herein or a vaccine or immunogenic composition as described herein, to a subject in need thereof.

[0246] Another embodiment of the present invention relates to the method of treatment as described herein, wherein the subject suffers from a viral disease or a cancer disease.

[0247] The findings that EF-Tu, LDH, PF and domains and fragments thereof can be used to guide and significantly adjuvate the immune response of an antigen that is the source of an adaptive immune response open the possibility to improve immune responses of many known antigens that are already being utilised in vaccine strategies. By incorporation of such antigens in the immunogenic construct described herein, strong and proven antigens may be repurposed in a new and improved format.

[0248] Accordingly, an aspect of the present invention relates to use of a targeting moiety for targeting an antigenic moiety to an immune cell, wherein said targeting moiety comprises a protein or polypeptide selected from the group consisting of elongation factor thermo unstable (EF-Tu), L-lactate dehydrogenase (LDH), Protein F (PF), or single domains, or shorter fragments thereof. The targeting moiety may also be used as a stand-alone adjuvant that activates antigen-presenting cells through receptors on their surface. This activation amongst others initiates upregulation of co-stimulatory proteins essential for priming naive T cells, thereby facilitating an enhanced immune response.

[0249] Therefore, an aspect of the present invention relates to use of a protein, peptide or polypeptide comprising an amino acid sequence selected from the group consisting of: a) SEQ ID NO: 1 (EF-Tu domain I), SEQ ID NO:2 (EF-Tu domain II), SEQ ID NO:3 (EF-Tu domain 3), and combinations thereof, b) SEQ ID NO:4 (LDH), c) SEQ ID NO:5 (PF), and d) an amino acid sequence having at least 90% sequence identity to the full- length sequence of any one of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, and combinations thereof, or SEQ ID NO:4, or SEQ ID NO: 5, as an adjuvant.

[0250] Another aspect of the present invention relates to an adjuvant comprising an amino acid sequence selected from the group consisting of: a) SEQ ID NO: 1 (EF-Tu domain I), SEQ ID NO:2 (EF-Tu domain II), SEQ ID NO:3 (EF-Tu domain 3), and combinations thereof, b) SEQ ID NO:4 (LDH), c) SEQ ID NO:5 (PF), and d) an amino acid sequence having at least 90% sequence identity to the full- length sequence of any one of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, and combinations thereof, or SEQ ID NO: 4, or SEQ ID NO: 5.

[0251] A further aspect of the present invention relates to a vaccine or immunogenic composition comprising an adjuvant as described herein.

[0252] It is to be understood that all aspect relating to the adjuvant when described as a stand-alone adjuvant, or as part or a vaccine or immunogenic composition, may be provided in the same variants as described for the targeting moiety.

[0253] The immunogenic constructs, vaccines and immunogenic compositions described herein may be provided in a kit. This is convenient for the end user and allows any special instructions to be enclosed. Therefore, an aspect of the present invention relates to a kit comprising: i) an immunogenic construct as described herein or a vaccine or immunogenic composition as described herein, and ii) optionally, instructions for use.

[0254] Immunogenic constructs in the form of fusion proteins as described herein may be produced recombinantly by introduction of an expression vector comprising a nucleic acid encoding the fusion protein or its components in a suitable host cell. The vector may be a plasmid that is introduced into a prokaryotic or eukaryotic cell.

[0255] Accordingly, an aspect of the present invention relates to a nucleic acid comprising a sequence encoding a fusion protein as described herein.

[0256] Another aspect of the present invention relates to a recombinant expression vector comprising a nucleic acid as described herein operatively linked to one or more control sequences suitable for directing the production of the fusion protein in a suitable host.

[0257] Production of the fusion protein may be performed by providing nucleic acid(s) encoding at least the targeting moiety and antigenic moiety. It is to be understood that all embodiments described in the context of the targeting moiety and antigenic moiety as well as for the nucleic acids, vectors and host cells also applies to the method for providing the fusion protein.

[0258] Thus, an aspect of the present invention relates to a method for providing an immunogenic construct as described herein, said method comprising the steps of: i) provision of one or more nucleic acids at least encoding: a) the targeting moiety, and b) the antigenic moiety; and ii) expression of said one or more nucleic acids, thereby providing the immunogenic construct as described herein.

[0259] The fusion protein may be expressed either as a single construct or as separate constructs wherein the antigen and targeting moiety parts are connected via a linker moiety subsequent to expression. Expression of the fusion protein as a single construct is the preferred method, but it is contemplated that expression of separate constructs and subsequent connection may be preferred for fusion proteins for personalized medicine. The immunogenic construct, vaccine or immunogenic composition may be designed for personalized treatment. To this end, a tumour sample from a human may be obtained and screened for the presence of tumour-specific antigens that can serve as a suitable antigen in the immunogenic construct. Such individualized screening may identify so- called neo-epitopes, i.e. unique antigenic epitopes created from tumour specific mutations, that can be used for very specific targeting of the immune system against tumour diseased tissue. If any neo-epitopes are identified, their sequences may be used to produce the antigen part of an immunogenic construct specifically designed to treat the individual from which the tumour sample was obtained.

[0260] Therefore, another embodiment of the present invention relates to the method as described herein, wherein step (i) is preceded by the following steps: i) obtaining a sample from a subject, ii) identifying an antigen within said sample, and iii) obtaining the nucleic acid sequence of said antigen.

[0261] Thus, another embodiment of the present invention relates to the method as described herein, wherein the antigen within said sample is a neo-epitope antigen.

[0262] Of particular importance to personalized treatment is high mutational burden types of tumours, such as non-small cell lung cancer, melanoma, and renal cell cancer.

[0263] However, the sample may in principle be obtained from an individual suffering from any type of cancer. Thus neo-epitopes may be derived from cancers including, but not limited to, ovarian carcinomas, female genital tract malignancy, breast carcinomas, prostatic adenocarcinoma, pancreatic adenocarcinoma, lung cancer (e.g. squamous cell or bronchial cell carcinoma), hepatocellular carcinoma, colorectal adenocarcinoma, gastric adenocarcinoma, cholangiocarcinoma, bladder cancer, tumours of the central nervous system, and malignancies related to the lymphatic system or bone marrow including leukemia, multiple myeloma or lymphoma.

[0264] It is appreciated that the immunogenic construct of the present invention may be obtained from a method as described herein in any of its variants.

[0265] Thus, an aspect of the present invention relates to an immunogenic construct as described herein obtained from the method as described herein. The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method for referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0266] The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0267] Preferences, options, and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options, and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the immunogenic construct, vaccine and immunological composition and all its features, which may readily be relayed to the method and adjuvant as described herein. Embodiments and features of the present invention are also outlined in the following items and also illustrated by the following non-limiting examples.

[0268] Items

[0269] 1. An immunogenic construct comprising a targeting moiety and an antigenic moiety.

[0270] 2. The immunogenic construct according to item 1, wherein the targeting moiety has affinity for antigen-presenting cells.

[0271] 3. The immunogenic construct according to any one of items 1 or 2, wherein the targeting moiety has affinity for a pattern recognition receptor (PRR). 4. The immunogenic construct according to any one of the preceding items, wherein the targeting moiety has affinity for Dectin-1.

[0272] 5. The immunogenic construct according to any one of the preceding items, wherein the targeting moiety has a binding affinity (I<D) for Dectin-1 in the range of about 1.0 x 10'9M to about 1.0 x 10'5M, such as about 5.0 x 10'8M to about 1.0 x 10'6M.

[0273] 6. The immunogenic construct according to any one of items 4 or 5, wherein said Dectin-1 comprises an amino acid sequence selected from SEQ ID NO:7 or SEQ ID NO:8, or an amino acid sequence having at least 90% sequence identity to the full- length sequence of SEQ ID NO:7 or SEQ ID NO:8.

[0274] 7. The immunogenic construct according to any one of the preceding items, wherein the targeting moiety comprises an amino acid sequence originating from a bacterium.

[0275] 8. The immunogenic construct according to any one of the preceding items, wherein the targeting moiety comprises an amino acid sequence originating from a bacterium genus selected from the group consisting of Aggregatibacter, Avibacterium, Enterobacter, Escherichia, Haemophilus, Gallibacterium, Brenneria, Caldithrix, Lactococcus, Listeria, Pasteurella, Porphyromonas, Rodentibacter, Salmonella, Shigella, Staphylococcus, and Streptococcus.

[0276] 9. The immunogenic construct according to any one of the preceding items, wherein the targeting moiety comprises an amino acid sequence originating from a bacterium species selected from the group consisting of Haemophilus influenzae, Escherichia coli, Streptococcus pneumoniae, Listeria monocytogenes, Aggregatibacter aphrophilus (Haemophilus aphrophilus), Aggregatibacter segnis, Avibacterium paragallinarum (Haemophilus gallinarum), Brenneria alni, Calditrichaeota bacterium, Enterobacter hormaechei, Escherichia albertii, Escherichia fergusonii, Gallibacterium anatis, Haemophilus aegyptius, Haemophilus haemolyticus, Haemophilus parainfluenzae, Haemophilus pittmaniae, Lactococcus chungangensis, Lactococcus hodotermopsidis, Lactococcus insecticola, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus reticulitermitis, Listeria booriae, Listeria cornellensis, Listeria fleischmannii, Listeria grandensis, Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria riparia, Listeria rocourtiae, Listeria seeligeri, Listeria weihenstephanensis, Listeriaceae bacterium, Pasteurella multocida, Porphyromonadaceae bacterium, Rodentibacter pneumotropicus, Salmonella enterica, Salmonella houtenae, Salmonella infantis, Salmonella typhimurium, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Streptococcus acidominimus, Streptococcus agalactiae, Streptococcus alactolyticus, Streptococcus anginosus, Streptococcus australis, Streptococcus azizii, Streptococcus bovimastitidis, Streptococcus canis, Streptococcus chenjunshii, Streptococcus chosunense, Streptococcus constellatus, Streptococcus criceti, Streptococcus cristatus, Streptococcus cuniculi, Streptococcus downei (Streptococcus sobrinus), Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equi subsp. equi, Streptococcus equi subsp. ruminatorum, Streptococcus equi subsp. Zooepidemicus, Streptococcus equinus (Streptococcus bovis), Streptococcus ferus, Streptococcus gallolyticus, Streptococcus gordonii, Streptococcus gwangjuense, Streptococcus halitosis, Streptococcus henryi, Streptococcus hyointestinalis, Streptococcus ictaluri, Streptococcus infantarius, Streptococcus infantis, Streptococcus iniae (Streptococcus shiloi), Streptococcus intermedius, Streptococcus lactarius, Streptococcus lutetiensis, Streptococcus macacae, Streptococcus macedonicus, Streptococcus massiliensis, Streptococcus merionis, Streptococcus milleri, Streptococcus minor, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus ovis, Streptococcus pantholopis, Streptococcus parasanguinis, Streptococcus parasuis, Streptococcus parauberis, Streptococcus pasteurianus, Streptococcus penaeicida, Streptococcus periodonticum, Streptococcus peroris, Streptococcus pharyngis, Streptococcus phocae, Streptococcus pluranimalium, Streptococcus porcinus, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus rubneri, Streptococcus ruminantium, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sinensis, Streptococcus sobrinus, Streptococcus suis, Streptococcus thermophilus, Streptococcus troglodytae, Streptococcus uberis, Streptococcus urinalis, Streptococcus varani, Streptococcus vestibularis, Streptococcus viridans and Streptococcus xiaochunlingii.

[0277] 10. The immunogenic construct according to any one of the preceding items, wherein the targeting moiety comprises an amino acid sequence originating from Haemophilus influenzae.

[0278] 11. The immunogenic construct according to any one of the preceding items, wherein the targeting moiety comprises a protein or polypeptide selected from the group consisting of elongation factor thermo unstable (EF-Tu), L-lactate dehydrogenase (LDH), Protein F (PF), or single domains, or shorter fragments thereof. Ila. The immunogenic construct according to any one of the preceding items, wherein the targeting moiety has affinity for a pattern recognition receptor (PRR), and wherein the targeting moiety comprises a protein or polypeptide selected from the group consisting of elongation factor thermo unstable (EF-Tu), L-lactate dehydrogenase (LDH), Protein F (PF), or single domains, or shorter fragments thereof.

[0279] 12. The immunogenic construct according to any one of the preceding items, wherein the targeting moiety comprises an amino acid sequence selected from the group consisting of: a) SEQ ID NO: 1 (EF-Tu domain I), SEQ ID NO:2 (EF-Tu domain II), SEQ ID NO:3 (EF-Tu domain III), and combinations thereof, b) SEQ ID NO:4 (LDH), c) SEQ ID NO:5 (PF), and d) an amino acid sequence having at least 90% sequence identity to the full- length sequence of any one of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, and combinations thereof, or SEQ ID NO:4, or SEQ ID NO: 5.

[0280] 13. The immunogenic construct according to any one of the preceding items, wherein the targeting moiety comprises an amino acid sequence selected from the group consisting of: a) SEQ ID NO: 1 (EF-Tu domain I), b) SEQ ID NO:4 (LDH), c) SEQ ID NO:5 (PF), and d) an amino acid sequence having at least 90% sequence identity to the full- length sequence of any one of SEQ ID NO: 1, SEQ ID NO:4, or SEQ ID NO:5.

[0281] 14. The immunogenic construct according to any one of the preceding items, wherein the targeting moiety comprises an amino acid sequence selected from SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity to the full-length sequence of SEQ ID NO: 1.

[0282] 15. The immunogenic construct according to any one of the preceding items, wherein the targeting moiety comprises an amino acid sequence selected from SEQ ID NO:6 (EF-Tu) or an amino acid sequence having at least 90% sequence identity to the full- length sequence of SEQ ID NO:6.

[0283] 16. The immunogenic construct according to any one of items 1-13, wherein the targeting moiety comprises an amino acid sequence selected from SEQ ID NO:4 (LDH) or an amino acid sequence having at least 90% sequence identity to the full-length sequence of SEQ ID NO:4.

[0284] 17. The immunogenic construct according to any one of items 1-13, wherein the targeting moiety comprises an amino acid sequence selected from SEQ ID NO: 5 (PF) or an amino acid sequence having at least 90% sequence identity to the full-length sequence of SEQ ID NO:5.

[0285] 18. The immunogenic construct according to any one of the preceding items, wherein said antigenic moiety comprises one or more antigens.

[0286] 19. The immunogenic construct according to any one of the preceding items, wherein the one or more antigens are selected from the group consisting of proteins, peptides, polypeptides and nucleic acids, and combinations thereof.

[0287] 20. The immunogenic construct according to any one of items 18 or 19, wherein said one or more antigens are connected by linker moieties.

[0288] 21. The immunogenic construct according to any one of items 18-20, wherein said one or more antigens originate from a virus, a bacterium or a mammal.

[0289] 22. The immunogenic construct according to item 21, wherein said mammal is a human.

[0290] 23. The immunogenic construct according to any one of items 18-21, wherein said one or more antigens are cancer-specific antigens or virus-specific antigens.

[0291] 24. The immunogenic construct according to item 23, wherein said cancer-specific antigens originate from a cancer selected from the group consisting of ovarian carcinomas, female genital tract malignancy, breast carcinomas, prostatic adenocarcinoma, pancreatic adenocarcinoma, lung cancer (e.g. squamous cell or bronchial cell carcinoma), hepatocellular carcinoma, colorectal adenocarcinoma, gastric adenocarcinoma, cholangiocarcinoma, bladder cancer, tumours of the central nervous system, and malignancies related to the lymphatic system or bone marrow including leukemia, multiple myeloma or lymphoma.

[0292] 25. The immunogenic construct according to any one of items 23 or 24, wherein said cancer-specific antigens are from a primary cancer. 26. The immunogenic construct according to any one of items 23-25, wherein said cancer-specific antigens are tumour-specific antigens, preferably solid tumour-specific antigens.

[0293] 27. The immunogenic construct according to item 26, wherein the tumour-specific antigens are neo-epitope antigens, preferably from a high mutational burden tumour or metastases.

[0294] 28. The immunogenic construct according to any one of items 23-27, wherein said virus-specific antigens originate from a virus selected from the group consisting of Adenovirus, SARS-CoV-1 virus, SARS-CoV-2 virus, Corona virus, Norovirus, Papillomavirus, Polyomavirus, Herpes simplex virus (HSV), Alpha herpesvirinae human herpesvirus 1, 2, 3, Human gamma herpesvirus 4, 8 (Kaposi sarcoma), Betaherpesvirinae 5, 6, 7, Varicella zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Picornavirus, Enterovirus, Rhinovirus, Hepatovirus, Cardiovirus, Aphthovirus, Coxsackie virus, Echovirus, Paramyxovirus, Measles virus, Parainfluenza virus, Mumps virus, Respiratory syncytial virus (RSV), Metapneumovirus, Nipah virus, Hendra viruses, Orthomyxoviruses, Influenza virus, Rhabdovirus, Filovirus, Marburg virus, Ebola virus, Bornavirus, Rabies virus, Reovirus, Rotavirus, Coltivirus, Orbivirus, Norwalk virus, Calicivirus, Rubella virus, Togavirus, Flavivirus, Arbovirus, Bunyavirus, Arena virus, Poxvirus, Parvovirus, Retrovirus, Human immunodeficiency virus (HIV), Human-T cell leukemia virus, and Hepatitis A, B, C, D, G, and E viruses, and combinations thereof.

[0295] 29. The immunogenic construct according to any one of items 23-28, wherein the virusspecific antigens originate from SARS-CoV-2 virus.

[0296] 30. The immunogenic construct according to item 29, wherein the SARS-CoV-2 antigen is selected from the spike (S) protein, the nucleocapsid (N) protein, or fragments thereof.

[0297] 31. The immunogenic construct according to item 30, wherein said spike (S) protein comprises an amino acid sequence represented by SEQ ID NO:9 (full length spike (S) protein), or an amino acid sequence having at least 90% sequence identity to the full- length sequence of SEQ ID NO:9. 32. The immunogenic construct according to any one of items 30 or 31, wherein said nucleocapsid (N) protein comprises an amino acid sequence represented by SEQ ID NO: 10 (full length nucleocapsid (N) protein), or an amino acid sequence having at least 90% sequence identity to the full-length sequence of SEQ ID NO: 10.

[0298] 33. The immunogenic construct according to any one of items 30-32, wherein said fragment of the spike (S) protein comprises an amino acid sequence represented by SEQ ID NO: 11 and said fragment of the nucleocapsid (N) protein comprises an amino acid sequence represented by SEQ ID NO: 12.

[0299] 34. The immunogenic construct according to any one of the preceding items, wherein the antigenic moiety comprises a particle.

[0300] 35. The immunogenic construct according to item 34, wherein the particle is selected from the group consisting of a lipid-based particle, a polymer-based particle, a peptide- based particle, a bead-based particle and a virus-like particle.

[0301] 36. The immunogenic construct according to any one of items 34 or 35, wherein the particle is a lipid-based particle.

[0302] 37. The immunogenic construct according to any one of items 34-36, wherein the particle is a lipid nanoparticle (LNP).

[0303] 38. The immunogenic construct according to any one of items 34-37, wherein the particle comprises one or more nucleic acids.

[0304] 39. The immunogenic construct according to item 38, wherein said one or more nucleic acids are encapsulated or trapped in the particle.

[0305] 40. The immunogenic construct according to any one of items 19-39, wherein said one or more nucleic acids are selected from ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and analogues thereof.

[0306] 41. The immunogenic construct according to any one of items 19-40, wherein said one or more nucleic acids are selected from the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), single stranded DNA (ssDNA), and double stranded DNA (dsDNA). 42. The immunogenic construct according to any one of items 19-41, wherein said one or more nucleic acids are mRNA.

[0307] 43. The immunogenic construct according to any one of items 34-42, wherein the targeting moiety is attached to the outer surface of the particle.

[0308] 44. The immunogenic construct according to any one of items 34-43, wherein the targeting moiety is coated or covalently bound to the surface of the particle.

[0309] 45. The immunogenic construct according to any one of items 34-44, wherein the targeting moiety is coated onto the surface of the particle.

[0310] 46. The immunogenic construct according to any one of items 34-45, wherein the targeting moiety is coated onto the surface of the particle by electrostatic interactions.

[0311] 47. The immunogenic construct according to items 1-33, wherein the immunogenic construct is a fusion protein.

[0312] 48. The immunogenic construct according to item 47 further comprising a linker moiety.

[0313] 49. The immunogenic construct according to item 48, wherein the linker moiety is positioned in between the targeting moiety and the antigenic moiety.

[0314] 50. The immunogenic construct according to any one of items 48 or 49, wherein the linker moiety is attached to the C-terminal of the targeting moiety and the N-terminal of the antigen(s) of the antigenic moiety.

[0315] 51. The immunogenic construct according to any one of items 48- 50, wherein the linker moiety is a peptide linker.

[0316] 52. The immunogenic construct according to item 51, wherein the peptide linker is selected from the group consisting of (GGGGS)n, (GGGS)n, SGGGSGGGS and AAY, wherein n is an integer selected from 1, 2 or 3.

[0317] 53. The immunogenic construct according to any one of items 51 or 52, wherein the peptide linker is a (GGGGS)2 linker. 54. A vaccine or immunogenic composition comprising an immunogenic construct according to any one of the preceding items.

[0318] 55. The vaccine or immunogenic composition according to item 54, wherein the vaccine or immunogenic composition further comprises one or more adjuvants.

[0319] 56. The vaccine or immunogenic composition according to item 55, wherein the one or more adjuvants are selected from the group consisting of Freund 's Adjuvants, 3 De- O-acylated monophosphoryl lipid A (3D-MPL), QS21, unmethylated CpG sequences, non-toxic oil-in-water emulsions and aluminium salts.

[0320] 57. The vaccine or immunogenic composition according to any one of items 54-56, wherein the vaccine or immunogenic composition further comprises a pharmaceutically acceptable carrier.

[0321] 58. An immunogenic construct according to any one of items 1-53 or a vaccine or immunogenic composition according to any one of items 54-57 for use as a medicament.

[0322] 59. An immunogenic construct according to any one of items 1-53 or a vaccine or immunogenic composition according to any one of items 54-57 for use in vaccination or immunization of a subject against viral and / or bacterial infections and / or cancer.

[0323] 60. The immunogenic construct, vaccine or immunogenic composition for use according to any one of items 58 or 59, wherein said immunogenic construct, vaccine or immunogenic composition is administered for prevention, inhibition or treatment of a viral or bacterial infection or cancer.

[0324] 61. The immunogenic construct, vaccine or immunogenic composition for use according to item 60, wherein the viral infection is caused by SARS-CoV-2.

[0325] 62. The immunogenic construct, vaccine or immunogenic composition for use according to any one of items 59 or 60, wherein the cancer is a primary cancer (non- metastatic cancer).

[0326] 63. The immunogenic construct, vaccine or immunogenic composition for use according to any one of items 58-62, wherein the subject is a mammal. 64. The immunogenic construct, vaccine or immunogenic composition for use according to item 63, wherein the mammal is a human.

[0327] 65. The immunogenic construct, vaccine or immunogenic composition for use according to any one of items 58-64, wherein the route of administration is selected from the group consisting of parenteral, intravenous, subcutaneous, intramuscular, intradermal, intranasal and oral.

[0328] 66. The immunogenic construct, vaccine or immunogenic composition for use according to any one of items 58-65, wherein said immunogenic construct, vaccine or immunogenic composition is administered in a single dose or in multiple doses separated by a period of time.

[0329] 67. A method of treatment comprising administration of an immunogenic construct according to any one of items 1-53 or a vaccine or immunogenic composition according to any one of items 54-57 to a subject in need thereof.

[0330] 68. The method of treatment according to item 67, wherein the subject suffers from a viral disease or a cancer disease.

[0331] 69. Use of a targeting moiety for targeting an antigenic moiety to an immune cell, wherein said targeting moiety comprises a protein or polypeptide selected from the group consisting of elongation factor thermo unstable (EF-Tu), L-lactate dehydrogenase (LDH), Protein F (PF), or single domains, or shorter fragments thereof.

[0332] 70. A kit comprising: i) an immunogenic construct according to any one of items 1-53 or a vaccine or immunogenic composition according to any one of items 54-57, and ii) optionally, instructions for use.

[0333] 71. A nucleic acid comprising a sequence encoding an immunogenic construct according to any one of items 47-53.

[0334] 72. A recombinant expression vector comprising a nucleic acid according to item 71 operatively linked to one or more control sequences suitable for directing the production of the immunogenic construct in a suitable host. 73. The recombinant expression vector according to item 72, wherein the vector is a pET26(b) vector.

[0335] 74. A recombinant host cell comprising a recombinant expression vector according to any one of items 72 or 73.

[0336] 75. The recombinant host cell according to item 74, wherein the recombinant host cell is Escherichia coli.

[0337] 76. The recombinant host cell according to item 74, wherein the recombinant host cell is a eukaryotic expression system, preferably a Chinese Hamster Ovarian (CHO) or Human Embryonic Kidney (HEK) cell line.

[0338] 77. A method for providing an immunogenic construct according to any one of items 47-53, said method comprising the steps of: i) provision of one or more nucleic acids at least encoding: a) the targeting moiety, and b) the antigenic moiety; and ii) expression of said one or more nucleic acids, thereby providing the immunogenic construct according to any one of items 47-53.

[0339] 78. The method according to item 77, wherein step (i) comprises provision of a nucleic acid according to item 71.

[0340] 79. The method according to any one of items 77 or 78, wherein said one or more nucleic acids encode a linker moiety.

[0341] 80. The method according to item 79, wherein only a single nucleic acid is provided and said single nucleic acid encodes the targeting moiety, the antigenic moiety, and the linker moiety.

[0342] 81. The method according to any one of items 77-80, wherein said expression of step (ii) is recombinant expression.

[0343] 82. The method according to item 81, wherein said recombinant expression is performed using a recombinant expression vector according to items 72 or 73. 83. The method according to any one of items 81 or 82, wherein said recombinant expression is performed using a recombinant host cell according to any one of items 74-76.

[0344] 84. The method according to any one of items 77-83, wherein step (i) is preceded by the following steps: i) obtaining a sample from a subject, ii) identifying an antigen within said sample, and iii) obtaining the nucleic acid sequence of said antigen.

[0345] 85. The method according to item 84, wherein the antigen within said sample is a neoepitope antigen.

[0346] 86. An immunogenic construct according to any one of items 47-53 obtained from the method according to any one of items 77-85.

[0347] The invention will now be described in further detail in the following non-limiting examples.

[0348] Examples

[0349] The following examples are included to demonstrate certain embodiments of the invention. The examples will address the potential of utilizing EF-Tu, LDH and PF in the stimulation of human and murine immune cells, both alone and as a part of an immunogenic construct with vaccine antigens. To evaluate this potential, SARS-CoV-2 N and S-proteins, B16F10 murine melanoma cell line expressing OVA (B16.OVA), A549 cell lines, HEK cell line, C57BL / 6J mice, OT-I mice and human MoDCs were used as model systems.

[0350] Example 1: The stimulatory capacity of EF-Tu on human immune cells

[0351] The purpose of this example is to assess the stimulatory capacity of EF-Tu on human immune cells; causing maturation, cytokine secretion, and upregulation of costimulatory surface proteins essential for T cell priming.

[0352] Methods

[0353] Reagents:

[0354] LPS, curdlan (Beta-1, 3-glucan from Alcaligenes faecal is), and carboxylflourescein succinimidyl ester (CFSE) were purchased from InvivoGen (San Diego, CA). Production of recombinant proteins:

[0355] The open reading frame of the gene encoding full-length NTHi 3655 EF-Tu (EDJ92442.1), EF-Tu domain 1 (EF-Tu_Dl; M1-D208 of NTHi 3655 EF-Tu), S. pneumoniae R6 (AAL00147.1), E. coli K-12 (WP_000031783.1) and L. monocytogenes EGD-e (CAD00866.1) were amplified from genomic DNA using the following primer pairs:

[0356] NTHi : 5'-GGGGCGGATCCGATGTCTAAAGAAAAATTTGAACGTA-3' (SEQ ID NO: 13) I 5'- GGCGGAAGC I I I I I GATGATTTTCGCAACAACGCCA-3' (SEQ ID NO: 14);

[0357] EF-Tu_Dl of NTHi : 5'-GGGGCGGATCCGATGTCTAAAGAAAAATTTGAACGTA-3' (SEQ ID NO: 15) I 5'-GGCGGAAGCTTTTAGTCGCCATTCTTCTTGTTCTTCTT-3' (SEQ ID NO: 16);

[0358] S. pneumoniae-. 5'-AAATAGGATCCGATGGCAAAAGAAAAATACGATCGTA-3' (SEQ ID NO: 17) I 5'-AATAAGCGGCCGCAGCTTCGATTTCTGTAACCATACC-3' (SEQ ID NO: 18);

[0359] E. coli: 5'-GAAGCGGATCCGGTGTCTAAAGAAAAATTTGAACGTAC-3' (SEQ ID NO: 19) I 5'-TAAAGAAGCTTGCCCAGAACTTTAGCAACAACGCCC-3' (SEQ ID NO:20);

[0360] L. monocytogenes-. 5'-AAATAGGATCCGATGGCAAAAGAAAAATTTGACCGCT-3' (SEQ ID NO: 21) I 5'-AATAAGCGGCCGCTTTGCTGATGTTAGAAACAACGCCA-3' (SEQ ID NO: 22). The open reading frame of the Idh gene encoding L-lactate dehydrogenase (LDH) from NTHi 3655 (BBF16182.1) was amplified from genomic DNA with 5'- GGGGCGGATCCATGAAAAAGAAAAATTATCAACGAC-3' (SEQ ID NO: 32) and 5'- GGCCGAAGCTTTTATTTCCCAGTTTTTCTTCTTCTC-3' (SEQ ID NO:33). The hpf gene encoding Protein F of NTHi 3655 (A4N8V8.1) was PCR-amplified with 5'- GGGGCGGATCCATGAATAAATTCGTTGCTACTCGC-3' (SEQ ID NO:34) and 5'- GGCCGAAGCTTTCA I I I I I CAGC I I l l i I CTTCTGC-3' (SEQ ID NO:35). Underlined are restriction enzyme sites BamHI and Hindlll, respectively. Following restriction enzyme digestion, the resulting DNA fragments were cloned into the expression vector pET26(b)+ (Novagen, Merck Darmstadt, Germany) for recombinant protein production. Briefly, the resulting plasmid was transformed into E. coli DH5o, followed by DNA sequencing. Recombinant proteins were thereafter produced in endotoxin-free E. coli BL21 (DE3) (ClearColi™, Research corporation technologies, Tucson, AZ) and purified by affinity chromatography using Ni-NTA agarose.

[0361] Generation of primary human monocvte-derived dendritic cells (MoDCs) and lymphocytes:

[0362] Monocytes were separated from human leucocyte concentrate from healthy donors by magnetic separation with positive CD14-selection by magnetic-activated cell sorting (MACS; Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer's instructions and incubated for 5 days with rhGM-CSF (800 lU / ml; R&D Systems, Minneapolis, MN) and rhIL-4 (500 lU / ml; R&D Systems). Cells were harvested and incubated with recombinant EF-Tu or EF-Tu domain 1 (5 |jg / ml), curdlan (50 |jg / ml), LPS (10 ng / ml), or culture medium alone for 48 h. CD14- cells after MACS separation were co-cultured for 48 h with stimulated MoDCs and analyzed by flow cytometry for intracellular IFN-y.

[0363] Enzyme-linked immunosorbent assay (ELISA):

[0364] Analyses of cytokine levels in cell culture supernatants were performed with commercially available kits from Invitrogen (hIL-12p40, hIL-8, hlFN-y, mlFN-y). For assessment of Dectin-1 binding, Nunc PolySorp (Sigma) plates were coated with 25nM fc-hDectin-la (Invivogen) in 0.1M Tris pH 9.0 overnight at 4°C, washed in PBST, and then blocked with 2.5% BSA / PBS for 1 h at RT. After washing, 25nM recombinant protein in 1% BSA / PBS was added and plates incubated for 1 h at RT. Upon washing, plates were incubated with 1:5,000 HRP-conjugated o-His antibody in 2.5% BSA + 0.05% Tween-20 for 1 h at RT. ELISA substrate was added after washing, and reactions were stopped 15 min later with H2SO4. The OD450nm was thereafter measured with a Tecan Sunrise plate reader (Tecan, Mannedorf, Switzerland). Sera from immunized mice were tested for antibody titers against truncated SARS-CoV-2 N protein by coating Nunc MaxiSporp plates (Sigma) with 0.5 pg protein / well in 0.1M Tris pH 9.0 overnight at 4°C, washed in PBST and then blocked with 1% BSA / PBST for 1 h at RT. After washing, sera were diluted in 1% BSA / PBST, added to the plates, and incubated for 1 h at RT. After washing, plates were incubated with 1: 1,000 rabbit o-mouse pAb (Dako Agilent, Santa Clara, CA), incubated for 1 h at RT, washed, and finally incubated with HRP-conjugated swine o-rabbit pAb in 1% BSA / PBST for 1 h at RT.

[0365] Flow cytometry:

[0366] Cells were washed in PBS supplemented with 0.5% BSA and incubated with human (Miltenyi Biotech) or murine (Invivogen) FcR blocking reagent followed by staining with the respective antibodies. Antibodies were purchased from Invitrogen: CDllc (N418), CD40 (5C3), CD40 (1C10), CD86 (IT2.2), CD86 (GL1), HLA-DR (LN3), MHC-II (M5 / 114.15.2), CD8a (53-6.7), anti-OVA257’264-peptide bound to H-2Kb (25-D1.16). IFN-y (B27) was purchased from Becton-Dickson (Franklin Lakes, NJ). Data was acquired using a FACSVerse flow cytometer (Becton-Dickson) and analyzed in R 3.6.2.

[0367] Results

[0368] Human monocyte-derived dendritic cells (MoDCs) from healthy donors were incubated with recombinant EF-Tu, EF-Tu domain 1 (EF-Tu_Dl), curdlan (P-l,3-glucan), or LPS. After 48 h of incubation, MoDCs incubated with any of the four different stimuli (recombinant EF-Tu, EF-Tu domain 1 (EF-Tu_Dl), curdlan (P-l,3-glucan), or LPS) had significantly lower antigen uptake (Fig. 1A). DC maturation leads to halted antigen (ovalbumin) uptake in vitro, and this can hence be used to assess maturation. In parallel, incubations were performed at 4°C to exclude nonspecific surface binding, and OVA uptake was, as expected, inhibited at the lower temperature (data not shown).

[0369] Upon stimulation with EF-Tu or EF-Tu domain 1 (DI), there was a significant increase in IL-23 levels in the cell culture supernatant (Fig. IB) which was far greater than what was seen with curdlan or LPS stimulation.

[0370] In order to prime naive T cells, APCs must express co-stimulatory proteins on their surface. Upon incubation of MoDCs with EF-Tu or EF-Tu domain 1, a significant upregulation was observed of CD40 (interacting with CD154 on T cells; Fig. 1C), CD86 (binds to CD28 on T cells; Fig. ID), and HLA-DR (an MHC class II surface receptor presenting antigen to the T cell receptor; Fig. IE).

[0371] Conclusion

[0372] Recombinant EF-Tu and EF-Tu domain 1 are both capable of stimulating human dendritic cells, as evidenced by decreased antigen uptake in vitro and upregulation of co-stimulatory proteins essential for priming naive T cells. An increased level of IL-23 secretion was seen compared with curdlan or LPS incubation, demonstrating that incubation with EF-Tu or EF-Tu domain 1 leads to preferential induction of Thl7 T helper cell subsets by comparison.

[0373] Example 2: EF-Tu orthologs from different bacterial species have conserved stimulatory capacity

[0374] The purpose of this example is to demonstrate that the conserved nature of EF-Tu across various bacterial species also transfers to the ability to stimulate human DCs.

[0375] Methods

[0376] Reagents, production of recombinant EF-Tu, generation of primary human monocyte- derived dendritic cells (MoDCs) and lymphocytes, and enzyme-linked immunosorbent assay (ELISA) were the same as in Example 1.

[0377] Results

[0378] Recombinant EF-Tu from Escherichia coli, Streptococcus pneumoniae, and Listeria monocytogenes in addition to Haemophilus influenzae was expressed. All four orthologs were able to induce IL-23 secretion upon stimulation of human MoDC. EF-Tu derived from S. pneumoniae and L. monocytogenes, which have the least sequence similarity with EF-Tu from Haemophilus influenzae, showed lower IL-23 inducing capacity, albeit still far greater than LPS or the negative control (Fig 2).

[0379] Conclusion

[0380] The highly conserved nature of EF-Tu and the fact that all four different orthologues had similar IL-23 inducing capacity demonstrates that the data can be extrapolated to all identical EF-Tu orthologs and other similar EF-Tu orthologs.

[0381] Example 3: The effect of EF-Tu stimulation of murine antigen-presenting cells and cross-priming of CD8+T cells in vitro and in vivo

[0382] The purpose of this example is to demonstrate the ability of EF-Tu, to induce a Thl- immune response and to improve cross-priming of CD8+T cells.

[0383] Methods

[0384] Reagents, production of recombinant EF-Tu, enzyme-linked immunosorbent assay (ELISA), and flow cytometry were the same as in Example 1.

[0385] Mice and tumour models:

[0386] Normal C57BL / 6J mice, Dectin-1 KO mice C57BL / 6J (C / e7a' / _), and OT I mice C57BL / 6- Tg (TcraTcrb) were purchased from the Jackson Laboratory (Bar Harbor, ME). The genotype and phenotype of C57BL / 6J(C / ec7a_ _) mice were confirmed by PCR and flow cytometry of sampled CD14+cells, respectively. For tumour challenge experiments, C57BL / 6J mice were injected s.c. with 100 pg OVA together with EF-Tu (20 pg / ml) or curdlan (100 pg / ml) day 1 and day 14. One week after the last dose, 105B16F10 murine melanoma cell line expressing full-length chicken ovalbumin (B16.OVA) were injected s.c. in the left flank. Tumour growth was then monitored continuously, and mice were euthanized when the tumour diameter exceeded 15 mm. OVA expression on B16F10 cells was assessed by flow cytometry before mouse challenge.

[0387] Generation of murine BMDCs and solenocytes:

[0388] Bone marrow-derived dendritic cells (BMDCs) were generated by culturing bone marrow cells from C57BL / 6J or C57BL / 6 Clec7a~ ~) mice in RPMI (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U penicillin, 100 mg / ml streptomycin (Gibco) and 20 ng / ml rmGM-CSF (Sigma-Aldrich, Saint Louis, MO). On day 5-6, semiadherent cells were harvested and included in downstream experiments. For stimulation experiments, BMDCs were incubated with recombinant protein (5 pg / ml unless another concentration is indicated), curdlan (50 pg / ml), or LPS (10 ng / ml) for 24-48 h. Supernatants were collected for cytokine analysis by ELISA, and cells were detached and analyzed by flow cytometry or co-cultured with splenocytes. CD8+T cells from OT-I mice (harboring transgenic T cell receptors specific for ovalbumin (OVA) residues 257-264 (H2-Kbrestricted) were purified from total splenocytes using MACS with negative selection according to the manufacturer's instructions (Miltenyi Biotech). T cells were then intracellularly stained with 5 pM CFSE for 20 min at 37°C, this enables monitoring if T cell expansion as intracellular CFSE will decrease with each cell division. For co-culture experiments, 2.5xl04BMDCs were pulsed for 24 h with rEF-Tu and CFSE-labeled CD8+T cells added in ratios 1:4 or 1:8 and cultured for additionally 48h followed by analysis by flow cytometry.

[0389] Ovalbumin uptake assay:

[0390] DC maturation was assessed by measuring the internalization of OVA after incubation for 48h with recombinant proteins (micropinocytosis is abrogated by DC maturation in vitro). Briefly, stimulated human MoDCs were pulsed with 50pg / ml Alexa488- conjugated OVA for 30 min at 37°C. Matched controls were kept at 4° to control for surface binding. Pulsed cells were washed three times with PBS, incubated with human FcR blocking reagent (Miltenyi Biotech), and anti-CDllc:PE (Invivogen) for 30 min at 4°C. OVA uptake in CDllc+cells were measured using flow cytometry.

[0391] IFN-v ELISoot:

[0392] The number of IFN-y secreting, restimulated, murine splenocytes following immunizations was assessed using IFN-y ELISpot assay kit (3420-4AST-10; Mabtech, Nacka, Sweden) according to the manufacturer's instructions. Mouse splenocytes were restimulated with truncated SARS-CoV-2 N protein (0.5 pg / ml) or Phytohemagluttinin- L (20 pl / ml) as a positive control for 36 h.

[0393] Production of recombinant model fusion protein vaccine:

[0394] A fusion protein consisting of full-length NTHi 3655 EF-Tu (EDJ92442.1) fused with a GGGS linker to a recombinant overlapping peptide (ROP) containing two repeats of the ovalbumin epitope SIINFEKL (SEQ ID NO:23) separated by a GGGS linker and an LMRK protease cutting site was designed and tested for stability and solubility in silico. Plasmids (pET26b(+)) containing the full fusion protein or the ROP only were then synthetically produced (GenScript Biotech, Piscataway, NJ) and transformed into E. coli DH5o, followed by DNA sequencing. Recombinant proteins were thereafter produced in endotoxin-free E. coli BL21 (DE3) (ClearColi™, Research corporation technologies, Tucson, AZ) and purified by affinity chromatography using Ni-NTA agarose. The recombinant EF-Tu used was produced as outlined in Example 1. Flow cytometry:

[0395] As described in Example 1. In addition, a synthetic SIINFEKL peptide (Invitrogen) was added at 1 pg / ml 30 min prior to staining as a positive control. Endotoxin-free ovalbumin (Invivogen) was also used as a control at 50 pg / ml. The SIINFEKL epitope presented in H-2Kb (MHC-I) was detected using anti-OVA257-264-peptide bound to H- 2Kb (25-D1.16) as in Example 1.

[0396] Results

[0397] Murine DCs derived from bone-marrow progenitors were incubated with EF-Tu or curdlan. Bone marrow was harvested from wild-type C57BL / 6J mice. Upon incubation with EF-Tu, a significant increase in IFN-y secretion, important for inducing Thl and CD8+T cell responses was observed (Fig. 3A). Similar to human DC stimulation, a significant upregulation of CD40 (Fig. 3B), CD86 (Fig. 3C), and MHC-II (Fig. 3D) could be seen in murine BMDCs after incubation with EF-Tu.

[0398] Cross-priming of CD8+T cells requires antigen presentation on MHC class I molecules, in addition to cytokine secretion and expression of co-stimulatory molecules as seen in Fig. 3. To evaluate the effect of EF-Tu on cross-priming of CD8+T cells, T cells derived from transgenic OT-I mice were used. These T cells all harbor uniform T cell receptors recognizing OVA residues 257-264 (SIINFEKL (SEQ ID NO:23); H2-Kbrestricted) specific for the C57BL / 6 mouse strain. Murine BMDCs from C57BL / 6 mice were pulsed with OVA together with EF-Tu, curdlan, LPS, or medium only for 24 h. CD8+T cells from OT-I mice were separated by magnetic cell separation (MACS), intracellularly stained with CFSE (cell proliferation stain), and co-cultured with BMDCs at 1 :4 and 1:8 DC to T cell ratio. Enhanced cross-priming of CD8+cells was seen upon pulsing with EF-Tu and OVA compared with OVA alone (Fig. 4A) as evidenced by the high proportion of CFSELOWCD8+cells (EF-Tu: 89% proliferating T cells; OVA only: 71%). Importantly, pulsing with EF-Tu resulted in improved T cell priming compared with curdlan or LPS (Fig. 4B). Some of the difference can be explained by increased cross-presentation of OVA257-264peptide (SIINFEKL) in MHC-I on the surface of pulsed BMDCs (Fig. 4C).

[0399] Further, the efficiency of the EF-Tu-OVA fusion protein to promote MHC class-I presentation was assessed by exposing murine bone marrow-derived dendritic cells to EF-Tu-OVA recombinant overlapping peptide (ROP) fusion protein, containing two repeats of the ovalbumin SIINFEKL peptide. Surface MHC-class I complexes with bound SIINFEKL peptide were detected by flow cytometry. Enhanced MHC class I presentation was seen when the murine dendritic cells were exposed to EF-Tu-OVA ROP fusion protein compared to EF-Tu and OVA ROP separately (Fig. 5). Lastly, the effect of EF-Tu stimulation in vivo was investigated by immunization of C57BL / 6J mice (n=6) with OVA together with recombinant EF-Tu, curdlan, or OVA only. One week after the immunization, the number of circulating CD8+T cells was assessed by flow cytometry (Fig. 6). In concordance with the in vitro data, immunization with EF-Tu together with OVA led to increased levels of CD8+T cells.

[0400] Conclusion

[0401] The effect of EF-Tu stimulation on murine DCs confirmed that of human DCs shown in Example 1. Furthermore, in the mouse, EF-Tu stimulation also induced enhanced crosspriming of cytotoxic T cells both in vitro and in vivo. Further, EF-Tu-OVA fusion protein leads to enhanced MHC-class I presentation in murine DCs compared to EF-Tu and OVA alone. Hence, it is advantageous to use EF-Tu in a fusion protein to promote crosspriming of CD8 T cells through increase MHC class I presentation on DCs

[0402] Example 4: Immunization with EF-Tu in conjunction with tumour antigen

[0403] The purpose of this example is to assess whether increased T cell priming, upon EF-Tu stimulation, results in enhanced antitumour immunity in C57BL / 6 mice.

[0404] Methods

[0405] Reagents and production of recombinant EF-Tu were the same as in Example 1. Mice and tumour models were the same as in Example 3.

[0406] Cell lines and cell culture:

[0407] B16F10 murine melanoma cell line expressing full-length chicken OVA (B16.OVA) cells were maintained in RPMI1649 Glutamax (Gibco; Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U penicillin, and 100 mg / ml streptomycin (Gibco). A549 cell lines (type II alveolar epithelial cells) transfected with the plasmids pUNOl (control vector) or pUNOl-hDectinla (bearing the human Dectin-1 gene [clec7A] isoform a) were maintained in DMEM high glucose with Glutamax (Giboc), supplemented with 10% fetal bovine serum (FBS) and 30pg / ml Blasticidin S (InvivoGen). Cultures were maintained in a humidified incubator at 37°C with 5% CO2.

[0408] Results

[0409] A mouse model of tumour prevention was used to assess the antitumour effect of T cells upon EF-Tu stimulation. C57BL / 6 mice were immunized with OVA, administered as a free component, in combination with either EF-Tu domain 1 (EF-Tu_Dl), or curdlan at two occasions, 14 days apart. One week after the last dose, mice were challenged with B16F10 melanoma cells expressing OVA (B16.OVA) by s.c. injection and tumour progression monitored until the experimental cut-off point was reached. Immunization with EF-Tu_Dl resulted in significantly increased survival of tumourbearing mice compared with curdlan (Fig. 7).

[0410] Conclusion

[0411] Immunization with EF-Tu in conjunction with tumour antigen led to improved antitumour immunity, resulting in increased survival.

[0412] Example 5: Fusion proteins of EF-Tu domain 1 with SARS-CoV-2 antigens used in human T cell priming in vitro

[0413] The purpose of this example is to assess whether the antigen-specific immunostimulatory effects of EF-Tu could be improved by delivering the antigen as part of a fusion protein comprising EF-Tu.

[0414] Methods

[0415] Reagents and production of recombinant EF-Tu, generation of primary human monocyte-derived dendritic cells (MoDCs) and lymphocytes, enzyme-linked immunosorbent assay (ELISA), and flow cytometry were the same as in Example 1.

[0416] Production of EF-Tu fusion proteins:

[0417] Truncated sequences of N and S proteins were fused to Domain 1 (DI) of EF-Tu from Haemophilus influenzae with a (GGGGS)2 linker. The resulting fusion proteins are denoted as DIN and DIS, respectively.

[0418] Briefly, the fusion proteins and control proteins were prepared as follows. All sequences were sent to Genscript (Piscataway, NJ) for synthesis. The sequences were PCR amplified as outlined in Table 1 followed by subcloning into pET26(b)+ (Novagen) using the primers shown in Table 2. Gene inserts were digested with restriction enzymes (Table 2) to ligate the DNA fragments into pET26(b)+. Subsequently, the plasmids were transformed into E. coli DH5o, followed by DNA sequencing. Recombinant proteins were thereafter produced in E. coli ClearColi BL21 (DE3) cells, harboring modified non-toxic LPS, and purified using His-tag affinity column chromatography, followed by size exclusion chromatography using an Akta purification system.

[0419] Table 1. Expand High Fidelity (Roche) PCR protocol used to amplify truncated N, truncated S, DIN and DIS gene inserts.aPrimer annealing temperatures used for each gene insert are as follows: N (61.9 °C), S (65 °C), DIN (59.9 °C) and DIS (59.1 °C).bElongation times that were used for each gene insert are as follows: truncated N and S (1 minute), DIN and DIS (1 minute and 30 seconds). Steps highlighted in light grey were repeated in 10 cycles and those in dark grey were repeated in 20 cycles.

[0420] Table 2. List of primers used. Restriction enzyme cutting sites are underlined. BamHI Notl2, Hindlll3.

[0421] Results

[0422] Fusion proteins were constructed (Fig. 8A), consisting of EF-Tu domain 1 (denoted EF- Tu_Dl) with an immunodominant 216 aa sequence from SARS-CoV-2 spike protein (denoted DIS) or an immunodominant 181 aa nucleocapsid protein (denoted DIN), both containing a (GGGS)2 linker. Incubation of human MoDCs with fusion proteins

[0423] DIS or DIN resulted in increased stimulation of autologous CD8+T cells compared with truncated N- or S protein alone (Fig. 8B), due to the upregulation of CD80 and CD86 on the MoDCs surface following incubation (Fig. 8C). Conclusion

[0424] Fusion proteins comprising EF-Tu domain 1 with SARS-CoV-2 antigens were able to induce an improved antigen-specific T cell response of human T cells in vitro.

[0425] Example 6: EF-Tu, LDH and Protein F interaction with human Dectin-1 and immunostimulatory effects

[0426] The purpose of this example is to determine the binding affinity of EF-Tu to human Dectin-1 and to assess the importance of Dectin-1 for the immunostimulatory effects of EF-Tu on dendritic cells.

[0427] Methods

[0428] Production of recombinant EF-Tu, LDH and Protein F, enzyme-linked immunosorbent assay (ELISA), and flow cytometry were the same as in Example 1. Mice and tumour models, generation of murine BMDCs and splenocytes, and ovalbumin uptake assay were the same as in Example 3. Cell lines and cell culture were the same as in Example 4.

[0429] EF-Tu antibody preparation:

[0430] Rabbit o-EF-Tu serum was prepared by immunization of rabbits subcutaneously with 200 pg of recombinant EF-Tu in 0.5 ml saline with 0.5 ml incomplete Freund's adjuvant. Animals were boosted three times every four weeks with alum used as an adjuvant. Blood was drawn two weeks after the last immunization. Rabbit pAbs against EF-Tu was then further affinity purified using EF-Tu coupled to CNBr-activated Sepharose TM (GE Healthcare Biosciences, Chicago, IL).

[0431] Measurement of protein-protein interactions by biolaver interferometry:

[0432] Kinetic analyses of the interaction between EF-Tu orthologs, domains and peptide fragments, and human Dectin-1 were performed by biolayer interferometry using a forteBio OctetRed96 platform (Pall, Menlo Park, CA). Recombinant hDectin-1 (R&D Systems) was immobilized on an amine-reactive (AR2G) sensor (Pall). The analytes were serially diluted in running buffer (PBS) ranging from 1.25 to 50 pM. The experiments were conducted at 30°C. Data analysis was performed using the forteBio Data Analysis software 8.1 (Pall). Curves were fitted with 1: 1 binding kinetics, and affinity (ko) was calculated.

[0433] Results

[0434] EF-Tu, LDH and Protein F (Fig. 14) from Haemophilus influenzae, as well as EF-Tu orthologs from E. coli, S. pneumoniae, and L. monocytogenes, all readily bind to human Dectin-1 (Fig. 9) with similar binding affinity, as assessed by biolayer interferometry (Table 3). The dissociation constant (kD) was calculated as 79 nM for EF-Tu from Haemophilus influenzae. Domain 1 of EF-Tu, when isolated, is able to bind Dectin-1 as well as two different surface-exposed peptide fragments derived from domain 3. Moreover, the EF-Tu orthologs bind to the carbohydrate recognition domain (CRD) of Dectin-1 (Table 4).

[0435] Table 3. Binding affinity to full length human Dectin-1 showing affinities of EF-Tu orthologs from Haemophilus influenzae, S. pneumoniae, L. monocytogenes, and E. coli; domain 1 of EF-Tu from Haemophilus influenzae and two 25 aa fragments from domain 3 of EF-Tu from Haemophilus influenzae; SARS-CoV-2 fusion proteins DIN, DIS and Protein F.

[0436] Table 4. Binding affinity of EF-Tu from different species to the carbohydrate recognition domain (CRD) of Dectin-1.

[0437] To further explore the EF-Tu and Dectin-1 interaction, A549 cells stably transfected with Dectin-1 were used. An evident increase in EF-Tu binding to cells overexpressing Dectin-1 could be seen (Fig. 10A). Furthermore, an increased IL-8 secretion was seen in response to EF-Tu stimulation of Dectin-1 transfected cells, compared with vector control (Fig. 10B), indicating that ligation results in downstream signaling.

[0438] The relative importance of Dectin-1 for the immunostimulatory effects of EF-Tu was examined. BMDCs derived from wild-type C57BL / 6J mice and Dectin-1 KO C57BL / 6J (Clec7a~ ~) were stimulated with EF-Tu. Following 48 h incubation, an apparent decrease in IFN-y secretion from cells devoid of Dectin-1 could be seen (Fig. 11). However, the effect was not completely abolished, and the drop lower than seen for the selective Dectin-1 agonist, curdlan. This indicates that Dectin-1, albeit important is not the only immune receptor important for the stimulatory effects of EF- Tu.

[0439] Conclusion

[0440] LDH and Protein F from Haemophilus influenzae, as well as EF-Tu orthologs from S. pneumoniae, E. coli, L. monocytogenes, and Haemophilus influenzae can bind to Dectin-1. EF-Tu can bind and activate Dectin-1 expressed on A549 cells, demonstrated by increased IL-8 secretion following EF-Tu stimulation of Dectin-1 transfected cells. Furthermore, EF-Tu stimulated BMDCs, derived from wild-type C57BL / 6J mice and Dectin-1 KO C57BL / 6J(C / ec7a_ / '), demonstrated that Dectin-1 is important for the effect of EF-Tu on dendritic cells. Example 7: Immunogenic constructs comprising particles

[0441] The purpose of this example is to assess the ability of the targeting moiety to direct a particle comprising an antigen to an immune cell. A model system with empty beads as well as lipid nanoparticles loaded with mRNA were used as model system.

[0442] Methods

[0443] Reagents:

[0444] CleanCap® enhanced green fluorescent protein (eGFP) mRNA fully substituted with 5- methoxyuridine (1 mg / mL in 1 mM sodium citrate buffer, pH 6.4) were acquired from TriLink Biotechnologies (San Diego, CA, USA). l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), and l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2000) were acquired from Avanti® Polar Lipids (Alabaster, AL, USA). Cholesterol and Triton X- 100™ were obtained from Sigma-Aldrich (St. Louis, MO, USA). Heptadecan-9-yl 8-((2- hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102) was obtained from MedChemTronica (Sollentuna, Sweden). Quant-iT™ RiboGreen® RNA Reagent and Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0) (TE buffer) were acquired from Molecular Probes, Invitrogen (Paisley, UK). RNase-free water was used for procedures pertaining to mRNA-LNP preparation.

[0445] Formulation of mRNA-loaded lipid nanoparticles:

[0446] LNPs were prepared by microfluidic mixing using the NanoAssemblr® Ignite™ microfluidic mixer system (Precision Nanosystems Inc., Vancouver, Canada). The aqueous phase consisted of mRNA dissolved in sodium citrate buffer (50 mM, pH = 3). C12-200, DOPE, cholesterol, and DMPE-PEG were dissolved in an organic phase consisting of absolute ethanol. The mRNA and lipid solutions were mixed at a flow rate ratio of aqueous to organic solvent phase of 3: 1 with a total flow rate of 12 mL / min. The formulations were dialyzed using Slide-A-Lyzer® G3 dialysis cassettes with a molecular weight cutoff (MWCO) of 3.5 kDa (Thermo Fisher Scientific, Waltham, MA) against IX Dulbecco's phosphate buffered saline at pH 7.4 for 3.5 h under constant stirring. LNPs were concentrated in IX D-PBS by centrifugation at 3000x g using either a 10, 30, or 100 kDa MWCO centrifugal filter (MilliporeSigma, Burlington, MA, USA). LNPs were sterile filtered using Minisart® Regenerated Cellulose sterile filters (Sartorius AG, Darmstadt, Germany). HEK reporter cell line:

[0447] Secreted embryonic alkaline phosphatase (SEAP) reporter cells expressing the human Dectin-la gene (HEK-Blue™ hDectin-la) and parental NF-KB inducible SEAP cell line (HEK-Blue™ Nulll-v), both derived from human embryonic kidney (HEK) 293 cells were acquired from InvivoGen (San Diego, CA). Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) high glucose (4.5 g / l glucose) supplemented with 10% (v / v) fetal bovine serum, 100 U / ml penicillin, 100 pg / ml streptomycin, 2 mM L-glutamine and selection antibiotics 1 pg / ml puromycin and IX HEK-BlueTM CLR Selection (hDectin-la) or 100 pg / ml Normocin (Nulll-v). Dectin-1 surface expression was confirmed by flow cytometry.

[0448] Incubation of Dectin-1 expressing HEK cells with protein-coated nickel beads:

[0449] HIS-tagged proteins were expressed and purified as described above. Magnetic nickel beads (Thermo Fisher Scientific, Waltham, MA, USA) were thoroughly washed, equilibrated with PBS and incubated with recombinant protein overnight under constant mixing. Beads were subsequently washed to remove unbound protein and resuspended in PBS. Coating efficiency was assessed by SDS-PAGE, and concentrations of unbound protein were measured using Bicinchoninic acid assay (BCA). For the stimulation experiments, cells were grown to 80% confluency, detached with PBS, and seeded at 2.6 x 105cells / mL in complete DMEM without selection antibiotics. Cells were subsequently incubated with protein-coated or empty beads at 37°C with 5% CO2 for 24h. NF-KB activation was assessed using a colorimetric enzyme assay to assess alkaline phosphatase (AP) activity. In a transparent 96-well plate 50pl conditioned culture media were mixed with 180pl QANTI-Blue™ Solution (InvivoGen), incubated at 37°C for 5h and optical density at 620nm read using a Tecan Spark® (Tecan Group Ltd) plate reader.

[0450] Transfection of Dectin-1 expressing HEK cells:

[0451] For the transfection experiments, LNPs were formulated as described above and coated by incubation with protein at a protein-to-lipid ratio (w / w) of 1:24 in PBS for 20 min at 37°C immediately prior to transfection. Cells were grown to 80% confluency, detached with PBS, and seeded at 2.6 x 105cells / mL in complete DMEM without selection antibiotics. Cells were subsequently transfected with eGFP mRNA-loaded LNPs and incubated at 37°C with 5% CO2. The eGFP expression was assessed by fluorescence measurements using a Tecan Spark® (Tecan Group Ltd, Mannedorf, Switzerland) plate reader at an excitation wavelength of 485 nm and emission of 535 nm at the indicated time points. NF-KB activation was assessed using a colorimetric enzyme assay to assess alkaline phosphatase (AP) activity. In a transparent 96-well plate 50pl conditioned culture media were mixed with 180pl QANTI-Blue™ Solution (InvivoGen), incubated at 37°C for 5h and optical density at 620nm read using a Tecan Spark® (Tecan Group Ltd) plate reader. Conditioned media from unstimulated cells was included as a negative control.

[0452] Result

[0453] EF-Tu was shown to induce Dectin-l-dependent downstream signalling when coated on a bead surface (Fig. 12). Additionally, domain 1 of EF-Tu was capable of inducing Dectin-1 activation comparable to full-length EF-Tu when coated on a bead surface (Fig. 15). It is clear from these data that coating the full-length or domain 1 of EF-Tu onto a bead surface led to increased NF-KB activation compared to empty beads as control.

[0454] Further, coating either of EF-Tu, LDH or PF onto the surface of LNPs carrying a mRNA cargo resulted in increased NF-KB activation through downstream signalling from binding to Dectin-1 (Fig. 13A) and effective delivery of mRNA cargo to target cells with sufficiently maintained translation levels as evidenced by increased eGFP expression (Fig. 13B).

[0455] Conclusion

[0456] Targeting moieties as described herein may be used for efficiently guiding antigencontaining particles to immune cells and simultaneously induce an adjuvating effect. Hence, coating of particles, such as LNPs, with bacterial targeting moieties, such as EF-Tu, LDH or PF, can be used as a platform technology to facilitate effective delivery of mRNA cargo as part of an advantageous vaccine strategy.

[0457] References

[0458] 1. Lin Y, Cheng Q, Wei T. Biophys Rep. 2023

Claims

Claims1. An immunogenic construct comprising a targeting moiety and an antigenic moiety, wherein the targeting moiety has affinity for a pattern recognition receptor (PRR), and wherein the targeting moiety comprises a protein or polypeptide selected from the group consisting of elongation factor thermo unstable (EF-Tu), L-lactate dehydrogenase (LDH), Protein F (PF), or single domains, or shorter fragments thereof.

2. The immunogenic construct according to claim 1, wherein the targeting moiety has affinity for Dectin- 1.

3. The immunogenic construct according to any one of claims 1-2, wherein the targeting moiety has a binding affinity (I<D) for Dectin-1 in the range of about 1.0 x 10'9M to about 1.0 x 10'5M, such as about 5.0 x 10'8M to about 1.0 x 10'6M.

4. The immunogenic construct according to any one of claims 2 or 3, wherein said Dectin-1 comprises an amino acid sequence selected from SEQ ID NO:7 or SEQ ID NO:8, or an amino acid sequence having at least 90% sequence identity to the full- length sequence of SEQ ID NO:7 or SEQ ID NO:8.

5. The immunogenic construct according to claim 1, wherein the targeting moiety comprises an amino acid sequence selected from the group consisting of: a) SEQ ID NO: 1 (EF-Tu domain I), SEQ ID NO:2 (EF-Tu domain II), SEQ ID NO:3 (EF-Tu domain III), and combinations thereof, b) SEQ ID NO:4 (LDH), c) SEQ ID NO:5 (PF), and d) an amino acid sequence having at least 90% sequence identity to the full- length sequence of any one of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, and combinations thereof, or SEQ ID NO:4, or SEQ ID NO: 5.

6. The immunogenic construct according to any one of the preceding claims, wherein said antigenic moiety comprises one or more antigens selected from the group consisting of proteins, peptides, polypeptides and nucleic acids, and combinations thereof.

7. The immunogenic construct according to claim 6, wherein said one or more antigens are connected by linker moieties.

8. The immunogenic construct according to any one of claims 6-7, wherein said one or more antigens originate from a virus, a bacterium or a mammal.

9. The immunogenic construct according to claim 8, wherein said mammal is a human.

10. The immunogenic construct according to any one of claims 6-9, wherein said one or more antigens are cancer-specific antigens or virus-specific antigens.

11. The immunogenic construct according to any one of the preceding claims, wherein the antigenic moiety comprises a particle selected from the group consisting of a lipid- based particle, a polymer-based particle, a peptide-based particle, a bead-based particle and a virus-like particle, and wherein the particle comprises one or more nucleic acids.

12. The immunogenic construct according to claim 11, wherein the particle is a lipid nanoparticle (LNP).

13. The immunogenic construct according to any one of claims 11-12, wherein the particle comprises one or more nucleic acids.

14. The immunogenic construct according to claim 13, wherein said one or more nucleic acids are encapsulated or trapped in the particle.

15. The immunogenic construct according to any one of claims 6-14, wherein said one or more nucleic acids are selected from the group consisting of messenger RIMA (mRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), single stranded DNA (ssDNA), and double stranded DNA (dsDNA).

16. The immunogenic construct according to any one of claims 6-15, wherein said one or more nucleic acids are mRNA.

17. The immunogenic construct according to any one of claims 11-16, wherein the targeting moiety is attached to the outer surface of the particle.

18. The immunogenic construct according to any one of claims 11-17, wherein the targeting moiety is coated onto the surface of the particle.

19. The immunogenic construct according to any one of claims 1-6, wherein the immunogenic construct is a fusion protein.

20. The immunogenic construct according to claim 19 further comprising a linker moiety, wherein the linker moiety is positioned in between the targeting moiety and the antigenic moiety.

21. The immunogenic construct according to claim 20, wherein the linker moiety is attached to the C-terminal of the targeting moiety and the N-terminal of the antigen(s) of the antigenic moiety.

22. The immunogenic construct according to any one of claims 19-21, wherein the linker moiety is a peptide linker.

23. The immunogenic construct according to claim 22, wherein the peptide linker is selected from the group consisting of (GGGGS)n, (GGGS)n, SGGGSGGGS and AAY, wherein n is an integer selected from 1, 2 or 3.

24. The immunogenic construct according to any one of claims 22 or 23, wherein the peptide linker is a (GGGGS)2 linker.

25. A vaccine or immunogenic composition comprising an immunogenic construct according to any one of claims 1-24.

26. An immunogenic construct according to any one of claims 1-24 or a vaccine or immunogenic composition according to claim 25 for use as a medicament.

27. An immunogenic construct according to any one of claims 1-24 or a vaccine or immunogenic composition according to claim 25 for use in vaccination or immunization of a subject against viral and / or bacterial infections and / or cancer.

28. Use of a targeting moiety for targeting an antigenic moiety to an immune cell, wherein said targeting moiety comprises a protein or polypeptide selected from the group consisting of elongation factor thermo unstable (EF-Tu), L-lactate dehydrogenase (LDH), Protein F (PF), or single domains, or shorter fragments thereof.

29. A kit comprising:i) an immunogenic construct according to any one of claims 1-24 or a vaccine or immunogenic composition according to claim 25, and ii) optionally, instructions for use.

30. A nucleic acid comprising a sequence encoding an immunogenic construct according to any one of claims 19-24.

31. A recombinant expression vector comprising a nucleic acid according to claim 30 operatively linked to one or more control sequences suitable for directing the production of the immunogenic construct in a suitable host.

32. The recombinant expression vector according to claim 31, wherein the vector is a pET26(b) vector.

33. A recombinant host cell comprising a recombinant expression vector according to any one of claims 31 or 32.

34. The recombinant host cell according to claim 33, wherein the recombinant host cell is Escherichia coli.

35. The recombinant host cell according to claim 33, wherein the recombinant host cell is a eukaryotic expression system, preferably a Chinese Hamster Ovarian (CHO) or Human Embryonic Kidney (HEK) cell line.

36. A method for providing an immunogenic construct according to any one of claims 19-24, said method comprising the steps of: i) provision of one or more nucleic acids at least encoding: a) the targeting moiety, and b) the antigenic moiety; and ii) expression of said one or more nucleic acids, thereby providing the immunogenic construct according to any one of claims 19-24.

37. The method according to claim 36, wherein step (i) comprises provision of a nucleic acid according to claim 30.

38. The method according to any one of claims 36 or 37, wherein said one or more nucleic acids encode a linker moiety.

39. The method according to claim 38, wherein only a single nucleic acid is provided and said single nucleic acid encodes the targeting moiety, the antigenic moiety, and the linker moiety.

40. The method according to any one of claims 36-39, wherein said expression of step (ii) is recombinant expression.

41. The method according to claim 40, wherein said recombinant expression is performed using a recombinant expression vector according to claims 31 or 32.

42. The method according to any one of claims 40 or 41, wherein said recombinant expression is performed using a recombinant host cell according to any one of claims 33-35.

43. The method according to any one of claims 36-42, wherein step (i) is preceded by the following steps: i) obtaining a sample from a subject, ii) identifying an antigen within said sample, and iii) obtaining the nucleic acid sequence of said antigen.

44. The method according to claim 43, wherein the antigen within said sample is a neo-epitope antigen.

45. An immunogenic construct according to any one of claims 19-24 obtained from the method according to any one of claims 36-44.

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