Field of Invention

In particular the invention relates to recombinant immunogenic polypeptides presenting inserted epitopes from zoonotic viruses and vaccine compositions comprising said recombinant molecule. The invention relates to recombinant immunogenic polypeptides presenting inserted epitopes from SARS-CoV-2 and vaccine compositions comprising said recombinant molecules. In particular, the invention relates to such compositions for eliciting an immune response against the virus responsible for Covid-19 infection.

Background of the invention

Zoonotic diseases are infectious diseases that are naturally transmitted from vertebrate animals to humans and vice versa. They are caused by all types of pathogenic agents, including bacteria, parasites, fungi, viruses and prions. Specific examples of zoonotic viruses include betacoronaviruses, including HCoV-NL63, MERS-CoV, SARS-Cov and SARS-CoV2. More than 65% of emerging infectious diseases in humans have been reported to originate from zoonotic pathogens. Among infectious agents associated with the emerging infectious diseases that are zoonotic, viruses are the most likely to pose the greatest threat. The great similarity of genetic, physiological, and behavioural characteristic in human and non-human primates makes the development of effective therapeutics to prevent/control cross-species transmission an area of high interest to investigators worldwide.

COVID-19 is primarily a respiratory-transmitted infection that is highly infectious and can be spread by asymptomatic as well as symptomatic individuals. SARS-CoV-2 enters host cells through binding of the spike glycoprotein (S protein) on its envelope to its receptor, angiotensin-converting enzyme 2 (ACE2), on the surface of human cells. The severity of illness in those infected varies considerably, with the more severe disease being seen in the elderly, the obese, and those with common co-morbidities, such as hypertension and diabetes mellitus. In more severe cases, it causes immune-mediated lung injury and acute respiratory distress syndrome (ARDS). The inflammatory response is systemic and contributes to the involvement of other organs. There is no effective cure, although dexamethasone, a corticosteroid, has been shown to reduce mortality in the most severely ill, and remdesivir, an anti-viral drug, may shorten time to recovery in hospitalized adults.

The development of safe and effective vaccines to prevent infection with SARS-CoV-2 infection is the leading goal of the current research strategy, and around 200 vaccine candidates of different types are at different stages of development. It is too soon to say which if any of these will prove successful since phase 3 trials are just beginning for the leading candidates.

Also, in development as a potential therapeutic strategy in those with active infection is the use of antibody treatments. This is being explored through a number of approaches including transfusion of convalescent serum from recently recovered individuals with high antibody titre responses, and the development of monoclonal antibodies that alone or, more likely, in combination will neutralize SARS-CoV-2 and, for example, prevent its binding to and entry into host cells.

Given the unprecedented scale of the health and economic crisis caused by Covid-19, the rapid development, evaluation, and implementation of successful preventive and therapeutic strategies to combat this disease is an urgent global imperative.

WO 01/57210 describes the production of several bifunctional molecules using a protein scaffold such as dendroaspin. Dendroaspin is an RGD-containing venom protein, isolated from the venom of Dendroaspis jamesonii. Dendroaspin is a three-looped structure with a well-defined fold. The orientation of RGD-containing loop III is completely different from that in other short chain neurotoxins such as erabutoxin b, and is displaced from the other 2 loops (Figure 1). The loop II and III have been used for the insertion of different protein domains and shown different functional features with structural modulation.

Many proteins from a variety of snake venoms have been identified as potent inhibitors of platelet aggregation and integrin-dependent cell adhesion. The majority of these proteins which belong to the so-called "disintegrin" family share a high level of sequence homology, are small (4-8 kDa), cysteine-rich and contain the sequence RGD (or KGD). In addition to the disintegrin family, a number of non-disintegrin RGD proteins of similar inhibitory potency, high degree of disulfide bonding, and small size have been isolated from both the venoms of the Elapidae family of snakes and from leech homogenates. All of these proteins are approximately 1000 times more potent inhibitors of the interactions of glycoprotein ligands with the integrin receptors than simple linear RGD peptides; a feature that is attributed to an optimally favourable conformation of the RGD motif held within the protein scaffold. The NMR structures of several inhibitors including kistrin, flavoridin, echistatin, albolabrin, decorsin, and dendroaspin have been reported, and the only common structural feature elucidated so far is the positioning of the RGD motif at the end of a solvent exposed loop, a characteristic of prime importance to their inhibitory action. Dendroaspin, a short chain neurotoxin analogue containing the RGD sequence, and the disintegrin kistrin, which show little overall sequence homology but have similar amino acids flanking the RGD sequence (PRGDMP), are both potent inhibitors of platelet adhesion to fibrinogen but poor antagonists of the binding of platelets to immobilised fibronectin. In contrast, elegantin, which has 65% sequence homology to kistrin but markedly different amino acids around RGD (ARGDNP), preferentially inhibited platelet adhesion to fibronectin as opposed to fibrinogen and binds to an allosterically distinct site on the α _llb βs complex.

Smith J W et al (1995) Journal of Biological Chemistry 270: 30486-30490 undertook protein "loop grafting" experiments to construct a variant of tissue-type plasminogen activator (t-PA) which bound platelet integrin α _llb βs. Amino acids in a surface loop of the epidermal growth factor (EGF) domain of t-PA were replaced with residues from a complementarity- dotermining region (CDR) forming one CDR of a monoclonal antibody reactive against the adhesive integrin receptor α _llb βs. The resulting variant of t-PA (loop-grafted-t-PA) bound α _llb βs with nanomolar affinity and had full activity to both synthetic and natural substrates. The effects and applicability of loop grafting are altogether unpredictable and uncertain.

The present inventors have now discovered that the dendroaspin scaffold lends itself to modification to incorporate immunogenic polypeptides capable of eliciting an anti SARS- CoV-2 response, with the resulting molecules being particularly useful as multifunctional immunogenic agents.

The dendroaspin scaffold lends itself to modification. When dendroaspin is modified to incorporate a first SARS-CoV-2 Spike protein epitope capable of eliciting an immune response against SARS-CoV-2, the resulting molecules are advantageously immunogenic.

Additionally, there is a need in the art for novel and improved adjuvants for vaccines. There is also a need in the art for alternative adjuvants that preferentially induce an anti-inflammatory Th2 type immune response, for use in a vaccination against inflammatory diseases.

Statement of invention

In one aspect the invention provides a recombinant polypeptide comprising: i) a dendroaspin scaffold; and ii) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a zoonotic pathogen protein.

In one embodiment, the zoonotic pathogen is bacteria, a parasite, a fungi, a virus and a prion.

In one embodiment, said recombinant polypeptide comprises a further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a zoonotic pathogen, characterised in that said first and second epitopes capable of eliciting an immune response against the zoonotic pathogen are distinct from one another.

In one embodiment the zoonotic pathogen is bacteria, a parasite, a fungi, a virus and a prion.

In one embodiment the polypeptide comprises a further non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a first pro-inflammatory protein. In one embodiment the first pro-inflammatory protein is selected from GM-CSF, TNFa, IL-6 IL-10 and soluble IL-2R.

In one embodiment the polypeptide comprises a further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a second pro-inflammatory protein, characterised in that said first and second pro- inflammatory protein are distinct from one another. In one embodiment the second pro- inflammatory protein is selected from GM-CSF, TNFa, IL-6 IL-10 and soluble IL-2R.

In one embodiment said first non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a zoonotic pathogen protein is incorporated into (a) loop I and/or loop II; (b) loop I and/or loop III; (c) loop II and/or loop III; or (d) loop I, loop II and loop III of the dendroaspin scaffold. In one embodiment said further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a zoonotic pathogen is incorporated into either loop I or loop II. In one embodiment said further non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a first pro-inflammatory protein is incorporated into either loop I or loop II. IN one embodiment said further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a second pro-inflammatory protein is incorporated into either loop I or loop II.

In one embodiment the RGD-containing loop is modified by insertion, deletion or substitution of one or more amino acid residues, preferably a maximum of 8 and a minimum of 1 amino acids can be modified within loop III of dendroaspin.

In one embodiment loop I and/or loop II are modified by insertion, deletion or substitution of one or more amino acid residues, preferably a number of amino acid residues in the range 14 to 36.

In one embodiment the said further sequence is inserted into the dendroaspin scaffold between amino acid residues selected from one or more of 2-16, 21-36, 21-31, 28-32, 9-13, 21-33, or at the end of the dendroaspin scaffold after residue 50.

In one aspect the invention provides an expression vector comprising a nucleic acid molecule encoding a recombinant polypeptide according to the invention.

In one aspect the invention provides a recombinant polypeptide according to the invention or the vector according to the invention for use as a medicament.

In one aspect the invention provides a vaccine comprising the recombinant protein according to the invention or the vector according to the invention.

In a further aspect the invention provides a recombinant polypeptide comprising: iii) a dendroaspin scaffold; and iv) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a zoonotic pathogen protein.

In one embodiment, the zoonotic pathogen is bacteria, a parasite, a fungi, a virus and a prion.

In one embodiment, said recombinant polypeptide comprises a further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a zoonotic pathogen, characterised in that said first and second epitopes capable of eliciting an immune response against the zoonotic pathogen are distinct from one another.

In one embodiment the zoonotic pathogen is bacteria, a parasite, a fungi, a virus and a prion. In one embodiment the polypeptide comprises a further non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a first pro-inflammatory protein. In one embodiment the first pro-inflammatory protein is selected from GM-CSF, TNFa, IL-6 IL-10 and soluble IL-2R.

In one embodiment the polypeptide comprises a further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a second pro-inflammatory protein, characterised in that said first and second pro- inflammatory protein are distinct from one another. In one embodiment the second pro- inflammatory protein is selected from GM-CSF, TNFa, IL-6 IL-10 and soluble IL-2R.

In one embodiment said first non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a zoonotic pathogen protein is incorporated into (a) loop I and/or loop II; (b) loop I and/or loop III; (c) loop II and/or loop III; or (d) loop I, loop II and loop III of the dendroaspin scaffold. In one embodiment said further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a zoonotic pathogen is incorporated into either loop I or loop II. In one embodiment said further non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a first pro-inflammatory protein is incorporated into either loop I or loop II. IN one embodiment said further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a second pro-inflammatory protein is incorporated into either loop I or loop II.

In one embodiment the RGD-containing loop is modified by insertion, deletion or substitution of one of more amino acid residues, preferably a maximum of 8 and a minimum of 1 amino acids can be modified within loop III of dendroaspin.

In one embodiment loop I and/or loop II are modified by insertion, deletion or substitution of one or more amino acid residues, preferably a number of amino acid residues in the range 14 to 36.

In one embodiment the said further sequence is inserted into the dendroaspin scaffold between amino acid residues selected from one or more of 2-16, 21-36, 21-31, 28-32, 9-13, 21-33, or at the end of the dendroaspin scaffold after residue 50.

In one aspect the invention provides an expression vector comprising a nucleic acid molecule encoding a recombinant polypeptide according to the invention.

In one aspect the invention provides a recombinant polypeptide according to the invention or the vector according to the invention for use as a medicament.

In one aspect the invention provides a vaccine comprising the recombinant protein according to the invention or the vector according to the invention.

In one aspect the invention provides a recombinant polypeptide comprising: i) a dendroaspin scaffold; and ii) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein.

In one embodiment said first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a SARS-CoV-2 S1 protein receptor binding motif. In one embodiment said first epitope capable of eliciting an immune response against a SARS- CoV-2 S1 protein comprises a polypeptide having at least 90% identity to SEQ ID NO: 2. In one embodiment said first epitope capable of eliciting an immune response against a SARS- CoV-2 S1 protein comprises a polypeptide having at least 90% identity to SEQ ID NO: 11. In one embodiment said first epitope capable of eliciting an immune response against a SARS- CoV-2 S1 protein comprises a polypeptide having at least 90% identity to SEQ ID NO:37.

In one embodiment said first non-dendroaspin amino acid sequence is inserted into dendroaspin loop II or is inserted into dendroaspin loop III.

In one embodiment the recombinant polypeptide further comprises a further non- dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, characterised in that said first and second epitopes capable of eliciting an immune response against a SARS-CoV-2 S1 protein are distinct from one another. In one embodiment said second epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a polypeptide having at least 90% identity to SEQ ID NO:3. In one embodiment, said one or more further epitopes capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises at least one polypeptide selected from: i) a polypeptide having at least 90% identity to SEQ ID NO:3; ii) a polypeptide having at least 90% identity to SEQ ID NQ:30; iii) a polypeptide having at least 90% identity to SEQ ID NO:31; iv) a polypeptide having at least 90% identity to SEQ ID NO:32; v) a polypeptide having at least 90% identity to SEQ ID NO:33; vi) a polypeptide having at least 90% identity to SEQ ID NO:34; vii) a polypeptide having at least 90% identity to SEQ ID NO:35; viii) a polypeptide having at least 90% identity to SEQ ID NO:36; or ix) a polypeptide having at least 90% identity to SEQ ID NO:55.

In one embodiment the recombinant polypeptide further comprises a further non- dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a SARS-CoV-2 S2 protein. In one embodiment said first epitope capable of eliciting an immune response against a SARS-CoV-2 S2 protein comprises a polypeptide having at least 90% identity to SEQ ID NO:5. In one embodiment, said one or more further epitopes capable of eliciting an immune response against a SARS-CoV-2 S2 protein comprises at least one polypeptide selected from: i) a polypeptide having at least 90% identity to SEQ ID NO:5; ii) a polypeptide having at least 90% identity to SEQ ID NO:38; iii) a polypeptide having at least 90% identity to SEQ ID NO:39; iv) a polypeptide having at least 90% identity to SEQ ID NQ:40; v) a polypeptide having at least 90% identity to SEQ ID NO:41; vi) a polypeptide having at least 90% identity to SEQ ID NO:42; or vii) a polypeptide having at least 90% identity to SEQ ID NO:43.

In one embodiment the recombinant polypeptide further comprises a further non- dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1/S2 protein. In one embodiment said epitope capable of eliciting an immune response against a SARS-CoV-2 S1/S2 protein comprises a polypeptide having at least 90% identity to SEQ ID NO:44.

In one embodiment the recombinant polypeptide further comprises a first epitope capable of eliciting an immune response against an IL6 protein. In one embodiment said first epitope capable of eliciting an immune response against an IL6 protein comprises a polypeptide having at least 90% identity to SEQ ID NO:7 or comprises a polypeptide having at least 90% identity to SEQ ID NO:8.

In one embodiment the recombinant polypeptide further comprises a second epitope capable of eliciting an immune response against an IL6 protein characterised in that said first and second epitopes capable of eliciting an immune response against an IL6 protein are distinct from one another. In one embodiment said second epitope capable of eliciting an immune response against eliciting an immune response against an IL6 protein comprises a polypeptide having at least 90% identity to SEQ ID NO:7 or comprises a polypeptide having at least 90% identity to SEQ ID NO:8.

In one aspect the invention provides an expression vector comprising a nucleic acid molecule encoding a recombinant polypeptide according to the invention.

In one aspect the invention provides the recombinant polypeptide according to the invention or the vector according to the invention for use as a medicament.

In one aspect the invention provides a method of eliciting an anti-SARS-CoV-2 response in a mammal comprising administering to said mammal the recombinant polypeptide according to the invention or the vector according to the invention.

In one aspect the invention provides a method of treating or preventing Covid-19 comprising administering to an individual in need thereof a therapeutically effective amount of the recombinant polypeptide according to the invention or the vector according to the invention.

In one aspect the invention provides the recombinant polypeptide according to the invention or the vector according to the invention for use in treating or preventing Covid-19.

In one aspect the invention provides an anti-SARS-CoV-2 vaccine comprising the recombinant peptide according to the invention or the vector according to the invention.

In one aspect the invention provides a method of reducing the viral load of SARS-CoV-2 comprising administering to said patient an effective amount of comprising the recombinant peptide according to the invention or the vector according to the invention.

Detailed description of the invention

The invention is described in detail with reference to the following figures.

Figure 1 shows a schematic structure of the peptide backbone of dendroaspin and its three exposed loops.

Figure 2 shows a schematic presentation of SARS-CoV-2. SARS-CoV-2 S protein binds to ACE2 through its RBD.

Figure 3 shows a schematic representation of SARS-CoV-2-driven signalling pathways and potential drug targets and host intracellular signalling pathways induced by SARS-CoV-2 infection. IKB, inhibitor of nuclear factor KB; NF-KB, p65-p50, nuclear factor KB; GSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage-colony stimulating factor; IP-10, IFN-y-induced protein 10; MCP-1 , monocyte chemoattractant protein 1 ; CCL3, chemokine (C-C motif) ligand 3; JAK, Janus kinase; STAT, signal transducer and activator of transcription; S1 P, sphingosine-1 -phosphate; S1 PRi, sphingosine-1 -phosphate receptor 1 ; MyD88, myeloid differentiation primary response gene 88; TRIF, TIR-domain-containing adapter-inducing IFN-p. Adopted from Catanzaro M, et al., 2020 Immune response in COVID- 19: addressing a pharmacological challenge by targeting pathways triggered by SARS-CoV- 2. Signal Transduct Target Ther. 2020;5:84.

Figure 4 shows the role of TMPRSS2 in SARS-CoV-2 infection (Adopted and modified from The University of Tokyo, 2020).

Figure 5 shows a schematic representation of representative recombinant immunogenic polypeptides of the invention.

Figure 6 shows a sequence alignment of representative recombinant immunogenic polypeptides of the invention (TRI88-V2-V5); The wild-type RBM of SARS-CoV-2 spike protein (TRI88-V1) and dendroaspin (Den).

Figure 7 shows a schematic presentation of molecular modelling (Swiss modelling) of the recombinant constructs. A. TRI88-V1 is a wild type receptor binding motif (RBM) of SARS- CoV-2 spike protein; B. TRI88-V2 has been introduced the Dendroaspin backbone (Dendroaspin scaffold); C. TRI88-V3 maintains V2 structure but with two additional epitopes derived from IL-6, which bind to the IL-6 beta receptor; D. TRI88-V4 has been introduced the other two binding sites beyond RBM in addition to V2; E. TRI88-V5 maintains V4 structure and sequence with additional IL-6 epitope which binds to IL-6 beta receptor. Strikingly, V4 and V5 show a homotrimeric structure similar to that of the receptor-binding domain (RBD) of SARS- CoV-2 spike protein and F. Homotrimeric structure of receptor-binding domain (RBD) of SARS-CoV-2 spike protein.

Figure 8 A shows cloning of recombinant proteins in pGEX-3X vector.

V1 ; B. V2; C. V3; D. V4; E. V5. The cloning sites identified as a shadow and B shows genes of recombinant constructs within pcDNa3.1 HisA plasmid DNAs. A. V1 ; B. V2; C. V4; D. V5. Figure 9 shows A. SDS-PAGE determination of protein purification. B. The purified recombinant protein binds to anti-SARS-CoV-2 S protein in a dose-dependent manner.

Figure 10 shows the purified recombinant protein binds to anti-SARS-CoV-2 S protein antibody in a dose-dependent manner and SDS-PAGE. (A). TRI88-V4; (B). TRI88-V5; (C). SARS-CoV-2 S1. V recognize the SARS-CoV-2 S protein antibody.

Figure 11 shows RIMMS immunization method with injection of antigen at 8 sites of mouse. Figure 12 shows immune response to the recombinant polypeptides of the invention IgG (Figure 12 A-D), and IgM (Figure 12E and F). Antigen-antibody titre was performed using protein constructs coated plate, and anti-sera were taken from mice immunized with TRI88-V1 , TRI88-V2, AGD-Den scaffold, and Alum, respectively (N=8 or 9).

Figure 13 shows A. Principle of Virus Neutralization Assay; B. ePass™ Neutralization Antibody Test. The SARS-CoV-2 Neutralizing Antibody Test Kit can detect circulating neutralizing antibodies against SARS-CoV-2 that blocks the interaction between the receptor-binding domain of the viral S protein (RBD), TRI88-V1 and TRI88-V2 (derived from RBD) with the ACE2 cell surface receptor. The assay detects any antibodies in serum and plasma that neutralize the RBD-ACE2 interaction. The test is both species and isotype Independent Figure 14 shows the results of a Neutralization Antibody Test. A and C. Neutralization using a ePass™ kit purchased from The GenScript; B and D. Inhibition of the interaction between the receptor-binding domain of the viral S protein (RBD) and receptor ACE2. The kit contains two key components: Horseradish peroxidase (HRP) conjugated recombinant SARS-CoV-2 RBD fragment (HRP-RBD) and the human ACE2 receptor (hACE2). If the neutralizing antibodies against SARS-CoV-2 RBD are sufficient in the serum or plasma, the protein-protein interaction between HRP-RBD and hACE2 can be blocked. The Optical Density (OD) of the final solution is read at 450 nm in a microtiter plate reader.

Figure 15 shows Inhibition of SARS-CoV-2 RBD binding to ACE by antisera (plasma) from immunized mice with TRI88-V1 , TRI88-V2, AGD-den, and Alum only. A. Overserved OD values; B. percentage of inhibition.

Figure 16 shows TRI88-V1 and V2 binding to ACE2. Doses of TRI889-V1 (A) and -V2 (B) binds to ACE2 (25ng/ml). TRI88-V1 binds to ACE2 (with different concentrations) (C). Protein with GST-tag is used to stick on the plate for binding assay.

Figure 17 shows TRI88-V4 and V5 binding to ACE2. Doses of TRI88-V4 (A) and -V5 (B) binding to ACE2 (25ng/ml); in different concentrations and comparison with TRI88-V1 (C). Figure 18 shows the purified recombinant protein TRI88-V5 binds to the anti-IL-6 antibody in a dose-dependent manner, A) compared with TRI88-V4 and IL-6 epitopes (P1 and P2). (B) Compared with SARS-CoV-2 RBD and SARS-CoV-2 S1.

Figure 19 shows TRI88-V4 and V5 binding to different doses of human ACE2 when compared with SARS-CoV-2 S1 protein.

Figure 20 shows RI88-V1 and V4 binding to ACE2 compared with SARS-CoV-2 and two epitopes derived from SARS-CoV-2 S1 and S2, respectively.

Figure 21 shows Immune response. IgG (A-D) and IgM (Fig.10 E-H). Antigen-antibody titre was performed using protein constructs coated plate, and anti-sera were taken from mice immunized with TRI88-V4, TRI88-V5, AGD-Den scaffold, and Alum, respectively (N=7 or 10). Figure 22 shows Immune responses. IgG (A-D). Antigen-antibody titre was performed using peptide epitope- coated plate, and anti-sera were taken from mice immunized with TRI88-V4, TRI88-V5, AGD-Den scaffold, and Alum, respectively (N=7 or 10).

Figure 23 shows Live virus neutralisation with antisera (plasma) from mice immunized with TRI88-V4.

Figure 24 shows Inhibition of SARS-CoV-2 RBD binding to ACE2 by plasma from immunized mice with TRI88-V4, TRI88-V5, AGD-den, and Alum, respectively. A. Observed OD values; B. Percentage of inhibition.

Figure 25 shows Plasma cytokines (IFN-y and IL-10) concentrations in immunized mice.

Figure 26 shows T cell responses from TRI88-V4 and -V5-immunized mice spleen stimulated by correspondent vaccine construct.

Figure 27 shows Modelling of Vaccine construct (TRI-V7, trimer) D614G (D150G), A222V (A45V) was not seen in this model (A); SARS-CoV-2 S protein (B) with A222V and D614G mutations.

Figure 28 shows Modelling of S protein with A222V, D614G mutations (enlarged).

Figure 29 shows Modelling of Vaccine construct (TRI-V8, monomer; V7+IL-6 epitope) A222V (A45V) D614G(D174G); B. Modelling of Vaccine construct (TRI-V9, monomer; V8+ MPRSS2 epitope) A222V (A45V); D614G (D174G).

Figure 30 shows Schematic presentation of SARS-CoV-2. (A) S protein domain map; (B) SARS-CoV-2 with three heads and legs. (C) Structure of SARS-CoV-2 nucleocapsid protein (Met1-Ala419). Structure of S glycoprotein with a single receptor-binding domain, using Swiss modeling PDB code: 6vsb, sequence identity 99.26%. (D) Homo-trimer of S protein (matching prediction); and (E) Homo-trimer of S protein 90-degree right turn from (D).

Figure 31 shows Schematic presentation of S protein S1. (A). S1 domain distributions, (B) S1 homo-trimer (matching prediction), PDB code 6vsb.

Figure 32 shows Binding of TRI88-V4 and -V7 to ACE2 in a dose-dependent manner.

Figure 33 shows TRI88-V7 Immune responses. CP-16 derived from S1 aa216-230; SB-2453- 1 derived from S1 aa553-570; SB-2453-2 derived from S2 aa809-826.

Figure 34 shows a Schematic presentation of TRI88-V10 model based on PDB code 6zgf.1.A. Spike glycoprotein: Spike Protein of RaTG13 Bat Coronavirus in Closed Conformation. B. Partial enlarged schematic presentation of spike protein with mutations found from Danish mink strain and 20A.EU1 , a strain accounted for more than eight out of 10 cases in the UK, 80 % of cases in Spain, 60 % in Ireland, and up to 40 % in Switzerland and France.

Figure 35 shows Swiss model of Vaccine constructs (Homo-trimer) compared with that of SARS-CoV-2 S protein. PDB code: 6zgf.1.A.

Figure 36 shows a Swiss model of V12 construct, Homo-trimer PDB code 7bbh.1.A Surface glycoprotein structure of coronavirus spike from smuggled Guangdong Pangolin (A). Cleaved by Furin PDB code: 6zqq.1.A (Spike glycoprotein Furin Cleaved Spike Protein of SARS-CoV-2 with One RBD Erect) (B). Figure 37 shows Predictions of antigenicity for (A)TRI88-V10, (B) V11 and (C) V12.

Figure 38 shows Binding of TRI88-V7(+GST), V11(+GST) and V12 (+GST) to ACE2, respectively compared with GST only (yellow).

Figure 39 shows Immune responses. IgG (A and B). CP-16 derived from S1 aa216-230; SB- 2453-1 derived from S1 aa553-570; CG-23 derived from S1 aa66-89.

Figure 40 shows Immune responses for TRI88-V12.

Figure 41 shows Neutralization Antibody Test for TRI88-V12.

Figure 42 shows Plasma cytokines (IFN-y and IL-10) concentrations in mice immunized with TRI88-V12.

Figure 43 shows T cell responses from TRI88-V12-immunized mice spleen stimulated by V12. Figure 44 shows Alignment of N proteins of CoVs, including SARS-CoV, SARS-CoV-2. MERS-CoV, HKU-CoV, NL63-CoV, 229E-CoV and OC43-CoV.

Figure 45 shows Alignment of N terminal domain of S1 protein of CoVs, including SARS-CoV, SARS-CoV-2. MERS-CoV, HKU-CoV, NL63-CoV, 229E-CoV and OC43-CoV. Showing little homology among these proteins.

Figure 46 shows Alignment of N proteins of CoVs, including SARS-CoV-2. MERS-CoV, NL63- CoV, and 229E-CoV.

Figure 47 shows high homology of N terminus of N protein from NL63-CoV, 229E-CoV and MERS-CoV.

Figure 48 shows high homology of C-terminus of N-protein of HKU1-CoV, OC43-CoV, SARS- CoV & SARS-CoV-2.

Figure 49 provides a schematic presentation of TRI88-V14 and -V15.

Figure 50 shows Modelling of TRI88-V14. Crystal structure of MERS-CoV N-NTD complexed with ligand P4-1. PDB code: 6lnn.1.A, identity: 75.38% (A). Nucleoprotein (Homodimer), Structure of SARS-CoV-2 Nucleocapsid dimerization domain, P1 form PDB code: 6wzo.2.A, identity: 92.86% (B). Spike glycoprotein (homotrimer) Furin Cleaved Spike Protein of SARS- CoV-2 with One RBD Erect, BDP code: 6zgg.1.A, identity: 89.39% (C).

Figure 51 shows Modelling of TRI88-V15. Crystal structure of MERS-CoV N-NTD complexed with ligand P4-1. PDB code: 6lnn.1.A, identity: 75.38% (A). Nucleoprotein (Homodimer), Structure of SARS-CoV-2 Nucleocapsid dimerization domain, P1 form PDB code: 6wzo.2.A, identity: 92.86% (B). Spike glycoprotein (homotrimer) Furin Cleaved Spike Protein of SARS- CoV-2 with One RBD Erect, BDP code: 6zgg.1.A, identity: 73.03% (C).

Figure 52 shows Prediction of antigenicity of TRI88-V14 (A) and -V15 (B).

Figure 53 shows TRI88-V14 (GST-Tagged) and V15 (GST-tagged) binding to human ACE2 in a dose dependent manor when compared with GST control, and sample in ELISA assay.

Figure 54 shows TRI88-V4 induced Chicken IgY Immune responses (Prepl and Prepll denote preparation of protein stage I and II, respectively). Figure 55 shows a schematic presentation of the solution structure of domain III (Dill) of Zika virus Envelope protein.

Figure 56 shows a sequence alignment of representative recombinant immunogenic polypeptides of the invention (TRI88-ZV1-ZV2 and dendroaspin (Den).

Figure 57 shows a schematic presentation of the ZIKV E motif.

Figure 58 shows the polypeptide sequence of the chain E Zika structural E protein.

Figure 59 shows a Schematic presentation of Envelope protein. (A). The overall structure of ZIKV. (B). Monomer. (C). Domain I. Crystal structure of domain I of the envelope glycoprotein ectodomain from dengue virus serotype 4 in complex with the Fab fragment of the chimpanzee monoclonal antibody 5H2

Figure 60 shows a bar chart displaying T cell activation and proliferation in response to antigen presentation by dendritic cells. The recombinant antigens used are LPs, BCg, HSP65 and dendroaspin.

Figure 61 shows a pie-chart comparison of cytokine (TNFa, I FNy, TGFp and IL10) production following T cell activation in response to LPS and in response to native dendroaspin (2F1). Figures 62A- F displays FACS data showing that in vitro culture with dendroaspin activates T reg cells.

Figure 63 shows a bar chart of the effect of dendroaspin pretreatment on TNF- a concentration in culture supernatant of macrophages activated with LPS in MDDC cells (Figure 63A) and J774 cells (Figure 63B).

Figure 64 shows a bar chart of the effect of dendroaspin on four IgG subtype responses to GST.

Figure 65 shows a bar chart of the increases in the percentage of Foxp3 positive T reg cells in lymphoid organs (lymph nodes and spleen) following oral dosing.

Figure 66 shows a bar chart of the reduction in percentage lesion area in total surface area (Figure 66A) and macrophage infiltration (Figure 66B) in DSP treated mice.

Figure 67 shows a bar chart of the increase in the percentage of Foxp3 positive T reg cells in the spleens of rabbits following oral dosing with dendroaspin.

Figure 68 shows a bar chart of the reduction in lesion area in total surface area in the aortic sinus of DSP treated mice.

Figure 69 shows a bar chart of the reduction in macrophage infiltration as a percentage of CD68 positive area in lesions of DSP treated mice.

Recombinant immunogenic polypeptides

The inventors have identified that a dendroaspin scaffold lends itself to the rapid generation of immunogenic recombinant polypeptides, capable of eliciting multiple different immune responses against a zoonotic infection. The broad applicability of the scaffold to rapid development of anti-zoonotic infection immunogenic recombinant polypeptides has been exemplified in the context of immunogenic recombinant polypeptides that produced neutralizing antibodies against Zika Virus E protein and also in the context of immunogenic recombinant polypeptides that produced neutralizing antibodies against SARS-CoV-2 that block the interaction between the receptor-binding domain of the viral S protein (RBD) and the ACE2 surface receptor.

In particular the inventors have developed an immunomodulatory Covid-19 therapy designed to stimulate anti-SARS-CoV-2 antibodies by administration of epitopes in a dendroaspin scaffold platform to a subject. Advantageously, the recombinant polypeptide can be expressed in E.coli with a high yield.

The inventors have demonstrated, in a murine model, that immunization with the recombinant polypeptides of the invention produced neutralizing antibodies against SARS- CoV-2 that blocked the interaction between the receptor-binding domain of the viral S protein (RBD) and the ACE2 surface receptor.

The recombinant immunogenic polypeptides comprise a SARS-CoV-2 S1 protein receptor binding motif (RBM) within a dendroaspin scaffold and are able to bind to human ACE2. The inventors have shown that such recombinant polypeptides are highly immunogenic and elicit high titre of anti-SARS-CoV-2 IgG and IgM antibodies. Moreover, the inventors have demonstrated that the recombinant polypeptides of the invention induce neutralizing antibodies in mice, and that said neutralizing antibodies can block SARS-CoV-2 binding to ACE2.

Advantageously, the molecular size of the recombinant polypeptide is smaller (9.6 kD) than either SARS-CoV-2 S1 (78.3 kD) or SARS-CoV-2 RBD (25 kD) and can thus be easily manipulated towards potent antibody neutralization.

Moreover, the use of the dendroaspin scaffold provides recombinant polypeptides that have multiple functionalities, such as multiple immunogenic functionalities. Accordingly, the inventors have demonstrated that recombinant polypeptides of the invention may be mono-, bi- or multi- functional in their immunogenic activities against Covid-19. The inventors have introduced additional anti-SARS-CoV-2 epitopes and/or anti-l L6 epitopes into the recombinant polypeptides which advantageously increase the potency of the immune response that the recombinant polypeptide elicits in a host. Because the S protein of SARS-CoV-2 is involved in receptor recognition, as well as virus attachment and entry (Figure 2), it represents one of the most critical targets for the design of vaccines or therapeutics.

Additionally, the nucleocapsid (N) protein of SARS-CoV-2 has been identified as an epitope target for inclusion in a vaccine of the invention. N protein has high homology between mutated variants of SARS-CoV-2, and so adding the N protein epitope might enhance immunogenicity of vaccine candidates targeting specific (variant) S protein epitopes in SARS-CoV-2. N protein also has high homology between different coronaviruses, as outlined in Figures 44, 46, 47 and 48 and so advantageously offers a foundation for a pan- coronavirus vaccine platforms. A comparison of the known coronavirus pathogens is provided in Table 9.

The CoV N protein N-Terminal Domain (NTD) fold consists of a five-stranded, antiparallel β- sheet with the topology P4-P2-P3-P1-P5, partially encapsulated within extended, intertwining loops and turns connecting the p-strands (PDB:5N4K).

The C-Terminal Domain (CTD) of CoV N protein exists in dimeric form. Each CoV N CTD monomer contains five a-helices, three 3(10) helices, and two p-strands and assumes an a1-4pip2a5 topology (PDB:5EPW). N protein is present in the helical nucleocapsid and the internal spherical/icosahedral core [35], The inner core consists of N protein, RNA, and the CTD of M protein. The N protein, therefore, plays an essential structural role in the CoV virion through a network of interactions with (i) the genomic RNA, (ii) M protein, and (iii) other N proteins.

The function of the N proteins of CoV, such as SARS-CoV, and of arenaviruses (such as the Lassa virus that can cause severe and lethal hemorrhagic fever infections in humans), is to support the progress of the polymerase during the elongation phase. NP is unidirectionally recruited to nascent ribonucleoprotein (RNP)s through homo-oligomerization and independently of RNA binding and plays a vital role in forming RNP complexes and in causing type I interferon (IFN) suppression.

Also, the Assembly of CoV virions not only requires CoV N protein dimerization and association with viral genomic RNA to form RNPs, but also protein-protein interactions amongst the four structural proteins (the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein), as well as a host membrane envelope obtained from the site of budding. More recently, however, it has become clear that some CoVs do not require the whole ensemble of structural proteins to form a complete infectious virion, suggesting that some structural proteins might be dispensable or that these CoVs might encode additional proteins with overlapping compensatory functions.

The SARS-CoV N protein's C-terminus could bind to a C-terminal PRYSPRY domain, a protein-protein interaction module. The SPRY domain of the cellular tripartite motif protein 25 (TRIM25) E3 ubiquitin ligase plays a vital role in post-translational modification of the N terminal caspase recruitment domains (CARDs) of the retinoic acid-inducible gene I (RIG-1) by ubiquitination. RIG-1, as a cytosolic pattern recognition receptor (PRR) is responsible for the type-1 interferon (IFN 1) response and for recognizing cells that have been infected with a virus in the innate immune system. CARDs are essential for interactions with mitochondrial antiviral signaling protein (MAVS) .

To date, most of the potent nAbs to SARS-CoV-2 target the RBD. In addition, nAbs targeting the NTD have been reported in SARS-CoV-2 and MERS-CoV infection, making it another potential target for inclusion in a vaccine. N-specific antibodies were reported to protect mice against mouse hepatitis virus, a mouse CoV, via Fc-mediated effector functions.

Immunization with N protein can also elicit CD4 ⁺ and CD8 ⁺ T cell responses in mice. N- specific CD8 ⁺ T cell epitopes are known to protect chickens against IBV infection.

Additionally, IL-6 plays a significant role in COVID-19. This infectious disease induces a pro- inflammatory generation and secretion of cytokines or a cytokine storm, leading to the acute respiratory distress syndrome (ARDS) in COVID-19 patients, which is more common in immune system -related diseases or immune-related therapy, such as virus infection. ARDS is a life-threatening condition in which lungs become so inflamed and filled with fluid that they struggle to provide enough oxygen to the body. To rescue the patients from this condition, it is vital to understand how SARS-CoV-2 triggers the cytokine storm, which leads to ARDS (Figure 3).

Furthermore, transmembrane serine protease 2 (TMPRSS2), a cell-surface protein that is expressed by epithelial cells of specific tissues such as those in the aerodigestive tract and the luminal side of the prostate epithelium. TMPRSS2 contains a type II transmembrane domain (aa 84-106), a receptor class A domain, a scavenger receptor cysteine-rich domain, and a protease domain. The results suggested that SARS-CoV-2 employed TMPRSS2 for SARS-CoV-2 S protein priming and S protein-driven cell entry (Figure 4). Camostat mesilate (CM), an inhibitor of TMPRSS2, blocked the spread and pathogenesis of SARS-CoV in a pathogenic mouse model and would be expected to show a similar effect in MERS-CoV. Virus entry is a multistep process. This protein also facilitates the entry of viruses into host cells by proteolytically cleaving and activating viral envelope glycoproteins. This process can be blocked by its inhibitors such as camostat mesylate (CM) demonstrated in a sample of SARS-CoV-2 virus isolated from a patient who was found the CM blocking the entry of the virus into the lung cells.

Additionally, the inventors have demonstrated that the recombinant immunogenic polypeptides of the invention are able to inhibit binding between SARS-CoV-2 spike protein and ACE2, offering a peptide therapeutic for treating COVID-19.

Accordingly, in a first aspect the invention provides a recombinant immunogenic polypeptide comprising: i) a dendroaspin scaffold; and ii) a non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope a first epitope capable of eliciting an immune response against a zoonotic pathogen protein.

Accordingly, in a further aspect the invention provides a recombinant immunogenic polypeptide comprising: i) a dendroaspin scaffold; and ii) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein.

As used herein the term "polypeptide" relates to a polymer comprising amino acids linked together by peptide bonds. Immunogenic polypeptides, preferably, elicit protective immune response when administered to a host, preferably, a mammal, more preferably a human. The recombinant immunogenic polypeptide of the invention, preferably, establishes or improves immunity to infection with SARS-CoV-2. Such recombinant immunogenic polypeptides are preferred reagents for vaccine compositions.

Dendroaspin scaffold

Dendroaspin is a short chain neurotoxin homologue from the venom of Elapidae snakes, which lacks neurotoxicity.

Unlike neurotoxins, it contains an Arg-Gly-Asp-(RGD)-motif and functions as an inhibitor of platelet aggregation and platelet adhesion with comparable potency to the disintegrins from the venoms of Viperidae. The structure of dendroaspin in solution has been determined using NMR spectroscopy. The structure contains a core similar to that of short chain neurotoxins, but with a novel arrangement of loops and a solvent-exposed RGD-motif. Dendroaspin is thus an integrin antagonist with a well defined fold different from that of the disintegrins, based on the neurotoxin scaffold.

The structure of dendroaspin consists of a core region from which three loops, denoted 1, 11 and III (residues 4-16, 23-36 and 40-50) extend outwards (Figure 1). The core contains the four disulphide bonds which are spatially close to each other and hold the loops together. Prior to inclusion of non-dendroaspin amino acid sequences the dendroaspin scaffold of the invention comprises a dendroaspin polypeptide of SEQ ID NO:9, or a fragment or variant thereof having dendroaspin activity. In one embodiment, excluding non-dendroaspin amino acid sequences, the dendroaspin variant comprises a polypeptide sequence having at least 70%, 75%, 80%, 85%, 90, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:9.

In one embodiment the dendroaspin scaffolds of the recombinant polypeptides of the invention may comprise a greater or lesser number of amino acid residues compared to the 59 amino acids of dendroaspin of SEQ D NO:9. For example, the dendroaspin scaffolds of the invention may comprise a number of amino acid residues in the range 45 to 159, preferably about 49 to 89, more preferably about 53 to 69, even more preferably about 57 to 61 of SEQ ID NO:9.

Loop I comprises amino acid residues 4-16, loop II residues 23-36 and loop III residues 40- 50 of SEQ ID NO:9. However, in certain embodiments the non-dendroaspin amino acid sequences incorporated into the dendroaspin scaffold may extend into or substitute regions external to the loops, i.e. residues 1-3, 17-22 and 37-39 such that residues of the non-loop regions are augmented or substituted for those of the further amino acid sequence or sequences being inserted.

In certain embodiments a non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein is incorporated into (a) loop I and/or loop II; (b) loop I and/or loop III; (c) loop II and/or loop III; or (d) loop I, loop II and loop III of the dendroaspin scaffold.

In certain embodiments a non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein is incorporated into (a) loop I and/or loop II; (b) loop I and/or loop III; (c) loop II and/or loop III; or (d) loop I, loop II and loop III of the dendroaspin scaffold.

In certain embodiments a non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a SARS-CoV-2 S2 protein is incorporated into (a) loop I and/or loop II; (b) loop I and/or loop III; (c) loop II and/or loop III; or (d) loop I, loop II and loop III of the dendroaspin scaffold.

In certain embodiments a non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against an IL-16 protein is incorporated into (a) loop I and/or loop II; (b) loop I and/or loop III; (c) loop II and/or loop III; or (d) loop I, loop II and loop III of the dendroaspin scaffold.

The amino acid sequence of dendroaspin Loop I is shown in SEQ ID NO: 18. The amino acid sequence of dendroaspin Loop II is shown in SEQ ID NO: 19. The amino acid sequence of dendroaspin Loop III is shown in SEQ ID NQ:20.

A preferred location for the inserted non-dendroaspin amino acid sequences is at a site in dendroaspin scaffold between amino acid residues: 4-16, 18-21, 23-36, or 52-59.

When two non-dendroaspin amino acid sequence are inserted into the dendroaspin scaffold the linear distance between each of the non-dendroaspin amino acid sequences is preferably in the range 1-35 amino acids, more preferably 1-14 amino acids. When more than two non-dendroaspin amino acid sequence are then there is preferably at least one native dendroaspin amino acid residue separating each further amino acid sequence.

Loop I and/or loop II and/or loop III of the dendroaspin scaffold may additionally be modified by insertion, deletion or substitution of one or more amino acid residues at amino acid residues 4-16 and/or amino acids 23-36 and/or loop III residues 40-50 of SEQ ID NO:9 respectively. Any suitable number of non-dendroaspin amino acid sequences can be inserted into the dendroaspin scaffold to give the desired mono-, bi- or multi-functional activity.

In certain embodiments, the dendroaspin scaffold may contain modifications in Loop I and/or loop II and/or loop III so as to counteract a steric hindrance effect resulting from insertion of a non-dendroaspin amino acid sequence.

Computer assisted molecular modelling using Insight II software (Molecular Simulations Inc) can be used to predict the structure of the "loop grafted" dendroaspins of this invention. In instances where steric effects between the loops may serve to cause loss of functionality, these effects can be "designed out" by modifying appropriate parts of the dendroaspin molecule in an appropriate way. In some embodiments this may involve inserting a number of suitable amino acid residues to extend one or more of the loop structures. Preferred modification includes the insertion of polyglycine into the loop or loops of the dendroaspin scaffold in order to extend them. Other modifications comprising repeat units of an amino acid residue or number of residues can be used. Computer modelling studies can be used to design the loop modifications needed in order to extend the loops of dendroaspin.

In some embodiments, non-dendroaspin amino acid sequences can be covalently attached to the dendroaspin scaffold via a linker. In some embodiments, the first linker and/or the second linker are independently a variable length poly Gly linker or Gly-Ser linker.

In the design of a bifunctional or multifunctional molecule in accordance with the invention, "fine tuning" of activity, stability or other desired biological or biochemical characteristic may be achieved by altering individual selected amino acid residues by way of substitution or deletion. Modification by an insertion of an amino acid residue or residues at a selected location is also within the scope of this "fine tuning" aspect of the invention. The site-directed mutagenesis techniques available for altering amino acid sequence at a particular site in the molecule will be well known to a person skilled in the art.

The polypeptide of the invention comprises at least one non-dendroaspin amino acid sequence encoding a first epitope capable of eliciting an immune response against a SARS- CoV-2 S1 protein. Preferably, the polypeptide of the invention comprises at least two non- dendroaspin amino acid sequences.

In one embodiment, where the recombinant polypeptide comprises at least two non- dendroaspin amino acid sequences, the at least two non-dendroaspin amino acid sequences are separated by at least one amino acid residue of dendroaspin. The two or more non- dendroaspin amino acid sequences may be transposed with respect to one another and to the linear order of amino acids in the native further amino acid sequence. In other words, the native order of the two or more amino acid sequence portions is altered although the actual sequences of each portion may not necessarily be altered.

Non-dendroaspin amino acid sequences

As used herein “non-dendroaspin amino acid sequence” refers to an amino acid sequence not found in the wild-type dendroaspin, i.e. a non-dendroaspin domain. Preferably, said non- dendroaspin amino acid sequence is an immunogenic sequence capable of eliciting an immune response in a mammalian host, preferably a human.

As used herein, the term "epitope" refers to a particular molecular surface feature of an antigen, for example a fragment of an antigen, which is capable of being bound by at least one antibody. Antigens usually present several surface features that can act as points of interaction for specific antibodies. Any such distinct molecular feature constitutes an epitope. On a molecular level, an epitope therefore corresponds to a particular molecular surface feature of an antigen (for example a fragment of an antigen) which is recognized and bound by a specific antibody.

As used herein “eliciting an immune response” refers to the ability a polypeptide sequence to elicit protective immune response in a mammalian host, preferably, in a human. Eliciting an immune response can involve establishing or improving immunity to infection. The immune response may be a cell mediated immune response or a humoral immune response or both. Eliciting a humoral immune response, as used herein, refers to the ability of a polypeptide sequence to cause cells of the immune system to produce antibodies that bind specified protein targets. Methods of determining the ability of a polypeptide to elicit an immune response are well known in the art and include Immunogenic analyses, Neutralization assays such as, ePass™ SARS CoV-2 Neutralization Antibody Detection Kit (as used in the Examples), live SARS-CoV-2 neutralization assays (Public Health England [PHE] plaque reduction neutralization test [PRNT I C50] , PHE microneutralization assay [MNA IC50, ICso, IC90], and Marburg virus neutralization [VN IC100]), and a pseudovirus neutralization assay (PseudoNA IC50). In one embodiment, immune response can be monitored using a SARS- CoV-2 Pseudovirus inhibition assay (PHE microneutralization assay), in which Pseudovirus- containing supernatants are incubated with serially diluted mouse sera at 37°C for 1 h before adding to target cells, 293T- cells expressing ACE2 or ACE-2 coated (pre-coated in 96-well culture plates at 10 ⁴ cells/well). Twenty-four hours later, cells are refed with fresh medium, be followed by lysing cells 72 h later using cell lysis buffer (Promega) and transfer of the lysates into 96-well luminometer plates. Luciferase substrate (Promega) is added to the plates, and relative luciferase activity determined by a Luminometer. The inhibition of SARS-CoV-2 pseudovirus is presented as % inhibition. Alternatively, immune response can be monitored by measuring neutralization activity, in which neutralization activity is measured by microneutralization assay in vitro. The virus microneutralization (MN) test is performed on Human embryonic kidney (HEK) 293T-ACE2 cells. 293 T-cells will be grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) FBS). 293 T-cells will be infected with SARS-CoV-2 (2019-nCoV) spike pseudovirus treated with serial dilutions of neutralizing antibody. The infection is neutralized by increasing concentrations of Anti-SARS-CoV-2 neutralizing antibody (mouse anti-sera compared with commercial antibody: Catalog # 40591 - MM43 purchased from http://www.sinobioloqical.com). 293T-cells alone expressing human ACE2 (hACE2) receptor (Human ACE2 Stable Cell Line, CSC-ACE01 - HEK293T purchased from info@creative-diaqnostics.com) (293T-cell without ACE2 expression are used as a control). Rate of inhibition is determined by comparing the Relative Light Unit (RLU) of Luciferase reporter in different antibody concentrations (luminometer is used).

As used herein, the terms “homology” and “identity” are used interchangeably. Calculations of sequence homology or identity between sequences are performed as follows.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSLIM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

Zoonotic pathogens

In a first aspect the invention provides a recombinant immunogenic polypeptide comprising: i) a dendroaspin scaffold; and ii) a non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope a first epitope capable of eliciting an immune response against a zoonotic pathogen protein.

As used herein, the term "zoonotic" refers to an infectious disease that may be transmitted between species. By the term "pathogen" is meant any virus, microorganism, or other substance causing disease A “zoonotic pathogen” may refer to a pathogenic antigen from a bacteria, a parasite, a fungi, a virus and a prion.

In one embodiment the zoonotic pathogen is a bacterial pathogen, such as Bacillus anthracis, Clostridium botulinum, a Brucella spp., a Campylobacter spp, Escherichia coli, a Leptospira spp., Yersinia pestis

In one embodiment the zoonotic pathogen is a viral pathogen such as a zoonotic influenza virus such as an avian influenza virus, for example A(H5N1) or A(H7N9), a Chikungunya virus, a Dengue virus, an Arenaviridae, a Bunyaviridae, such as a Nairovirus, a Filoviridae, a Flaviviridae, such as a Japanese encephalitis (JE) virus or a zika virus, an Ebolavirus, such as Zaire ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Bundibugyo ebolavirus, Reston ebolavirus, or Bombali ebolavirus, an Arenavirus, such as Lassa virus, a Marburg virus, a Phlebovirus, such as a Rift Valley fever virus, a Leishmaniavirus, a coronavirus, such as MERS-CoV, SARS-CoV, SARS-CoV-2, or Rabies lyssavirus.

In one embodiment the zoonotic pathogen is a parasite such as Trypanosoma cruzi, Echinococcus granulosus, Echinococcus multilocularis, a Clonorchis, an Opisthorchis, a Fasciola and a Paragonimus, Taenia saginata, Taenia solium, and Taenia asiatica, In one embodiment the zoonotic pathogen is a zika virus.

Zika virus (ZIKV) is a mosquito-borne flavivirus transmitted by the bite of an infected mosquito from the Aedes genus, mainly Aedes aegypti, in tropical and subtropical regions. The first recorded outbreak of Zika virus disease was reported from the Island of Yap (Federated States of Micronesia) in 2007. In March 2015, Brazil reported a massive outbreak of rash illness, soon identified as Zika virus infection. ZIKV was found to be associated with Guillain-Barre syndrome (GBS), a form of temporary paralysis in adults, that has links to other neurological complications, neuropathy and myelitis. Symptoms are generally mild and include fever, rash, conjunctivitis, muscle and joint pain, malaise or headache. Symptoms typically last for 2-7 days. Most people with Zika virus infection do not develop symptoms.

No vaccine is yet available for the prevention or treatment of Zika virus infection. The development of a Zika vaccine remains an active area of research.

In one embodiment, the recombinant polypeptide comprises a further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a zoonotic pathogen. In one embodiment said first and second epitopes capable of eliciting an immune response against a zoonotic pathogen are distinct from one another. In one embodiment said zoonotic pathogens against which said first and second epitopes are capable of eliciting an immune response against are distinct from one another.

In one embodiment, the recombinant polypeptide comprises at least two, three or four further non-dendroaspin amino acid sequences comprising a second, third and fourth epitope capable of eliciting an immune response against a zoonotic pathogen.

In this way the molecules of the invention may be rendered multifunctional so that they are active against more than just one epitope.

IL-6 is a pro-inflammatory cytokine and can be released in response to viral infections. In a study on 150 patients from Wuhan, China, IL-6, ferritin, and C reactive protein (CRP) were elevated in patients who died in comparison with those who survived). These elevated laboratory markers suggest the possibility of hypercytokinemia. in COVID-19 patients. Other pro-inflammatory factors include GM-CSF, TNFa, IL-10 and soluble IL-2R. In one embodiment, the recombinant polypeptide comprises a further non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a pro-inflammatory protein. In one embodiment the first epitope is capable of eliciting an immune response against a pro-inflammatory protein that inhibits or reduces that proteins pro-inflammatory activity. In one embodiment the pro-inflammatory protein is an IL-6 protein and the epitope elicits an immune response that inhibits or reduces IL-6 pro-inflammatory activity, for example by inhibiting or reducing binding to the IL6 receptor, CD126. In one embodiment the IL6 is human IL6. In one embodiment said first epitope capable of eliciting an immune response against an IL6 protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:7 or SEQ ID NO:8. In one embodiment said first epitope capable of eliciting an immune response against an IL6 protein comprises or consists of SEQ ID NO:7 or SEQ ID NO:8.

In one embodiment, the recombinant polypeptide comprises a further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a pro-inflammatory protein. In one embodiment the second epitope is capable of eliciting an immune response against a pro-inflammatory protein that inhibits or reduces that proteins pro-inflammatory activity. In one embodiment said first and second epitopes capable of eliciting an immune response against a pro-inflammatory protein are distinct from one another. In one embodiment said pro-inflammatory proteins against which said first and second epitopes are capable of eliciting an immune response against are distinct from one another.

In one embodiment, the second epitope is capable of eliciting an immune response against an IL6 protein. In one embodiment the second epitope elicits an immune response that inhibits or reduces IL-6 pro-inflammatory activity, for example by inhibiting or reducing binding to the IL6 receptor, CD126. In one embodiment the IL6 is human IL6. In one embodiment, the first and second epitopes capable of eliciting an immune response against a pro-inflammatory protein each elicit an immune response against IL6, characterised in that said first and second epitopes capable of eliciting an immune response against an IL6 protein are distinct from one another. In one embodiment, said second epitope capable of eliciting an immune response against an IL6 protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:7 or comprises a polypeptide comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:8. In one embodiment, said second epitope capable of eliciting an immune response against eliciting an immune response against an IL6 protein comprises or consists of SEQ ID NO:7 or comprises or consists of SEQ ID NO:8.

In one embodiment, the recombinant polypeptide comprises dendroaspin Loop I comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 18, dendroaspin Loop II comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 19, and dendroaspin Loop III comprising or consisting of the amino acid sequence as shown in SEQ ID NQ:20.

In one embodiment, the recombinant polypeptide comprises dendroaspin Loop I comprising or consisting of the amino acid sequence as shown in SEQ ID NO:24, dendroaspin Loop II comprising or consisting of the amino acid sequence as shown in SEQ ID NO:25, and dendroaspin Loop III comprising or consisting of the amino acid sequence as shown in SEQ ID NO:26.

In one embodiment the zoonotic pathogen is a coronavirus, such as MERS-CoV, SARS- CoV, or SARS-CoV-2.

SARS-CoV-2

In a further aspect the recombinant polypeptide comprises a dendroaspin scaffold and a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein.

The invention refers to certain positions of proteins. By "position" as used herein is meant a location in the sequence of a protein. Corresponding positions are determined alignment with other parent sequences. By " residue " as used herein is meant a position in a protein and its associated amino acid identity. Various epitopes used in the present invention are defined comprise amino acid modifications that compose them relative to a parent sequence, as used herein “modified” is meant a polypeptide sequence differs from that of a parent wild type sequence by virtue of at least one amino acid modification.

The modification can be an addition, deletion, or substitution. Thus, for example, AA ²⁰⁹ ' ²³⁰ SARS-COV-2 S1 protein (modified with A222V) is a SARS-CoV-2 S1 protein variant with the substitution A222V relative to the parent wild type SARS-CoV-2 S1 protein polypeptide. Reference to positions in the SARS-CoV-2 S1 protein is sequential, with residue 1 corresponding to amino acid 1 in SEQ ID NO:56. The numbering includes the signal peptide, amino acids 1 to 13 of SEQ ID NO:56. Reference to positions in the SARS-CoV-2 S2 protein is sequential, with residue 685 corresponding to amino acid 1 in SEQ ID NO:4 Host cell entry of coronaviruses is mediated by a transmembrane homotrimeric class I fusion glycoprotein, the spike protein (S protein), which two functional subunits: the S1 subunit for binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2) (also known as the Receptor Binding Domain(RBD)); and the S2 subunit for fusion of the viral and host cell membranes. The S1 subunit has an amino acid sequence as shown in SEQ ID NO:1. The S2 subunit has an amino acid sequence as shown in SEQ ID NO:4.

The S1 subunit comprises a Receptor Binding Motif (RBM) at amino acids 437 to 508 of SEQ ID NO:1. The S1 RBM has an amino acid sequence as shown in SEQ ID NO:2. In one embodiment the first epitope capable of eliciting an immune response against a SARS-CoV- 2 S1 RBM protein.

In preferred embodiments, the invention incorporates a S1 RBM modified to incorporate 6 cysteine residues, which facilitate an advantageous disulfide bridge pattern.

In one embodiment the first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO:11. In one embodiment, the first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises or consists of SEQ ID NO: 11.

In one embodiment the first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO:37. In one embodiment, the first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises or consists of SEQ ID NO: 37.

In one embodiment the first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a Receptor Binding Motif (RBM), amino acids 437 to 508 of SEQ ID NO:1 or variant thereof having having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2. In one embodiment, the first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises or consists of SEQ ID NO: 2. In one embodiment the first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a Receptor Binding Motif (RBM) sequence of SEQ ID NO:11. In one embodiment the first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a Receptor Binding Motif (RBM) at amino acids of SEQ ID NO:37. In certain embodiments the first epitope comprising the aforementioned RBM is modified to include a disulfide bond, e.g. a disulfide bond between two cysteine residues in the RBM. In one embodiment the recombinant polypeptide comprises one or more additional non-dendroaspin amino acid sequences comprising one or more further epitopes capable of eliciting an immune response against a SARS-CoV-2 S1 protein, characterised in that said first and one or more further epitopes capable of eliciting an immune response against a SARS-CoV-2 S1 protein are distinct from one another.

In some embodiments, the recombinant polypeptide comprises 2, 3, 4, 5, 6, 7 or 8 further epitopes capable of eliciting an immune response against a SARS-CoV-2 S1 protein are distinct from one another.

Said one or more further epitopes capable of eliciting an immune response against a SARS- CoV-2 S1 protein comprises at least one polypeptide is selected from: i) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3; ii) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NQ:30; iii) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:31 ; iv) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:32; v) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:33; vi) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:34; vii) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:35; viii) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:36; or ix)a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:55.

In one embodiment, the recombinant polypeptide comprises one or more additional non- dendroaspin amino acid sequence comprising one or more epitopes capable of eliciting an immune response against a SARS-CoV-2 S2 protein. In some embodiments, the recombinant polypeptide comprises 2, 3, 4, 5, 6 or 7 additional epitopes capable of eliciting an immune response against a SARS-CoV-2 S2 protein that are distinct from one another.

Said one or more additional epitopes capable of eliciting an immune response against a SARS-CoV-2 S2 protein comprises at least one polypeptide is selected from: i) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5; ii) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:38; iii) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:39; iv) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NQ:40; v) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:41 ; vi) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:42; vii) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:43 or viii) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:44.

In one embodiment, an epitope capable of eliciting an immune response against a SARS- CoV-2 S1 protein comprises an antigenic fragment of SEQ ID NO:1.

In one embodiment, an epitope capable of eliciting an immune response against a SARS- CoV-2 S2 protein comprises an antigenic fragment of SEQ ID NO:4.

The S protein (S1 N-Terminal Domain, NTD) does not generalise between different CoVs: it has low homology and a high degree of mutation, as illustrated in Figure 45.

SARS-CoV-2 mutations in the gene encoding Spike (S) protein are continuously reported. The first SARS-CoV-2 genome reported on January 5, 2020 has about 80% sequence identity with that of SARS-CoV. However, compared with SARS-CoV, SARS-CoV-2 S protein has 725 mutations over its 1273 residues. Their sequence identity is only 76%. Among 725 mutations on the SARS-CoV-2 S protein, 89 were on the RBD (AA319-541), which has a total of 194 residues, suggesting that the RBD is subject to more mutations.

Recent studies using over 15,000 genome samples show that the SARS-CoV-2 S protein is among the most non-conservative ones in its genome. Recently, the study suggested that the increased fatality rate may be linked with the most dominant variant D614G, which was a particularly high frequency 20/23 (87%) among Italian SARS-CoV-2 sequenced specimens, which was then emerging as the most severely affected country outside of China, with an overall case fatality rate of 7.2%. Presumably, this change may have induced a conformational change in the S protein, resulting in the increased infectivity.

Experiments show that S protein mutation D614G also has made SARS-CoV-2 more infectious. However, it remains largely unclear whether these reported variants could influence viral infectivity, transmissibility, or reactivity with neutralizing antibodies.

Strikingly, an international team of scientists that has been tracking the virus through its genetic mutations has described the extraordinary spread of the variant, called 20A.EU1, a mutation of A222V in the spike protein. The research showed that this strain accounted for more than eight out of 10 cases in the UK, 80% of cases in Spain, 60% in Ireland, and up to 40% in Switzerland and France. This was identified (as Spanish-coronavirus-strain) in 12 European countries. The A222V is located within the B cell immunodominant epitopes. As such, Spike protein A222V is a potential site eliciting CD4 ⁺ T cell responses.

In Denmark, there have been five clusters of mink variants of SARS-CoV-2; the Danish State Serum Institute (SSI) has designated these as clusters 1-5 (Danish: cluster 1-5). There have been changes to the H69del/V70del, Y453F, I692V, and M1229I amino acids.

World Health Organization (WHO) routinely assesses if variants of SARS-CoV-2 result in changes in transmissibility, clinical presentation, and severity, or if they impact on countermeasures, including diagnostics, therapeutics, and vaccines. As mentioned above, the spike D614G substitution was demonstrated to enhance viral replication in the upper respiratory tract and increases the susceptibility of the virus to neutralization by antibodies.

In addition, a distinct phylogenetic cluster (named lineage B.1.1.7) was detected within the COG-UK surveillance dataset, including N501Y, which increases ACE2 receptor affinity and P681H, one of 4 residues comprising the insertion that creates a furin cleavage site between S1 and S2 in spike. The S1/S2 furin cleavage site of SARS-CoV-2 has been shown to promote entry into respiratory epithelial cells and transmission in animal models. The deletion of two amino acids at sites 69-70 in the S protein has been observed in multiple lineages linked to several RBD mutations. This includes the mink-associated outbreak in Denmark (in the background of the Y453F RBD mutation), and in humans in association with the N439K RBD mutation, accounting for its relatively high frequency in the global genome data (-3000 sequences). Variants with Y453F, unrelated to the Danish and Dutch variants, have also been reported sporadically from other countries (the Russian Federation, South Africa, Switzerland, and the United States) in the GISAID EpiCoV sequence database. On 5 November 2020, the Danish public health authorities reported the detection of a mink- associated SARS-CoV-2 variant with a combination of mutations not previously observed (referred to as “Cluster 5”) in 12 human cases in North Jutland, detected from August to September 2020. The Cluster 5 variant strains carry I692V, M 12291 for a total of four S protein mutations. M 12291 is present throughout much of Europe, with a peak in the Czech Republic.

A new SARS-CoV-2 virus variant referred to in the UK as SARS-CoV-2 VUI 202012/01 is defined by multiple S protein mutations (deletion 69-70, deletion 144, N501Y, A570D, D614G, P681 H, T716I, S982A, D1118H) as well as mutations in other genomic regions [33], The South African variant ‘501 ,V2’ contains N501Y, E484K, and K417N mutations in the S protein - so it shares the N501Y mutation with the UK variant, but the other two mutations are not found in the UK variant. Similarly, the South African variant does not contain the 69- 70 deletion mutation found in the UK variant.

For all the spike variants where decreased neutralization was observed, there is a possibility that the mutations might restore the ability of the virus to replicate and spread in people who have antibodies following a first infection or vaccination. This could eventually lead to a requirement for updated vaccine candidates.

Exemplary S Protein mutations are outlined in Table 6. In one embodiment, the antigenic fragment of SEQ ID NO:1 or SEQ ID NO:4 comprises one or more (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) insertions, deletions or substitutions, for example one or more or the insertions, deletions or substitutions outline in Table 6.

In one embodiment, the recombinant polypeptide comprises one or more further non- dendroaspin amino acid sequence comprising one or more epitopes capable of eliciting an immune response against a CoV N protein.

In some embodiments, the recombinant polypeptide comprises 2, 3, 4, 5, 6 or 7 epitopes capable of eliciting an immune response against a CoV N protein that are distinct from one another.

Said one or more further epitopes capable of eliciting an immune response against a CoV N protein may comprise an antigenic fragment of a MERS, HKU1-CoV, OC43-CoV, SARS- CoV or SARS-CoV-2 N protein.

The SARS CoV-2 N protein has an amino acid sequence as shown in SEQ ID NO:27.

In some embodiments the recombinant polypeptide comprises one or more additional non- dendroaspin amino acid sequences comprising one or more further epitopes capable of eliciting an immune response against a SARS-CoV-2 N protein, characterised in that said first and one or more further epitopes capable of eliciting an immune response against a SARS-CoV-2 N protein are distinct from one another.

Said one or more further epitopes capable of eliciting an immune response against a SARS- CoV-2 N protein comprises at least one polypeptide is selected from: i) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:54.

The MERS N protein has an amino acid sequence as shown in SEQ ID NO:52.

In some embodiments the recombinant polypeptide comprises one or more additional non- dendroaspin amino acid sequences comprising one or more further epitopes capable of eliciting an immune response against a MERS N protein, characterised in that said first and one or more further epitopes capable of eliciting an immune response against a MERS N protein are distinct from one another.

Said one or more further epitopes capable of eliciting an immune response against a MERS N protein comprises at least one polypeptide is selected from: i) a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:53.

IL-6 is a pro-inflammatory cytokine and can be released in response to viral infections. In a study on 150 patients from Wuhan, China, IL-6, ferritin, and C reactive protein (CRP) were elevated in patients who died in comparison with those who survived/ Ruan Q, Yang K, Wang W, et al. Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China. Intensive Care Med. Epub ahead of print 3 March 2020. DOI: 10.1007/s00134-020-05991 -x> These elevated laboratory markers suggest the possibility of hypercytokinemia. in COVID-19 patients. (Mehta P, McAuley DF, Brown M, et al. COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet. Epub ahead of print 16 March 2020. DOI: 10.1016/S0140-6736(20)30628-0) Other pro- inflammatory factors include GM-CSF, TNFa, IL-10 and soluble IL-2R.

In one embodiment, the recombinant polypeptide comprises a further non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a pro-inflammatory protein. In one embodiment the first epitope is capable of eliciting an immune response against a pro-inflammatory protein that inhibits or reduces that proteins pro-inflammatory activity epitopes capable of eliciting an immune response against a pro- inflammatory protein. In one embodiment the pro-inflammatory protein is an IL-6 protein and the epitope elicits an immune response that inhibits or reduces IL-6 pro-inflammatory activity, for example by inhibiting or reducing binding to the IL6 receptor, CD126. In one embodiment the IL6 is human IL6. In one embodiment said first epitope capable of eliciting an immune response against an IL6 protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:7 or SEQ ID NO:8. In one embodiment said first epitope capable of eliciting an immune response against an IL6 protein comprises or consists of SEQ ID NO:7 or SEQ ID NO:8.

In one embodiment, the recombinant polypeptide comprises a further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a pro-inflammatory protein. In one embodiment the second epitope is capable of eliciting an immune response against a pro-inflammatory protein that inhibits or reduces that proteins pro-inflammatory activity. In one embodiment said first and second epitopes capable of eliciting an immune response against a pro-inflammatory protein are distinct from one another. In one embodiment said pro-inflammatory proteins against which said first and second epitopes are capable of eliciting an immune response against are distinct from one another.

In one embodiment, the second epitope is capable of eliciting an immune response against an IL6 protein. In one embodiment the second epitope elicits an immune response that inhibits or reduces IL-6 pro-inflammatory activity, for example by inhibiting or reducing binding to the IL6 receptor, CD126. In one embodiment the IL6 is human IL6. In one embodiment, the first and second epitopes capable of eliciting an immune response against a pro-inflammatory protein each elicit an immune response against IL6, characterised in that said first and second epitopes capable of eliciting an immune response against an IL6 protein are distinct from one another. In one embodiment, said second epitope capable of eliciting an immune response against an IL6 protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:7 or comprises a polypeptide comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:8. In one embodiment, said second epitope capable of eliciting an immune response against eliciting an immune response against an IL6 protein comprises or consists of SEQ ID NO:7 or comprises or consists of SEQ ID NO:8.

Transmembrane serine protease 2 (TMPRSS2), is a cell-surface protein that is expressed by epithelial cells of specific tissues such as those in the aerodigestive tract and the luminal side of the prostate epithelium. TMPRSS2 contains a type II transmembrane domain, a receptor class A domain, a scavenger receptor cysteine-rich domain and a protease domain. Serine proteases are known to be involved in many physiological and pathological processes. The protease domain of this protein is thought to be cleaved and secreted into cell media after autocleavage.

SARS-CoV-2 also employs TMPRSS2 for SARS-CoV-2 S protein priming and S protein- driven cell entry. TMPRSS2 also facilitates entry of viruses into host cells by proteolytically cleaving and activating viral envelope glycoproteins. The polypeptide sequence of TMPRSS2 is shown in SEQ ID NO:15

In one embodiment, the recombinant polypeptide comprises one or more further non- dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a TMPRSS2. In one embodiment the first epitope is capable of eliciting an immune response against TMPRSS2 that inhibits or reduces TMPRSS2 cleavage activity. In one embodiment said first epitope capable of eliciting an immune response against a TMPRSS2 protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:16 or SEQ ID NO:17. In one embodiment said first epitope capable of eliciting an immune response against a TMPRSS2 protein comprises or consists of SEQ ID NO:16 or SEQ ID NO:17.

In a preferred embodiment of the present invention the recombinant polypeptide comprises a dendroaspin scaffold: i) a first non-dendroaspin amino acid sequence wherein said first non- dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, optionally wherein said first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a SARS-CoV-2 S1 protein receptor binding motif, such as a modified S1 RBM; and ii) a first epitope capable of eliciting an immune response against an IL6 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises dendroaspin scaffold and: i) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, optionally wherein said first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a SARS-CoV-2 S1 protein receptor binding motif, such as a modified S1 RBM; ii) a first epitope capable of eliciting an immune response against an IL6 protein; and iii) a second epitope capable of eliciting an immune response against an IL6 protein characterised in that said first and second epitopes capable of eliciting an immune response against an IL6 protein are distinct from one another.

In a preferred embodiment of the present invention the recombinant polypeptide comprises dendroaspin scaffold and: i) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, optionally wherein said first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a SARS-CoV-2 S1 protein receptor binding motif, such as a modified S1 RBM; and ii) a further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, characterised in that said first and second epitopes capable of eliciting an immune response against a SARS-CoV-2 S1 protein are distinct from one another.

In a preferred embodiment of the present invention the recombinant polypeptide comprises dendroaspin scaffold and: i) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, optionally wherein said first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a SARS-CoV-2 S1 protein receptor binding motif, such as a modified S1 RBM; ii) a further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, characterised in that said first and second epitopes capable of eliciting an immune response against a SARS-CoV-2 S1 protein are distinct from one another; and iii) a further non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a SARS-CoV-2 S2 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises dendroaspin scaffold and: i) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, optionally wherein said first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a SARS-CoV-2 S1 protein receptor binding motif such as a modified S1 RBM; and ii) a further non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a SARS-CoV-2 S2 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprise dendroaspin scaffold and s: i) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, optionally wherein said first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a SARS-CoV-2 S1 protein receptor binding motif, such as a modified S1 RBM; ii) a further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, characterised in that said first and second epitopes capable of eliciting an immune response against a SARS-CoV-2 S1 protein are distinct from one another; iii) a first epitope capable of eliciting an immune response against an IL6 protein; and optionally iv) a second epitope capable of eliciting an immune response against an IL6 protein characterised in that said first and second epitopes capable of eliciting an immune response against an IL6 protein are distinct from one another.

In a preferred embodiment of the present invention the recombinant polypeptide comprises dendroaspin scaffold and: i) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, optionally wherein said first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a SARS-CoV-2 S1 protein receptor binding motif, such as a modified S1 RBM; ii) a further non-dendroaspin amino acid sequence comprising a second epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, characterised in that said first and second epitopes capable of eliciting an immune response against a SARS-CoV-2 S1 protein are distinct from one another; iii) a further non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a SARS-CoV-2 S2 protein; iv) a first epitope capable of eliciting an immune response against an IL6 protein; and optionally v) a second epitope capable of eliciting an immune response against an IL6 protein characterised in that said first and second epitopes capable of eliciting an immune response against an IL6 protein are distinct from one another.

In a preferred embodiment of the present invention the recombinant polypeptide comprises dendroaspin scaffold and: i) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, optionally wherein said first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a SARS-CoV-2 S1 protein receptor binding motif, such as a modified S1 RBM; ii) a further non-dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a SARS-CoV-2 S2 protein; iii) a first epitope capable of eliciting an immune response against an IL6 protein; and optionally iv) a second epitope capable of eliciting an immune response against an IL6 protein characterised in that said first and second epitopes capable of eliciting an immune response against an IL6 protein are distinct from one another.

In a preferred embodiment of the present invention the recombinant polypeptide comprises dendroaspin scaffold and: i) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, optionally wherein said first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a SARS-CoV-2 S1 protein receptor binding motif, such as a modified S1 RBM; ii) non- dendroaspin amino acid sequence comprising a first epitope capable of eliciting an immune response against a SARS-CoV-2 S2 protein; iii) a further non-dendroaspin amino acid sequence acid sequence comprising a first epitope capable of eliciting an immune response against a SARS-CoV-2 N protein; and optionally a further non-dendroaspin amino acid sequence acid sequence comprising a first epitope capable of eliciting an immune response against a MERS N protein .

In a preferred embodiment of the present invention the recombinant polypeptide comprises dendroaspin scaffold and: i) an epitope comprising or consisting of SEQ ID NO: 11 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; and ii) an epitope comprising or consisting of SEQ ID NO:7 or 8 and capable of eliciting an immune response against an IL6 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises dendroaspin scaffold and: i) an epitope comprising or consisting of SEQ ID NO: 11 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein,; ii) an epitope comprising or consisting of SEQ ID NO:7 or 8 and capable of eliciting an immune response against an IL6 protein; and iii) an comprising or consisting of SEQ ID NO:7 or 8 and capable of eliciting an immune response against an IL6 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises dendroaspin scaffold and: i) an epitope comprising or consisting of SEQ ID NO: 11 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; and ii) an epitope comprising or consisting of SEQ ID NO:3 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises dendroaspin scaffold and: i) an epitope comprising or consisting of SEQ ID NO: 11 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; ii) an epitope comprising or consisting of SEQ ID NO:3 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; and iii) an epitope comprising or consisting of SEQ ID NO:5, capable of eliciting an immune response against a SARS-CoV-2 S2 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises dendroaspin scaffold and: i) an epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprising or consisting of SEQ ID NO: 11 ; and ii) an epitope comprising or consisting of SEQ ID NO:5 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises dendroaspin scaffold and: i) an epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprising or consisting of SEQ ID NO: 11 ; ii) an epitope comprising or consisting of SEQ ID NO:3 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iii) an epitope comprising or consisting of SEQ ID NO:7 and capable of eliciting an immune response against an IL6 protein; and optionally iv) an epitope comprising or consisting of SEQ ID NO:8 and capable of eliciting an immune response against an IL6 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises a dendroaspin scaffold and: i) an epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprising or consisting of SEQ ID NO: 2; ii) an epitope comprising or consisting of SEQ ID NO:3 and capable of eliciting an immune response against a SARS- CoV-2 S1 protein; iii) an epitope comprising or consisting of SEQ ID NO:5 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; iv) an epitope comprising or consisting of SEQ ID NO:7 and capable of eliciting an immune response against an IL6 protein; and optionally v) an epitope comprising or consisting of SEQ ID NO:8 and capable of eliciting an immune response against an IL6 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises a dendroaspin scaffold and: i) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, wherein said first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises or consists of SEQ ID NO: 11 ; ii) a further non-dendroaspin amino acid sequence comprising a first epitope comprising or consisting of SEQ ID NO:5 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; iii) a first epitope, comprising or consisting of SEQ ID NO:7 and capable of eliciting an immune response against an IL6 protein; and optionally iv) a second epitope, comprising or consisting of SEQ ID NO:8 and capable of eliciting an immune response against an IL6 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises a dendroaspin scaffold and: i) a first non-dendroaspin amino acid sequence wherein said first non-dendroaspin amino acid sequence comprises a first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein, optionally wherein said first epitope capable of eliciting an immune response against a SARS-CoV-2 S1 protein comprises a SARS-CoV-2 S1 protein receptor binding motif; ii) a first epitope capable of eliciting an immune response against TMPRSS2, and optionally iii) a first epitope capable of eliciting an immune response against an IL6 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises a dendroaspin scaffold and: i) an epitope comprising or consisting of SEQ ID NO: 11 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; and ii) an epitope comprising or consisting of SEQ ID NO: 16 or 17 and capable of eliciting an immune response against an TMPRSS2 protein; and iii) an epitope comprising or consisting of SEQ ID NO:7 or 8 and capable of eliciting an immune response against an IL6 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises a dendroaspin scaffold and: i) an epitope comprising or consisting of SEQ ID NO: 3 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; and ii) an epitope comprising or consisting of SEQ ID NO: 30 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iii) an epitope comprising or consisting of SEQ ID NO: 31 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; and an epitope comprising or consisting of SEQ ID NO: 5 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises a dendroaspin scaffold and: i) an epitope comprising or consisting of SEQ ID NO: 3 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; and ii) an epitope comprising or consisting of SEQ ID NO: 30 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iii) an epitope comprising or consisting of SEQ ID NO: 31 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iv) an epitope comprising or consisting of SEQ ID NO: 5 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; and v) an epitope comprising or consisting of SEQ ID NO: 11 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises a dendroaspin scaffold and: i) an epitope comprising or consisting of SEQ ID NO: 3 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; and ii) an epitope comprising or consisting of SEQ ID NO: 30 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iii) an epitope comprising or consisting of SEQ ID NO: 31 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iv) an epitope comprising or consisting of SEQ ID NO: 5 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; v) an epitope comprising or consisting of SEQ ID NO: 11 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; and vi) an epitope comprising or consisting of SEQ ID NO: 17 and capable of eliciting an immune response against TMPRSS2.

In a preferred embodiment of the present invention the recombinant polypeptide comprises a dendroaspin scaffold and: i) an epitope comprising or consisting of SEQ ID NO: 3 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; and ii) an epitope comprising or consisting of SEQ ID NO: 30 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iii) an epitope comprising or consisting of SEQ ID NO: 31 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iv) an epitope comprising or consisting of SEQ ID NO: 5 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; v) an epitope comprising or consisting of SEQ ID NO: 11 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein;; vi) an epitope comprising or consisting of SEQ ID NO: 8 and capable of eliciting an immune response against an IL6 protein; and optionally vii) an epitope comprising or consisting of SEQ ID NO: 17 and capable of eliciting an immune response against TMPRSS2.

In a preferred embodiment of the present invention the recombinant polypeptide (V10) comprises a dendroaspin scaffold and: i) an epitope comprising or consisting of SEQ ID NO: 32 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; ii) an epitope comprising or consisting of SEQ ID NO: 33 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iii) an epitope comprising or consisting of SEQ ID NO:11 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iv) an epitope comprising or consisting of SEQ ID NO: 3 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; v) an epitope comprising or consisting of SEQ ID NO: 34 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; vi) an epitope comprising or consisting of SEQ ID NO: 38 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; vii) an epitope comprising or consisting of SEQ ID NO: 5 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; and viii) an epitope comprising or consisting of SEQ ID NO: 39 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein.

In a preferred embodiment of the present invention the recombinant polypeptide (V11) comprises a dendroaspin scaffold and: i) an epitope comprising or consisting of SEQ ID NO: 32 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; ii) an epitope comprising or consisting of SEQ ID NO: 35 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iii) an epitope comprising or consisting of SEQ ID NO: 33 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iv) an epitope comprising or consisting of SEQ ID NO: 11 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; v) an epitope comprising or consisting of SEQ ID NO: 36 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; vi) an epitope comprising or consisting of SEQ ID NO: 34 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; vii) an epitope comprising or consisting of SEQ ID NO: 44 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; viii) an epitope comprising or consisting of SEQ ID NO: 5 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; ix) an epitope comprising or consisting of SEQ ID NO: 40 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; x) an epitope comprising or consisting of SEQ ID NO: 43 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; and xi) an epitope comprising or consisting of SEQ ID NO: 39 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein.

In a preferred embodiment of the present invention the recombinant polypeptide (V12) comprises a dendroaspin scaffold and: i) an epitope comprising or consisting of SEQ ID NO: 32 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; ii) an epitope comprising or consisting of SEQ ID NO: 35 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iii) an epitope comprising or consisting of SEQ ID NO: 33 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iv) an epitope comprising or consisting of SEQ ID NO: 37 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; v) an epitope comprising or consisting of SEQ ID NO: 36 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; vi) an epitope comprising or consisting of SEQ ID NO: 34 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; vii) an epitope comprising or consisting of SEQ ID NO: 44 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; viii) an epitope comprising or consisting of SEQ ID NO: 5 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; ix) an epitope comprising or consisting of SEQ ID NO: 40 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; x) an epitope comprising or consisting of SEQ ID NO: 43 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; and xi) an epitope comprising or consisting of SEQ ID NO: 39 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein. In a preferred embodiment of the present invention the recombinant polypeptide (V14, V15) comprises a dendroaspin scaffold and: i) an epitope comprising or consisting of SEQ ID NO: 53 and capable of eliciting an immune response against a MERS N protein; ii) an epitope comprising or consisting of SEQ ID NO: 54 and capable of eliciting an immune response against a MERS N protein; iii) an epitope comprising or consisting of SEQ ID NO: 32 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; iv) an epitope comprising or consisting of SEQ ID NO: 35 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; v) an epitope comprising or consisting of SEQ ID NO: 33 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; vi) an epitope comprising or consisting of SEQ ID NO: 37 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; vii) an epitope comprising or consisting of SEQ ID NO: 36 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; viii) an epitope comprising or consisting of SEQ ID NO: 34 and capable of eliciting an immune response against a SARS-CoV-2 S1 protein; ix) an epitope comprising or consisting of SEQ ID NO: 44 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; x) an epitope comprising or consisting of SEQ ID NO: 5 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; xi) an epitope comprising or consisting of SEQ ID NO: 40 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; xii) an epitope comprising or consisting of SEQ ID NO: 43 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein; and xiii) an epitope comprising or consisting of SEQ ID NO: 39 and capable of eliciting an immune response against a SARS-CoV-2 S2 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:11 , wherein the polypeptide is capable of eliciting an immune response against a SARS-CoV-2 S1 protein. In a preferred embodiment of the present invention the recombinant polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:12, wherein the polypeptide is capable of eliciting an immune response against a SARS-CoV-2 S1 protein and eliciting an immune response against eliciting an immune response against an IL6 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:13, wherein the polypeptide is capable of eliciting an immune response against a SARS-CoV-2 S1 protein and eliciting an immune response against eliciting an immune response against SARS-CoV-2 S2 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14, wherein the polypeptide is capable of eliciting an immune response against a SARS-CoV-2 S1 protein; eliciting an immune response against eliciting an immune response against SARS-CoV-2 S2 protein; and eliciting an immune response against eliciting an immune response against an IL6 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:21 , wherein the polypeptide is capable of eliciting an immune response against a SARS-CoV-2 S1 protein and a SARS-CoV-2 S2 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:22, wherein the polypeptide is capable of eliciting an immune response against a SARS-CoV-2 S1 protein, a SARS-CoV-2 S1 protein and an IL6 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:23, wherein the polypeptide is capable of eliciting an immune response against a SARS-CoV-2 S1 protein, a SARS-CoV-2 S1 protein, an IL6 protein and TMPRSS2 .

In a preferred embodiment of the present invention the recombinant polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:24, wherein the polypeptide is capable of eliciting an immune response against a SARS-CoV-2 S1 protein and a SARS-CoV-2 S2 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:25, wherein the polypeptide is capable of eliciting an immune response against a SARS-CoV-2 S1 protein and a SARS-CoV-2 S2 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:26, wherein the polypeptide is capable of eliciting an immune response against a SARS-CoV-2 S1 protein and a SARS-CoV-2 S2 protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:28, wherein the polypeptide is capable of eliciting an immune response against a SARS-CoV-2 S1 protein, a SARS-CoV-2 S2 protein, a SARS-CoV-2 N protein and a MERS N protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:29, wherein the polypeptide is capable of eliciting an immune response against a SARS-CoV-2 S1 protein a SARS-CoV-2 S2 protein, a SARS-CoV-2 N protein and a MERS N protein.

In a preferred embodiment of the present invention the recombinant polypeptide comprises or consists of SEQ ID NO:11.

In a preferred embodiment of the present invention the recombinant polypeptide comprises or consists of SEQ ID NO:12.

In a preferred embodiment of the present invention the recombinant polypeptide comprises or consists of SEQ ID NO: 13.

In a preferred embodiment of the present invention the recombinant polypeptide comprises or consists of SEQ ID NO:14.

In a preferred embodiment of the present invention the recombinant polypeptide comprises or consists of SEQ ID NO:21.

In a preferred embodiment of the present invention the recombinant polypeptide comprises or consists of SEQ ID NO:22. In a preferred embodiment of the present invention the recombinant polypeptide comprises or consists of SEQ ID NO:23.

In a preferred embodiment of the present invention the recombinant polypeptide comprises or consists of SEQ ID NO:24.

In a preferred embodiment of the present invention the recombinant polypeptide comprises or consists of SEQ ID NO:25.

In a preferred embodiment of the present invention the recombinant polypeptide comprises or consists of SEQ ID NO:26.

In a preferred embodiment of the present invention the recombinant polypeptide comprises or consists of SEQ ID NO:28.

In a preferred embodiment of the present invention the recombinant polypeptide comprises or consists of SEQ ID NO:29.

Expression Vectors

In one aspect the invention provides an expression vector comprising an expressible nucleic acid encoding the recombinant proteins herein described. In one embodiment he vector may be any vector capable of transferring DNA to a cell. Preferably, the vector is an integrating vector or an episomal vector.

Preferred integrating vectors include recombinant retroviral vectors. A recombinant retroviral vector will include DNA of at least a portion of a retroviral genome which portion is capable of infecting the target cells. The term “infection” is used to mean the process by which a virus transfers genetic material to its host or target cell. Preferably, the retrovirus used in the construction of a vector of the invention is also rendered replication-defective to remove the effect of viral replication of the target cells. In such cases, the replication-defective viral genome can be packaged by a helper virus in accordance with conventional techniques. Generally, any retrovirus meeting the above criteria of infectiousness and capability of functional gene transfer can be employed in the practice of the invention.

Other vectors useful in the present invention include adenovirus, adeno-associated virus, SV40 virus, vaccinia virus, HSV and poxvirus vectors. A preferred vector is the adenovirus. Adenovirus vectors are well known to those skilled in the art and have been used to deliver genes to numerous cell types, including airway epithelium, skeletal muscle, liver, brain and skin and to tumours .

A further preferred vector is the adeno-associated (AAV) vector. AAV vectors are well known to those skilled in the art and have been used to stably transduce human T-lymphocytes, fibroblasts, nasal polyp, skeletal muscle, brain, erythroid and haematopoietic stem cells for gene therapy applications. International Patent Application WO 91/18088 describes specific AAV-based vectors.

Preferred episomal vectors include transient non-replicating episomal vectors and self- replicating episomal vectors with functions derived from viral origins of replication such as those from EBV, human papovavirus (BK) and BPV-1. Such integrating and episomal vectors are well known to those skilled in the art and are fully described in the body of literature well known to those skilled in the art. In particular, suitable episomal vectors are described in WO98/07876.

Mammalian artificial chromosomes can also be used as vectors in the present invention.

In a preferred embodiment, the vector of the present invention is a plasmid. The plasmid may be a non-replicating, non-integrating plasmid.

The term “plasmid” as used herein refers to any nucleic acid encoding an expressible gene and includes linear or circular nucleic acids and double or single stranded nucleic acids. The nucleic acid can be DNA or RNA and may comprise modified nucleotides or ribonucleotides, and may be chemically modified by such means as methylation or the inclusion of protecting groups or cap- or tail structures.

A non-replicating, non-integrating plasmid is a nucleic acid which when transfected into a host cell does not replicate and does not specifically integrate into the host cell’s genome (i.e. does not integrate at high frequencies and does not integrate at specific sites).

Replicating plasmids can be identified using standard assays including the standard replication assay of llstav et al (1991 EMBO J 10: 449-457).

The present invention also provides a host cell transformed or transfected with the vector of the present invention. The host cell may be any mammalian cell. Preferably the host cell is a rodent or human cell. Preferably the cell is an isolated cell.

Numerous techniques are known and are useful according to the invention for delivering the vectors described herein to cells, including the use of nucleic acid condensing agents, electroporation, complexing with asbestos, polybrene, DEAE cellulose, Dextran, liposomes, cationic liposomes, lipopolyamines, polyornithine, particle bombardment and direct microinjection.

A vector of the invention may be delivered to a host cell non-specifically or specifically (i.e. , to a designated subset of host cells) via a viral or non-viral means of delivery. Preferred delivery methods of viral origin include viral particle-producing packaging cell lines as transfection recipients for the vector of the present invention into which viral packaging signals have been engineered, such as those of adenovirus, herpes viruses and papovaviruses. Preferred non-viral based gene delivery means and methods may also be used in the invention and include direct naked nucleic acid injection, nucleic acid condensing peptides and non-peptides, cationic liposomes and encapsulation in liposomes.

Compositions

A composition is also provided, comprising the recombinant polypeptide described herein, together with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier. Compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.

As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected binding protein without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. an expression cassette, plasmid or virion), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like. Adjuvants are pharmacological and/or immunological agents that modify the effect of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art.

Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.

Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.

Vaccines

In one aspect the invention provides anti-zoonotic pathogen vaccines comprising the recombinant protein disclosed herein or the vectors disclosed herein.

In one aspect the invention provides anti-SARS-CoV-2 vaccines comprising the recombinant protein disclosed herein or the vectors disclosed herein.

Also provided is a method of eliciting an anti-SARS-CoV-2 immune response in a mammal comprising administering the recombinant protein or expression vector herein described, or an anti-SARS-CoV-2 vaccine or pharmaceutical composition comprising the recombinant protein or expression vector.

As used herein, "vaccine" is defined broadly to refer to any type of biological agent in an administrable form capable of stimulating an immune response in an animal inoculated with the vaccine that prevents or ameliorates the symptoms of zoonotic pathogen infection, in particular Covid-19. Thus, reduction in the incidence or severity of characteristic symptoms of zoonotic pathogen infection, in particular Covid-19 in comparison to non-immunized subjects may be termed a vaccine for the purposes of this invention.

The terms "subject" and "patient" are used interchangeably herein and will be understood to refer to a warm-blooded animal, particularly a mammal. Non-limiting examples of animals within the scope and meaning of this term include guinea pigs, dogs, cats, rats, mice, horses, goats, cattle, sheep, zoo animals, monkeys, non-human primates, and humans. In certain embodiments vaccines of the invention are administered prophylactically. In certain embodiments, a vaccine of the invention is administered therapeutically. In an embodiment, the vaccine is administered to a patient with an active zoonotic pathogen infection to both treat the active infection and/or prevent recurrence of the zoonotic pathogen infection.

The present invention provides novel vaccine compositions comprising the recombinant protein disclosed herein or the vectors disclosed herein effective for controlling SARS-CoV-2 infection in mammalian, most preferably human, subjects. The present vaccine compositions are useful in providing immune resistance against the strain(s) of SARS-CoV-2 used for preparation of the composition, as well as against strains which are different from those used in the preparation of the vaccine composition.

In certain embodiments vaccines of the invention are administered prophylactically. In certain embodiments, a vaccine of the invention is administered therapeutically. In an embodiment, the vaccine is administered to a patient with an active SARS-CoV-2 infection to both treat the active infection and/or prevent recurrence of a SARS-CoV-2.

Peptide therapeutic

The recombinant immunogenic polypeptides of the invention inhibit binding of the SARS- CoV-2 spike protein to ACE2. Accordingly, the recombinant immunogenic polypeptides of the invention provide ACE2 antagonists that can be used in the treatment of COVID-19 / SARS-CoV-2 infection.

Accordingly, the invention provides a method of treating or preventing COVID-191 SARS- CoV-2 in a patient in need thereof comprising administering to said patient an effective amount of recombinant immunogenic polypeptide of the invention.

Also provided are methods of reducing the viral load of SARS-CoV-2 comprising administering to said patient an effective amount of recombinant immunogenic polypeptide of the invention.

As used herein, “antagonist” refers to a biologically active ligand that binds to a biologically active receptor and inhibits the physiological response of the receptor. By way of example, as used herein, a “ACE2 receptor antagonist” and “ACE2 antagonist,” refers to a ligand that binds to an ACE2 receptor and inhibits a physiological response of that receptor, for example, inhibiting host cell entry of SARS-CoV-2. “Therapeutically effective amount,” as described herein, can refer to an amount of a therapeutic agent whose administration, when viewed in a relevant population, correlates with or is reasonably expected to correlate with achievement of a particular therapeutic effect, including for example, amelioration of disease or disorder or delay of progression of disease or disorder. The therapeutic effect may be objective (i.e. , measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic peptide, a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, or combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and/or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in the medical arts.

Also provided is a method of treating, preventing or ameliorating Covid19/ SARS-CoV-2 infection, comprising administering to an individual requiring such treatment a therapeutically effective amount of the recombinant protein, expression vector or pharmaceutical composition herein described.

“Preventing” a disease refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease.

As used herein, the term “therapeutically effective amount” refers to a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this may be the amount of an anti-SARS-CoV-2 composition useful for eliciting an immune response in a subject and/or for preventing Covid-19 caused by SARS-CoV-2.

Adjuvant The inventors have also found that dendroaspin elicits an immune response in a subject. More specifically, dendroaspin elicits an anti-inflammatory Th2 type immune response in a subject.

In general, the present invention also relates to immunogenic compositions comprising a dendroaspin adjuvant and methods of use to elicit a disease specific immune response.

Particularly, the invention relates to the application of immunogenic compositions comprising dendroaspin as an adjuvant that is safe for use in humans and non-human animals, which when administered in combination with antigenic and/or immunomodulating substance(s), enhances a specific mucosal immune response and/or a systemic immune response.

In a further aspect the invention provides an adjuvant comprising: a) a protein comprising an amino acid sequence as set forth in SEQ ID. NO. 9 b) a protein with at least 75% sequence identity to SEQ ID. NO. 9; c) a biologically active fragment of (a) or (b).

In certain embodiments said adjuvant comprises an amino acid sequence with at least 80% identity to the sequence set forth in SEQ ID. NO. 9, e.g. 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID. NO. 9. Preferably said composition comprises an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID. NO. 1.

In certain embodiments the invention relates to any one of the aforementioned compositions wherein the composition comprises dendroaspin or a fragment thereof.

In order that the application may be more completely understood, several definitions are set forth below. Such definitions are meant to encompass grammatical equivalents.

By "adjuvant," as used herein, refers to any substance or mixture of substances that increases or diversifies the immune response of a host to an antigenic compound.

By "amino acid" as used herein is meant one of the 20 naturally occurring amino acids or any non-natural analogues that may be present at a specific, defined position.

An "immunogenic composition" as used here in refers to a combination of two or more substances (e.g., an antigen and an adjuvant) that together elicit an immune response when administered to a host. As used herein, the term "antigenic compound" refers to any substance that can be recognized by the immune system (e.g., bound by an antibody or processed so as to elicit a cellular immune response) under appropriate conditions.

An "antigen" refers to a substance, including compositions in the form of a vaccine where the vaccine itself comprises an antigenic compound and may or may not comprise an adjuvant other than dendroaspin, which when administered by an appropriate route induces a specific immune response, for example, the formation of antibodies, including antibodies that specifically bind the antigen. Two of the characteristic features of antigens are their immunogenicity, that is, their capacity to induce a specific immune response in vivo, and their antigenicity, that is their capacity to be selectively recognized by the antibodies whose origins are the antigens.

By "antigen" as used herein includes but is not limited to cells; cell extracts; proteins; lipoproteins; glycoproteins; nucleoproteins; polypeptides; peptides; polysaccharides; polysaccharide conjugates; peptide mimics of polysaccharides; lipids; glycolipids; carbohydrates; viruses; viral extracts; bacteria; bacterial extracts; fungi; fungal extracts; multicellular organisms such as parasites; and allergens. Antigens may be exogenous (e.g., from a source other than the individual to whom the antigen is administered, e.g., from a different species) or endogenous (e.g., originating from within the host, e.g., a diseased element of body, a cancer antigen, a virus infected cell producing antigen, and the like). Antigens may be native (e.g., naturally-occurring); synthetic; or recombinant. Antigens include crude extracts; whole cells; and purified antigens, where "purified" indicates that the antigen is in a form that is enriched relative to the environment in which the antigen normally occurs and/or relative to the crude extract, for example, a cultured form of the antigen.

By “non-native antigen” as used herein is meant an antigen that is not derived from a polynucleotide or polypeptide that it is inserted into.

By "derived from" as used herein is intended to include the isolation (in whole or in part) of a polynucleotide segment from an indicated source or the purification of a polypeptide from an indicated source. The term is intended to include, for example, direct cloning, PCR amplification, or artificial synthesis from or based on a sequence associated with the indicated polynucleotide source.

By "gene(s)" or "polynucleotide(s)" or “nucleic acid(s)” as used herein are intended to include nucleic acid molecules, e.g., polynucleotides which include an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences, and introns. In addition, the terms are intended to include one or more genes that map to a functional locus. In addition, the terms are intended to include a specific gene for a selected purpose. The gene may be endogenous to the host cell or may be recombinantly introduced into the host cell, e.g., as a plasmid maintained episomally or a plasmid (or fragment thereof) that is stably integrated into the genome. In addition to the plasmid form, a gene may, for example, be in the form of linear DNA.

By "plasmid" as used herein refers to any polynucleotide encoding an expressible gene and includes linear or circular polynucleotides and double or single stranded polynucleotides. The polynucleotide can be DNA or RNA and may comprise modified nucleotides or ribonucleotides, and may be chemically modified by such means as methylation or the inclusion of protecting groups or cap- or tail structures.

By "protein(s)" or “peptide(s)” or “polypeptide(s)” or “oligopeptide(s)” as used herein is meant at least two covalently attached amino acids. The peptidyl group may comprise naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e. "analogs", such as peptoids. The amino acids may either be naturally occurring or non- naturally occurring; as will be appreciated by those in the art. For example, homo- phenylalanine, citrulline, and noreleucine are considered amino acids for the purposes of the invention, and both D- and L- (R or S) configured amino acids may be utilized. The variants of the present invention may comprise modifications that include the use of unnatural amino.

By "modification" as used herein is meant an alteration in the physical, chemical, or sequence properties of a polypeptide. Preferred modifications of the invention are substitution, deletion and/or insertion or one or more amino acids from and/or into a peptide sequence.

By "substitution" as used herein is meant the replacement of one or more amino acids at a particular position in a parent polypeptide sequence with one or more other amino acids. In some embodiments, from 1 , 2, 3, 4, 5, 6, 7, 8, 9 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions are done, with any range within being contemplated, in combination with one or more insertion(s) and/or one or more deletion(s).

By "insertion" as used herein is meant the addition of one or more amino acids at a particular position in a parent polypeptide sequence. For ease of reference, the original numbering after an insertion is not changed; that is, in a molecule containing an insertion, the amino acid normally found following the insertion site is still numbered as if the insertion did not occur, unless noted otherwise.

By "deletion" as used herein is meant the removal of one ore more amino acids at a particular position in a parent polypeptide sequence. For ease of reference, the original numbering after a deletion is not changed; that is, in a molecule containing a deletion, the amino acid normally found following the deletion site is still numbered as if the deletion did not occur, unless noted otherwise.

As is noted herein, any amino acid modification outlined herein or in the incorporated references can be combined with any other modification; the examples herein are not meant to be limiting. Thus, for example, it may be desirable to combine one or more deletions with one or more insertions and one or more substitutions; one or more deletions with one or more insertions; one or more deletions with one or more substitutions; one or more substitutions with one or more insertions, etc.

By “incorporated into” as used herein is meant any modification outlined herein which results in a non-native amino acid sequence being integrated into a polypeptide sequence or which results in a non-native nucleic acid sequence being integrated into a polynucleotide sequence.

By "position" as used herein is meant a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example the Ell index as in Kabat.

By "antibody" as used herein is meant a protein consisting of one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes. The recognized immunoglobulin genes, for example in humans, include the kappa (K), lambda (A), and heavy chain genetic loci, which together comprise the myriad variable region genes, and the constant region genes mu (u), delta (5), gamma (y), sigma (o), and alpha (a) which encode the IgM, IgD, IgG (lgG1 , lgG2, lgG3, and lgG4), IgE, and IgA (lgA1 and lgA2) isotypes respectively. Antibody herein is meant to include full length antibodies and antibody fragments, and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes. Additional description and definition of "antibody", including for example "humanized", "chimeric", etc., is outlined below. By "eliciting an immune response" is used herein generally to encompass induction and/or potentiation of an immune response.

By "inducing an immune response" refers to an immune response that is, stimulated, initiated, or induced.

By "potentiating an immune response" refers to a pre-existing immune response that is improved, furthered, supplemented, amplified, enhanced, increased or prolonged. The expression "enhanced immune response" or similar means that the immune response is elevated, improved or enhanced to the benefit of the host relative to the prior immune response status, for example, before the administration of an immunogenic composition of the invention.

The terms "mucosal immune response" and "mucosal immunity" are terms well understood in the art, and refers to an immune response characterized, at least in part, by production of secretory IgA and/or stimulation of a mucosal CTL response in mucosal tissues such as gastrointestinal tract tissues, including rectal tissues; vaginal tissues; and tissues of the respiratory tract.

The terms "humoral immunity" and "humoral immune response" refer to the form of immunity in which antibody molecules are produced in response to antigenic stimulation.

The terms "cell-mediated immunity" and "cell-mediated immune response" are meant to refer to the immunological defense provided by lymphocytes, such as that defense provided by T cell lymphocytes when they come into close proximity to their victim cells. A cell-mediated immune response normally includes lymphocyte proliferation. When "lymphocyte proliferation" is measured, the ability of lymphocytes to proliferate in response to a specific antigen is measured. Lymphocyte proliferation is meant to refer to B cell, T-helper cell or CTL cell proliferation.

The term "immunogenic amount" refers to an amount of antigenic compound sufficient to stimulate an immune response, when administered with a subject immunogenic composition, as compared with the immune response elicited by the antigen in the absence of the adjuvant.

The term "immunopotentiating amount" refers to the amount of the adjuvant needed to effect an increase in antibody titer and/or cell-mediated immunity when administered with an antigenic compound in a composition of the invention, as compared with the increase in antibody and/or cell mediated immunity level observed in the absence of the adjuvant. By "wild type" or "WT" as used herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A WT protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or a nucleotide sequence that has not been intentionally modified.

By "subject," used interchangeably herein with "host," "individual," and "animal," includes humans and all domestic e.g. livestock and pets and wild mammals and fowl, including, without limitation, cattle, horses, cows, swine, sheep, goats, dogs, cats, rabbits, deer, mink, chickens, ducks, geese, turkeys, game hens, and the like.

The term "mucosal" or "mucosal membrane" or "mucosal surface" refers to the surfaces, passages and cavities that are in contact directly or indirectly with the exterior environment, including the surfaces of the respiratory, digestive, sensory and genitourinary systems. "Mucosal surface of the gastrointestinal tract" is meant to include mucosa of the bowel (including the small intestine and large intestine), rectum, stomach (gastric) lining, oral cavity, and the like. The term "formulated for mucosal administration" refers to a composition that is adapted for and thus compatible with administration to the mucosa (e.g., to a mucosal surface or mucosal membrane). In some embodiments, the composition is formulated for mucosal administration by a route other than rectal, vaginal, nasal, oral, or opthamalic (e.g., the composition is formulated for administration to lung tissue, e.g., by pulmonary administration.

In certain embodiments the invention relates to any one of the aforementioned methods wherein the protein is dendroaspin or a fragment thereof.

In certain embodiments the invention relates to any one of the aforementioned methods wherein the composition is a vaccine composition.

In certain embodiments the invention relates to any one of the aforementioned methods wherein the method is for the treatment and/or prevention of an autoimmune disease, preferably the disease is selected from the group consisting of rheumatoid arthritis, atherosclerosis, diabetes and lupus.

In certain embodiments the invention relates to any one of the aforementioned methods wherein the method is for the treatment and/or prevention of an inflammatory disease, preferably the disease is selected from the group consisting of rheumatoid arthritis, atherosclerosis, diabetes and lupus.

In certain embodiments the invention relates to any one of the aforementioned methods wherein the composition further comprises at least one antigen.

In order that the application may be more completely understood, several definitions are set forth below. Such definitions are meant to encompass grammatical equivalents.

By "adjuvant," as used herein, refers to any substance or mixture of substances that increases or diversifies the immune response of a host to an antigenic compound.

By "amino acid" as used herein is meant one of the 20 naturally occurring amino acids or any non-natural analogues that may be present at a specific, defined position.

An "immunogenic composition" as used here in refers to a combination of two or more substances (e.g., an antigen and an adjuvant) that together elicit an immune response when administered to a host.

As used herein, the term "antigenic compound" refers to any substance that can be recognized by the immune system (e.g., bound by an antibody or processed so as to elicit a cellular immune response) under appropriate conditions.

An "antigen" refers to a substance, including compositions in the form of a vaccine where the vaccine itself comprises an antigenic compound and may or may not comprise an adjuvant other than dendroaspin, which when administered by an appropriate route induces a specific immune response, for example, the formation of antibodies, including antibodies that specifically bind the antigen. Two of the characteristic features of antigens are their immunogenicity, that is, their capacity to induce a specific immune response in vivo, and their antigenicity, that is their capacity to be selectively recognized by the antibodies whose origins are the antigens.

By "antigen" as used herein includes but is not limited to cells; cell extracts; proteins; lipoproteins; glycoproteins; nucleoproteins; polypeptides; peptides; polysaccharides; polysaccharide conjugates; peptide mimics of polysaccharides; lipids; glycolipids; carbohydrates; viruses; viral extracts; bacteria; bacterial extracts; fungi; fungal extracts; multicellular organisms such as parasites; and allergens. Antigens may be exogenous (e.g., from a source other than the individual to whom the antigen is administered, e.g., from a different species) or endogenous (e.g., originating from within the host, e.g., a diseased element of body, a cancer antigen, a virus infected cell producing antigen, and the like). Antigens may be native (e.g., naturally-occurring); synthetic; or recombinant. Antigens include crude extracts; whole cells; and purified antigens, where "purified" indicates that the antigen is in a form that is enriched relative to the environment in which the antigen normally occurs and/or relative to the crude extract, for example, a cultured form of the antigen.

By “non-native antigen” as used herein is meant an antigen that is not derived from a polynucleotide or polypeptide that it is inserted into.

By "derived from" as used herein is intended to include the isolation (in whole or in part) of a polynucleotide segment from an indicated source or the purification of a polypeptide from an indicated source. The term is intended to include, for example, direct cloning, PCR amplification, or artificial synthesis from or based on a sequence associated with the indicated polynucleotide source.

By "gene(s)" or "polynucleotide(s)" or “nucleic acid(s)” as used herein are intended to include nucleic acid molecules, e.g., polynucleotides which include an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences, and introns. In addition, the terms are intended to include one or more genes that map to a functional locus. In addition, the terms are intended to include a specific gene for a selected purpose. The gene may be endogenous to the host cell or may be recombinantly introduced into the host cell, e.g., as a plasmid maintained episomally or a plasmid (or fragment thereof) that is stably integrated into the genome. In addition to the plasmid form, a gene may, for example, be in the form of linear DNA.

By "plasmid" as used herein refers to any polynucleotide encoding an expressible gene and includes linear or circular polynucleotides and double or single stranded polynucleotides. The polynucleotide can be DNA or RNA and may comprise modified nucleotides or ribonucleotides, and may be chemically modified by such means as methylation or the inclusion of protecting groups or cap- or tail structures.

By "protein(s)" or “peptide(s)” or “polypeptide(s)” or “oligopeptide(s)” as used herein is meant at least two covalently attached amino acids. The peptidyl group may comprise naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e. "analogs", such as peptoids. The amino acids may either be naturally occurring or non- naturally occurring; as will be appreciated by those in the art. For example, homo- phenylalanine, citrulline, and noreleucine are considered amino acids for the purposes of the invention, and both D- and L- (R or S) configured amino acids may be utilized. The variants of the present invention may comprise modifications that include the use of unnatural amino.

By "modification" as used herein is meant an alteration in the physical, chemical, or sequence properties of a polypeptide. Preferred modifications of the invention are substitution, deletion and/or insertion or one or more amino acids from and/or into a peptide sequence.

By "substitution" as used herein is meant the replacement of one or more amino acids at a particular position in a parent polypeptide sequence with one ore more other amino acids. In some embodiments, from 1 , 2, 3, 4, 5, 6, 7, 8, 9 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions are done, with any range within being contemplated, in combination with one or more insertion(s) and/or one or more deletion(s).

By "insertion" as used herein is meant the addition of one or more amino acids at a particular position in a parent polypeptide sequence. For ease of reference, the original numbering after an insertion is not changed; that is, in a molecule containing an insertion, the amino acid normally found following the insertion site is still numbered as if the insertion did not occur, unless noted otherwise.

By "deletion" as used herein is meant the removal of one or more amino acids at a particular position in a parent polypeptide sequence. For ease of reference, the original numbering after a deletion is not changed; that is, in a molecule containing a deletion, the amino acid normally found following the deletion site is still numbered as if the deletion did not occur, unless noted otherwise.

As is noted herein, any amino acid modification outlined herein or in the incorporated references can be combined with any other modification; the examples herein are not meant to be limiting. Thus, for example, it may be desirable to combine one or more deletions with one or more insertions and one or more substitutions; one or more deletions with one or more insertions; one or more deletions with one or more substitutions; one or more substitutions with one or more insertions, etc.

By “incorporated into” as used herein is meant any modification outlined herein which results in a non-native amino acid sequence being integrated into a polypeptide sequence or which results in a non-native nucleic acid sequence being integrated into a polynucleotide sequence. By "position" as used herein is meant a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example the Ell index as in Kabat.

By "antibody" as used herein is meant a protein consisting of one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes. The recognized immunoglobulin genes, for example in humans, include the kappa (K), lambda (A), and heavy chain genetic loci, which together comprise the myriad variable region genes, and the constant region genes mu (u), delta (5), gamma (y), sigma (o), and alpha (a) which encode the IgM, IgD, IgG (lgG1 , lgG2, lgG3, and lgG4), IgE, and IgA (lgA1 and lgA2) isotypes respectively. Antibody herein is meant to include full length antibodies and antibody fragments, and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes. Additional description and definition of "antibody", including for example "humanized", "chimeric", etc., is outlined below.

By "eliciting an immune response" is used herein generally to encompass induction and/or potentiation of an immune response.

By "inducing an immune response" refers to an immune response that is, stimulated, initiated, or induced.

By "potentiating an immune response" refers to a pre-existing immune response that is improved, furthered, supplemented, amplified, enhanced, increased or prolonged. The expression "enhanced immune response" or similar means that the immune response is elevated, improved or enhanced to the benefit of the host relative to the prior immune response status, for example, before the administration of an immunogenic composition of the invention.

The terms "mucosal immune response" and "mucosal immunity" are terms well understood in the art, and refers to an immune response characterized, at least in part, by production of secretory IgA and/or stimulation of a mucosal CTL response in mucosal tissues such as gastrointestinal tract tissues, including rectal tissues; vaginal tissues; and tissues of the respiratory tract.

The terms "humoral immunity" and "humoral immune response" refer to the form of immunity in which antibody molecules are produced in response to antigenic stimulation.

The terms "cell-mediated immunity" and "cell-mediated immune response" are meant to refer to the immunological defense provided by lymphocytes, such as that defense provided by T cell lymphocytes when they come into close proximity to their victim cells. A cell-mediated immune response normally includes lymphocyte proliferation. When "lymphocyte proliferation" is measured, the ability of lymphocytes to proliferate in response to a specific antigen is measured. Lymphocyte proliferation is meant to refer to B cell, T-helper cell or CTL cell proliferation.

The term "immunogenic amount" refers to an amount of antigenic compound sufficient to stimulate an immune response, when administered with a subject immunogenic composition, as compared with the immune response elicited by the antigen in the absence of the adjuvant.

The term "immunopotentiating amount" refers to the amount of the adjuvant needed to effect an increase in antibody titer and/or cell-mediated immunity when administered with an antigenic compound in a composition of the invention, as compared with the increase in antibody and/or cell mediated immunity level observed in the absence of the adjuvant.

By "wild type" or "WT" as used herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A WT protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or a nucleotide sequence that has not been intentionally modified.

By "subject," used interchangeably herein with "host," "individual," and "animal," includes humans and all domestic e.g. livestock and pets and wild mammals and fowl, including, without limitation, cattle, horses, cows, swine, sheep, goats, dogs, cats, rabbits, deer, mink, chickens, ducks, geese, turkeys, game hens, and the like.

Examples

1.1 Epitope selection

The inventors have developed recombinant proteins comprising epitopes capable of eliciting an immune response against SARS-CoV-2 within a Dendroaspin scaffold as immunotherapeutics for the prevention or treatment of COVID-19.

Table 1 summarizes the properties of the tested recombinant proteins (designated TRI88- V1-5). Figures 5 and 6 show the schematic presentation of the recombinant polypeptides TRI88-V2-V5 and alignment of these recombinant proteins, together with a wild type RBM of SARS-CoV-2 spike protein and dendroaspin. Table 2 summarizes the identities between the tested recombinant polypeptides TRI88-V1-V5. Table 3 summarizes the immunogenic epitopes derived from Sars-CoV-2 anlL-6 that are incorporated in to dendroaspin scaffolds in the recombinant immunogenic polypeptides of the invention.

To demonstrate the applicability of the invention to Sars-Cov-2 mutants, the inventors have generated vaccine constructs to target the mutated SARS-CoV-2-induced COVID-19, containing representative mutations A222V and D614G (Table 4).

In addition, vaccine constructs have been designed containing Danish mink SARS-CoV-2 strain S protein mutations, together with A222V and D614G (Table 5). Vaccine constructs have additionally been designed containing Danish S protein mutations (Table 7).

In addition, two recombinant protein constructs combining N protein epitopes with S protein (RBD) epitopes have been created TRI88-V14 and TRI88-V15. This approach builds on our development of a range of multi-epitope constructs targeting S protein (TRI88-V1-V12, as described earlier). (See Table 10). The three main parts of these constructs are the RBD (S protein) core, N-terminus of N protein and C-terminus of N protein. The sequence of N- terminal of N protein derived from MERS is selected as it has high homology with those of 229E and NL63. In addition, sequence of C-terminal of N protein derived from CoV-2 is introduced as it has high homology with those of CoV, OC43, and HKLI1. TRI88-V15 is designed using our dendropaspin scaffold carrying a modified RBD. TRI88-V14 is designed for use as a comparator in studies of TRI88-V15 and includes the same N protein epitopes but does not use the dendroaspin scaffold, see Figure 49.

1.2 Swiss Modelling

In the design of the recombinant proteins comprising epitopes capable of eliciting an immune response against SARS-CoV-2 within the Dendroaspin scaffold (TRI88-V2-V5), the SWISS- MODEL template library was searched with the primary local alignment search tool (BLAST) and HMM-HMM-based lightning-fast iterative sequence search (HHBIits) (Remmert M, et al., lightning-fast iterative protein sequence searching by HMM -HMM alignment. Nat Methods. 2012;9:173-175) for related evolutionary structures matching the target sequence of the SARS-CoV-2 spike protein (Figure 7).

Swiss modelling in the design of the recombinant proteins comprising epitopes capable of eliciting an immune response against SARS-CoV-2 mutants within the Dendroaspin scaffold is illustrated in Figures 27 to 31 (TRI88-V7-V9), in Figure 34 (TRI88-V10), in Figure 35 (TRI88-V7, V10 and V11), in Figure 36 (TRI88-V12), in Figure 50 (TRI88-V14) and in Figure 50 (TRI88-V15) .

Predictions of antigenicity for TRI88-V10, V11 and V12 are illustrated in Figure 37. Predictions of antigenicity for TRI88-V14 and V15 are illustrated in Figure 52. Predictions of antigenic epitopes of TRI88-V14 and V15 are shown in Tables 11 and 12 respectively.

1.3 Synthesis of the genes of the designed constructs

Epitopes selected from potential proteins or antigens identified as outlined above were inserted into the Dendroaspin scaffold through amino acid residue substitution and deletion (Figure 1). The inventors used a proprietary Dendroaspin mutation database, including protein solubility, expression yield, and stability, to aid design.

Gene sequences of recombinant molecules were designed using Laser gene version 13 software and synthesized in GeneScrpt and cloned in pGEX-3X vector with GST-Tag (for affinity purification) and a Xa cleavage site (for separating GST-tag) (Figure 8). The plasmid DNA pGEX-3X containing inserted protein gene was transformed into DH5a (for transformation) and BL-21 (for protein expression) strains, respectively. The proteins were purified by the affinity column (Sepharose 4B), ion exchange, and gel filtration chromatography.

The purity of the recombinant proteins was determined by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (PAGE), which showed a single band as shown in Figure 9A.

Purified protein was identified by binding to the anti-SARS-CoV-2 S protein antibody as shown in Figure 9B. The constructs recognize the SARS-CoV-2 S protein antibody (purchased from Abeam, Cambridge UK). The constructs TRI88-V4 and TRI88-V5 recognize the SARS-CoV-2 S1 protein antibody (purchased from Abeam, Cambridge UK) as shown in Figure 10A and 10B, respectively, when compared with SARS-CoV-2 S1 protein in Figure 10C. binding to S1 antibody by V4 and V5 is even better than that by S1 protein itself).

Additionally, TRI88-V5 contains an epitope (P2) derived from IL-6. The result showed that TRI88-V5 can bind to IL-6 (whole molecule-induced) antibody. In contrast, TRI88-V4 did not bind to IL-6 antibody as it did not contain any IL-6 epitope. Furthermore, two epitopes (P1 and P2 derived from IL6), only P2 showed positive binding (Figure 18A). Moreover, there is no binding to IL-6 antibody by either SARS-Cov2 RBD or SARS-CoV2 S1 (Figure 18B).

1.4 Mouse immunization, serum, and tissue or organ collection

Mice were subcutaneously immunized with 20 pg/mouse of the recombinant proteins formulated with Alum adjuvant (Sigma, UK) and a booster with 10 pg/mouse of the immunogen (recombinant protein TRI88-V1 , V2-V5 and V7-V12) and Alum. Repetitive immunizations multiple sites (RIMMs) immunization method was used (injection of 8 sites (Figure 11), 5 times injection on day 0, 2, 5, 7, and 9). 14 days after the first injection, the blood was collected.

At the end of experiment, mice were sacrificed, and plasma was prepared from the blood. Spleens were divided, and 1/3 spleen of each mice were embedded in Optimal cutting temperature (OCT) compound and frozen on dry ice, 2/3 part of the spleen were homogenized into complete RPMI medium (pre-incubation at 37 °C) to obtain spleen cells, then frozen in 90% FCS and 10% DMSO at -80°C O/N, then transferred into liquid nitrogen and then transfer into dry ice for delivery.

Homogenization procedure: tissue was placed onto a plate with complete RPMI medium or PBS, homogenized by hand using a syringe plunger’ piston gently tap or rub the tissues before the filter.

Lymph nodes were taken and homogenized into complete RPMI medium (pre-incubation at 37 °C), then frozen in 90% FCS and 10% DMSO at -80°C O/N, then transferred into liquid nitrogen and moved into dry ice for delivery. Two whole nodes collected from each mouse were embedded in OCT, the remaining nodes were separated into lymphocyte cells. The liver and kidney were collected, and embedded in OCT, to check any damage of liver and kidney functions. 1.5 Immune response assay

The immune response assay was conducted using Thermo Scientific Pierce Maleimide Activated Plates, which are ideal for binding sulfhydryl-containing molecules that are difficult to coat onto polystyrene plates, such as peptides that contain a terminal cysteine. These coated plates are an especially useful tool for assessing specific anti-hapten antibody titers during antibody production. Plates are available in clear for colorimetric assays, white for chemiluminescent assays, and black for fluorescent assays.

A. Materials Required

• Binding Buffer: 0.1M sodium phosphate, 0.15M sodium chloride, 10mM EDTA; pH 7.2

• Wash Buffer: 0.1M sodium phosphate, 0.15M sodium chloride, 0.05% Tween®-20 Detergent; pH 7.2

• Cysteine*HCI (Product No. 44889)

• Primary antibody

• Enzyme-conjugated secondary antibody

• Enzyme substrate (e.g., for HRP use the Thermo Scientific TMB Substrate Kit, Product No. 34021)

B. Method

1. Wash plate wells three times with 200pL each of Wash Buffer.

2. Prepare the sulfhydryl-containing peptide at 1-50pg/mL in Binding Buffer.

3. Add 100-150pL of the peptide solution to each well and incubate for > 2 hours at room temperature or overnight at 4°C. For best results, use a plate mixer during incubation.

4. Wash wells three times using 200pL of Wash Buffer for each wash.

5. Immediately before use, prepare cysteine solution at 10pg/mL Add 200pL and incubate for 1 hour at room temperature to inactivate excess maleimide groups.

6. Wash wells three times using 200pL of Wash Buffer for each wash.

7. Add 100pL of primary antibody per well and incubate for 1 hour at room temperature.

8. Wash wells three times using 200pL of Wash Buffer for each wash.

9. Add 100pL per well of secondary antibody. Incubate for 1 hour at room temperature.

10. Wash wells three times using 200pL of Wash Buffer for each wash.

11. Add the enzyme substrate and develop according to manufacturer’s recommendations.

Using the above described methodology, antisera collected 14 days after the first immunization with the recombinant proteins was measured for the immune responses, including IgG (Figure 12 A-D; Figure 21 A-D), and IgM (Figure 12 E and F; Figure 21 E-H). The cross-reaction activity between TRI88-V1 and TRI88-V2 was also detected (antigen V1 reacted with antibody of V2; antigen V2 reacted with antibody of M2) as they contain 97% amino acid sequence homologies.

The results showed that TRI88-V1 .TRI88-V2, TRI88-V4 and TRI88-V5induced significant immune responses. Additionally, the results showed that TRI88-V1 and TRI88-V2 have high cross-reaction activity as they contain 97% amino acid sequence homologies. The antibody isotypes IgA and IgM were measured.

The results also showed epitopes (beyond RBM) derived from either S1 or S2 (TRI88-V4 and V5) or IL-6 (TRI88-V5) can generate immune responses when compared to the controls (AGD-den and Alum) (Figure 22), which may contribute to antibody neutralisation.

Antigen-antibody titre was additionally performed using V7 construct as V7-contained peptide epitope derived from SARS-Cov-2 S1 - coated plate, and anti-sera were taken from mice (8 weeks female Balb/C) immunized with TRI88-V7, GST (unrelated protein used as a protein control and Alum used as an adjuvant, respectively (N=6 or 10) (Figure 33).

Antigen-antibody titre was performed using V11 construct or V11-contained peptide epitopes derived from SARS-Cov-2 S - coated plate, and anti-sera were taken from mice (8 weeks female Balb/C) immunized with TRI88-V11 , GST, a protein-tag used as a control and Alum used as an adjuvant, respectively (N=6 or 10) (Figure 39).

Antigen-antibody titre was performed using V12 construct or peptide epitopes derived from SARS-Cov-2 S-coated plate, and anti-sera were taken from mice (8 weeks female Balb/C) immunized with TRI88-V12 and GST, an unrelated protein used as a control or alum used as a control respectively (N=8 or 10) (Figure 40)

1.6 Neutralization assay by ELISA ePass™ SARS CoV-2 Neutralization Antibody Detection Kit was used for the detection of antibody neutralization. The test is a blocking ELISA assay approved by FDA as an Emergency Use Authorization (EUA) for emergency using product (6/11/2020), which mimics the virus neutralization process. Plasma from immunized mice with TRI88-V1 , TRI88-V2, TRI88-V4, TRI88-V5, TRI88-V12, AGD-Den, and Alum was tested. Plasma from immunized mice with TRI88-V1 , TRI88-V2, AGD-Den, and Alum was tested (Figure 13).

Using SARS-CoV-2 surrogate virus neutralization test kit (Genscript), the inventors detected that the plasma from mice immunized with TRI88-V1 , V2, V4, V5 and V12 contained circulating neutralizing antibodies against SARS-CoV-2 that block the interaction between the receptor-binding domain of the viral S protein (RBD) and ACE2. The percentage of inhibition by plasma from immunized mice with TRI88-V1 and TRI88-V2 was shown 52.5% and 55.7% at 1 :2 dilution of plasma, respectively, when compared to those of either with AGD-den and Alum only (no inhibition was detected) (Figure 14 A and B). Immunization with TRI88-V4 and TRI88-V5 achieved 71.5% and 68.3% inhibition, respectively, at 1 :4 dilution of plasma [Figure 14 C and D], Based on the criteria of the ELISA kit function, inhibition is defined as [1-(OD) value of Sample/ (OD value of negative control] 100%. When inhibition is >30%, the sample is positive for notarizing antibodies, our constructs show the potent neutralizing IgG activity against authentic SARS-CoV-2, which was in agreement with that reported in a recent publication [12],

Thus, the recombinant polypeptides of the invention show potent neutralizing IgG activity against SARS-CoV-2 based on the criteria, reported in Kreer C., et al. Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients Cell. 2020;S0092-8674(20)30821-7.

As shown in Figure 23, at 1 :5 in the sera of vaccinated animals at 25 of ID50 values samples are 1-4, 1-5, 1-6, 1-7 and 1-8 (N=5). Controls are 3-4, 3-5, 3-6 (AGD-den) and 4-4, 4-5 (Alum only). Assay was performed as follows: Vero-E6 cells were seeded at a concentration of 20K cells per 100 μl per well in 96-well plates and allow to stick onto the plates O/N. Serial dilutions of plasma samples (heat inactivated at 56 °C for 30min) were prepared with DMEM (2% FBS and 1%PBS) and incubated with authentic SARS-CoV-2 for 1h at 37 °C. The medium was removed from the pre-plated Vero-E6 cells and the plasma- virus mixture were added to the Vero-E6 cells and incubated at 37 °C for 24H, This plasma- virus mixture was aspirated, and each well was fixed with 150 pl of 4% formalin at room temperature for 30min and then topped up to 300 pl using PBS. The cells were washed once with PBS and permeabilized with 0.1% Triton-X in PBS at room temperature for 15min. The cells were washed twice with PBS and blocked using 3% milk in PBS at room temperature for 15min. The blocking solution was removed and an N-protein specific Mab (marinized CR3009) was added at 2 pg/ml (diluted using 1% milk in PBS) at room temperature for 45 min. The cells were washed twice with PBS and horse anti-mouse IgG-conjugated to HRP was added (1:2000 in milk in PBS, Cell signal Technology, catalogue no, S7076) at room temperature for 45min. The cell was washed twice with PBS, developed using TMB substrate for 30min and quenched using 2M H2SO4, before reading at 450nm.

Measurement were performed in duplicate and triplicates used to calculate the ID50. The results showed positive antibody neutralisation (ID50 = 25 at 1 :5 dilution).

For TRI88-V12, neutralization was shown using a ePass TM kit purchased from The GenScript; B. Enlargement of A and C. Inhibition of the interaction between the receptor- binding domain of the viral S protein (RBD) and receptor ACE2. The kit contains two key components: Horseradish peroxidase (HRP) conjugated recombinant SARS-CoV-2 RBD fragment (HRP-RBD) and the human ACE2 receptor (hACE2). If the neutralizing antibodies against SARS-CoV-2 RBD are sufficient in the serum or plasma (mice), the protein-protein interaction between HRP-RBD and hACE2 can be blocked. The Optical Density (OD) of the final solution is read at 450 nm in a microtiter plate reader (Figure 41).

1.1 Inhibition of ACE2 (Biotinylated) SARS-CoV-2 RBD by plasma from mice immunized with TRI88-V1 and TRI88-V2

Serial dilutions of anti-sera (plasma) were added into Biotinylated ACE2: SARS-CoV2 RBD. The assay was performed as follows: (a). Coating the plate with SARS-CoV2 RBD. (b). After O/N incubation, washing plate wells and adding the negative control or plasma, incubation for 1h. (c) Washing plate wells, and add 100μL biotinylated human ACE2 and incubation for 1h. (d). Washing plate wells and add HRP conjugated anti-biotin antibody and incubation for 1 h, then incubation with 200 μl TMB buffer for 20 mins, stopping TMB reaction by adding 50 pl of 2 M H2SO4. (e). Checking OD at 450 nm wavelength. The ability of antisera (plasma) to inhibit the binding between ligand S protein RBD and biotinylated ACE2 can be determined by comparing OD values in different experimental groups.

The results showed that 46 and 47% inhibition at 1:4 dilution of plasma were obtained when compared to the control without plasma (Figure 15A), or plasma from immunized mice with ether AGD-den or Alum only (no inhibition) (Figure 15B).

TRI88-V4 and V5 were additionally measured; the results showed that 60.4% and 63.7% inhibition for TRI88-V4 and V5 respectively, at 1 :4 dilution of plasma, were obtained when compared to the control without plasma or plasma from immunized mice with ether AGD-den or Alum only (no inhibition) (Figure 24 A and B).

1.8 Functional assay by ELISA to measure binding to Biotinylated human ACE2 Functional assay by ELISA was used to measure the binding of constructs to Biotinylated human ACE2. Immobilized SARS-CoV-2 Spike Protein (RBD), TRI88-V1, and TRI88-V2 at ~1.5 pg/ml (100 pl/well) (-6 pg/ml with GST-tag) can bind human ACE2 protein (biotinylated). It was measured by its binding ability in a functional ELISA. Anti-biotin horseradish peroxidase (HRP) conjugated antibody or streptavidin-HRP can bind biotin. 3’, 3', 5,5'- Tetramethylbenzidine (TMB) was used as an HRP substrate and OD450nm was used to measure the OD values by a spectrometer.

(Figures 16, 17 and 20)

The results show that saturation of binding to ACE2 by TRI88-V4 and TRI88-V5 can be reached at can be reached at 2-3 μg/ml (without GST-tag) (OD value at 0.8) versus TRI88-V1 (OD value at 0.65).

100 μl was coated onto the plate.

Additionally, the results have shown that TRI88-V4 and TRI88-V5 binding to different concentrations of human ACE2 can be saturated at OD 8-1.0 whereas at 1.2 for S1 protein as shown in Figure 19, indicating the potency of both of V4 and V5 is closely similar to that of S1 protein in binding to human ACE2.

The ability of TRI88-V4 and TRI88-V7 to bind to human ACE2 in a dose dependant manor is shown in Figure 32.

The ability of TRI88-V7, TRI88-V11 and TRI88-V7 to bind to human ACE2 in a dose dependant manor is shown in Figure 38.

The ability of TRI88-V14 and TRI88-V15 to bind to human ACE2 in a dose-dependent manor is shown in Figure 53.

1.9 T-cell analysis

CD4 ⁺ and CD8 ⁺ T-cell responses to vaccination can be assessed according to the secretion of I FNy, interleukin-2 (IL-2), IL-17, and tumor necrosis factor a (TN Fa). The response is measured by intracellular cytokine staining assays in spleen cells labelled with cell staining after the stimulation with corresponding constructs for approximately 6 h and detected by a CytoFlex flow cytometry analyser (Beckman Coulter). (CD4 ⁺ IFNy, CD4 ⁺ IL2, CD41L-17, CD4 ⁺ TNFa; CD8 ⁺ IFNy, CD8 ⁺ IL2, CD8 ⁺ IL-17, CD8 ⁺ TNFa). 1.10 Plasma cytokine analysis

IFN-y serves as a Th1 pathway marker thought to be a pro-inflammatory cytokine, significantly elevated in the late stage of severe COVID-19 illness [13], IL-10 is a Th2 pathway marker, which is generally considered to be an anti-inflammatory cytokine. However, clinical studies have shown that it was only elevated in severe but not mild cases after the virus infection. Similarly, IL-1 receptor antagonist (IL-1RA) levels in severe cases were significantly higher than those in mild cases in the first two weeks [14], which suggests the increase might be an attempt to moderate the immune response. As such, TRI88-V4 and V5 can be used as Immunotherapeutics used to modulate the immune system towards COVID- 19 patient recovery.

The CD4 ⁺ and CD8 ⁺ T-cell responses to vaccination were assessed according to the secretion of IFN-y, interleukin-2 (IL-2), and tumor necrosis factor a (TNF-a). These were measured by intracellular cytokine staining assays in spleen cells after the stimulation with our constructs for approximately 6 hours and detected by flow cytometry (e.g., CD4 ⁺ IFN-y ⁺ , CD4 ⁺ TNF-a ⁺ ; CD8 ⁺ IFN-y ⁺ , CD8 ⁺ IL-2 ⁺ , CD8 ⁺ TNF-a ⁺ ) (Figures 25 and 26).

Plasma cytokines (IFN-y and IL-10) concentrations in immunized mice were measured by ELISA following the manufacturer’s instruction (R&D system, Abingdon, UK). Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm. Average the triplicate readings for each standard, control, and sample and subtract the average zero standard optical density (O.D.). Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. If samples have been diluted, the attention read from the standard curve must be multiplied by the dilution factor. Collect plasma using heparin as an anticoagulant — centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at < -20 °C (Avoid repeated freeze-thaw cycles).

T cell responses from TRI88-V4 and -V5-immunized mice spleen stimulated by correspondent vaccine construct. The data from the flow cytometry. FACS analysis of T cells from spleens of mice immunized with V4, V5, AGD, or naive. Total populations were analysed on FL1 (Forward Light 1) for FITC-CD4 or CD8 versus FL2 (PE-IFN-y, IL-2 or TNF- a). Two-dimensional plots determine the expression of cytokines IFN-y, IL-2, or TNF-a in CD8+ and CD4+ T cell subsets. For each sample, 1x10 ⁴ cells gated events were analysed.

The results showed that the expressions of CD4 ⁺ IL-2 ⁺ , CD4 ⁺ IFN-y ⁺ , CD8 ⁺ IFN-y ⁺ , CD4 ⁺ TNF- a ⁺ were significantly increased compared to those of in control groups. These results are in agreement with other vaccine candidates reported recently [15],

Plasma cytokines (IFN-y and IL-10) concentrations in TRI88-V12 immunized mice were measured by ELISA following the manufacturer’s instruction (R&D system, Abingdon, UK). Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm. Average the triplicate readings for each standard, control, and sample and subtract the average zero standard optical density (O.D.). Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic (4-PL) curve- fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. If samples have been diluted, the attention read from the standard curve must be multiplied by the dilution factor. Collect plasma using heparin as an anticoagulant — centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at < -20 °C (Avoid repeated freeze-thaw cycles) (Figure 42).

T cell responses from TRI88-V12-immunized mice spleen stimulated by V12. The data from the flow cytometry. FACS analysis of T cells from spleens of mice immunized with V12(c), GST or naive. Total populations were analysed on FL1 (Forward Light 1) for FITC-CD4 or CD8 versus FL2 (PE-IFN-y, TNF-a and IL-2 or Two-dimensional plots determine the expression of cytokines IFN-y, IL-2, or TNF-a in CD8+ and CD4+ T cell subsets. For each sample, 1x10 ⁴ cells gated events were analysed (Figure 43).

The properties of the tested vaccine constructs are summarised in Table 8.

2. Antigen-induced specific polyclonal IgY therapeutics

The immunization of hens represents an excellent alternative for efficiently generating polyclonal antibodies. Also, the yolk immunoglobulin has several intrinsic biochemical advantages, particularly no cross-reaction with mammalian immunoglobulins [48], Because IgY immunoglobulins do not activate mammalian complement and show no interaction with mammalian Fc receptors or other polyvalent antibodies that could mediate an inflammatory response in the gastrointestinal tract, IgY antibodies are attractive for peroral or aerosol immunotherapy [49], Immunized birds naturally deposit protective antibodies (IgY) in the yolk of their eggs. Specific anti-viral IgY was previously tested in several designs.

TRI88-V4-induced specific IgY was performed by Davids Biotechnologie GmbH (www.davids-bio.com). The ELISA data of IgY is shown in Figure 54.

3. Zika Virus

3.1 Epitope selection

Zika virus has recently emerged as an important human pathogen. It is a member of the flavivirus genus within the family Flaviviridae. This group of approximately 70 viruses are primarily transmitted by mosquito or tick vectors and include members such as dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus.

Zika virus is a positive sense, single-strand ribonucleic acid (RNA) virus. The RNA is translated into a single polyprotein (3423 amino acids in length SEQ ID NO:45) encoding 3 structural proteins — capsid (C); membrane (M), which is generated from its precursor premembrane (prM); and envelope (E) (SEQ ID NO:46) — as well as 7 nonstructural proteins (NS1 , NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The nonstructural proteins appear to assist in replication and packaging of the genome as well as in subverting the host pathways in favor of the virus. The structural proteins form the virus particle. The generation of the 10 individual proteins from the polyprotein is regulated by viral and host proteases, and the efficiency of one of the host proteases (furin) in cleaving the viral targets (prM) is variable and may play a role in pathogenesis.

Studies have revealed that ZIKV E (aa 291-791) shares a three-domain architecture with the E proteins of DENV and other related flaviviruses (Figure 57 and Figure 58). Domain III (Dill, aa296-393, SEQ ID NO:24) of the ZIKV envelope (E) protein is a vital vaccine target, and a vaccine developed using a mutant Dill of E (EDIII) protein protects adult and pregnant mice, and unborn offspring, against ZIKV infection. The domain III of flavivirus E protein (EDIII) contains the cellular receptor-binding motifs and importantly, the majority of the type- specific neutralizing epitopes that induce strong host antibody responses and/or protective immunity are mapped to this domain (Figure 55). Recently, EDIII of ZIKV (zEDIII) is targeted by several different ZlKV-specific antibodies with distinct yet potent neutralizing activities. Since neutralizing antibodies have been considered to be correlated with protection for approved vaccines against fever (YFV) and tick-borne encephalitis virus (TBEV), as well as having been demonstrated to play essential roles in the protection against infection by many flaviviruses. The potential of zEDIII in inducing potent neutralizing antibodies renders it a prime candidate as an effective subunit vaccine against ZIKV.

Upon entry into host cells via endocytosis, the acidic endosomal environment triggers an irreversible conformational change in the E protein and a transition from a dimer to trimer formation that leads to the membrane fusion event. In the ER, newly assembled virus progeny from immature virions and exhibit a spiky surface anchored with 60 trimeric protrusions of the E and precursor-membrane (prM) heterodimers. During virus maturation, a low pH environment in the trans-Golgi network (TGN) induces the reorganization of the E- prM heterodimers into E homodimers. During the maturation process, E-prM heterodimer can be cleaved by the furin, and then uncleaved E-prM heterodimer will revert back to a spiky immature trimeric structure.

Flavivirus E protein is responsible for virus entry and represents a major target for neutralizing antibodies. Nevertheless, the molecular basis of the mAb-E interaction is still elusive.

The overall structure of ZIKV has three distinct domains [Figure 59A]: a central p-barrel (domain I) which is folded into an eight-stranded p-barrel with an additional N-terminal Ao strand; an elongated finger-like structure [(domain II, residues 52-131 (between strands Do and Eo) and residues 193-279 (between strands Ho and Io)], and a C-terminal immunoglobulin-like module (domain III, residues 296-393) [Figure 59B], The central domain I is folded into an eight-stranded p-barrel with an additional N-terminal Ao strand [Figure 59C],

3.2 Synthesis of the genes of the designed constructs

Epitopes selected from potential proteins or antigens identified as outlined above were inserted into the Dendroaspin scaffold through amino acid residue substitution and deletion), followed by further modification through Swiss modelling. The inventors used a proprietary Dendroaspin mutation database, including protein solubility, expression yield, and stability, to aid design.

Once obtaining preferred protein sequence, the gene sequences of recombinant molecules were designed through DNASTAR Lasergene 13 software. The epitopes capable of eliciting an immune response against Zika structural E protein were incorporated into the dendroaspin scaffold as shown in Figure 56.

Details of the selected proteins are provided in Table 13. Details of the dendroaspin and zika vaccine constructs are provided in Table 14.

3.3 Neutralization Assay

Neutralizing activity of antisera is measured using a standard plaque reduction neutralization with BHK21 cells as previously described [9], Briefly, 5-fold serial dilutions of antisera are added to approximately 200 PFU of ZIKV SZ01 strain [10] and incubated for 1 hr at 37°C. Then, the mixture will be added to BHK21 cell monolayers in a 12-well plate in duplicate and incubated for 1 hr at 37 °C. The mixture is removed, and 1 ml of 1.0% (w/v) LMP agarose (Promega) in DMEM plus 4% (v/v) FBS is layered onto the infected cells. After further incubation at 37°C for 4 days, the wells are stained with 1% (w/v) crystal violet dissolved in 4% (v/v) formaldehyde to visualize the plaques. PRNT50 values are determined using non- linear regression analysis.

4. Th2-TYPE IMMUNE RESPONSE

T helper cells (also known as Th cells) are a sub-group of lymphocytes that play an important role in establishing and maximizing the capabilities of the immune system. Mature Th cells are believed to always express the surface protein CD4. T cells expressing CD4 are also known as CD4 ⁺ T cells. Regulatory T cells are normally anergic and do not proliferate in vitro unless stimulated by antigen primed dendritic cells (DC) or in the presence of cytokine cocktail. Antigen presenting cells (A PCs) such as DC present antigens to Th cells. Helper T cells recognize these and are activated by them. The activation of a resting helper T cell causes it to release cytokines and other stimulatory signals that stimulate the activity of macrophages, killer T cells and B cells. Proliferating helper T cells that develop into effector T cells differentiate into two major subtypes of cells known as Th1 and Th2 cells (also known as Type 1 and Type 2 helper T cells, respectively).

The Type 1/ Th1 response stimulates the cellular immune system. Cytokines associated with the Type 1 response include but are not limited to interferon-y (IFN-y) and interleukin-12 (IL12).

The Type 2/ Th2 response stimulates the Humoral immune system. Cytokines associated with the Type 2 response include but are not limited to interleukin-10 (IL10), interleukin-4 (IL4) and transforming growth factor (TGFP). The inventors have found that dendroaspin is an immunogenic protein that can stimulate a switch from the Th1 response to TH2 response. This switch which may have important consequences for the inflammatory/immune process in certain diseases, particularly inflammatory and/or allergic diseases. Thus, dendroaspin is a Th2 cell adjuvant. Dendroaspin issues a Th2-biased immune response. Dendroaspin can stimulate Th2 response leading to lgG1 secretion. Dendroaspin induces an anti-inflammatory immune response.

Dendroaspin can be used as an adjuvant in a vaccine to treat allergic diseases, autoimmune diseases or infection-induced immunopathological reactions.

The following Examples demonstrate that immunisation with dendroaspin induces a higher level of IL10 compared to TNFa indicating that Dendroaspin induces a Th2-biased immune response. Dendroaspin induces the secretion of anti-inflammatory cytokines which are associated with the Th2 response. Dendroaspin activates the regulatory T cells in vitro. Thus, dendroaspin has the potential to be developed as an adjuvant for inducing anti inflammatory immune response.

4.1 EXAMPLE A

Monocyte derived dendritic cells (MDDC) were generated from peripheral blood mononuclear cells (PBMCs) cultured for 6 days in media supplemented with 5ng/ml each of recombinant IL-4 and GM-CSF. MDDCs were activated with LPS (10 pg/ml), HSP65 (10 ug/ml) BCG (10 ug/ml) for 24 hours and co-cultured with autologous T cells for 5 days in serum free X-vivo 20 media. T cell proliferation was monitored by the reduction in CFSE fluorescence in the CD4 positive T cells for 5 days. Proliferation index was calculated by the formulae No of proliferating cells in test - No of proliferating cells in control/Total number of proliferating cells. Proliferation index of >0.15 is considered positive. Cytokines in the MDDC: T cell culture supernatant was measured by the Cytometric bead assay (CBA, BD Biosciences). FOXP3 expression in T cells was assessed at using the anti-human FOXP3 Staining Kit (Becton-Dickinson).

4.2 EXAMPLE B

Groups of mice (n= 10) were immunized with dendroaspin or recombinant protein containing different peptide epitopes. ELISA was carried out to determine serum antibody concentrations. Mean absorbance ± SD are given in Table A. Table A shows IgG response to dendroaspin following immunization with dendroaspin and other recombinant molecules. Immunization with dendroaspin as well as recombinant molecules containing antigenic epitopes induced a high level of antibody response to the dendroaspin protein. OD at a serum dilution of 1:100.

Table A

Dendroaspin immunized mice produced antibodies of IgG 1 isotype indicating a Th2 response (Table B). Table B shows the isotype of the antibody response to dendroaspin. able B

Groups of mice were immunized with dendroaspin or control GST. Cytokines were detected in the plasma of immunized mice 6 weeks after the immunizations by ELISA. Table C shows the levels of cytokines in the plasma of immunized mice. The level of IL10 in plasma was very high in the dendroaspin immunized mice compared to GST control (781.161 pg/ml compared to the detectable limit).

Table C The results displayed in Tables A,B,C indicate a potential of dendroaspin to induce Th2 response which is anti-inflammatory.

Regulatory T cell (T regs) are normally anergic and do not proliferate in vitro unless stimulated by antigen primed DC or in the presence of cytokine cocktail. Therefore, the number of these cells normally is found to be lower after 5 days in culture, unless they are driven to proliferation by the antigen pulsed DC. We looked at the number of T regs on day 5 and compared it to the day 0 to enumerate the increase in the number of the cells, if any. The number of regulatory T cells was found to increase in the DC T cell co culture experiments upon stimulation with dendroaspin. T cell proliferation was monitored for 5 days by CFSE dilution. The recombinant antigens were used at a concentration of 10 ug/ml. Proliferation index was calculated by the following formulae: number of proliferating cells in the test = No of proliferating cells in control/Total number of proliferating cells. Proliferation index of >0.15 is considered positive. Figure 60 shows T cell activation and proliferation in response to antigen presentation by dendritic cells. Dendroaspin stimulated dendritic cells (DC)/T cell co-culture induced T cell proliferation and cytokine production in vitro (Figure 60 and 61 B).

Cytokine production was monitored following T cell activation in response to antigen presentation by dendritic cells. Cytokines were measured in the dendritic cell (DC)/T cell co- culture supernatants after 5 days of incubation. The cytokine levels were estimated by CBA_Flex kits from BD pharmingen. Figures 61 A (LPS) and 61 B (dendroaspin) shows secretion of inflammatory and anti-inflammatory cytokines by the activated T cells. C / T cell co-culture induced cytokine production in vitro.

The CD4+ CD25+ and FOXP3 + cells were enumerated as the Regulatory T cells. The percentages given are for the total lymphocyte population. Table D shows increase in the Regulatory T cells compared to Day 0 after co-culture with DC activated with dendroaspin and HSP65.

Table D

The concentration of both TGFp and IL10 in the culture supernatants stimulated with dendroaspin were found to be significantly higher (p=0.04 and 0.02) than the control. The T- test was used to calculate the significant increase in the concentration on antigen stimulation. The concentrations are given in pg/ml. Table E shows cytokine concentration in the culture supernatant in the co-culture with dendritic cells (DC) activated with dendroaspin and HSP65. 2F1 is dendroaspin without any cloned peptide molecule. IL10 concentration was higher in the culture supernatants stimulated with HSP65 and HSP65+2F1(0.04 and 0.00) but TGFp concentrations were not significantly higher than control. IFNy concentration was significantly higher (p=0.0007) on LPS stimulation which is as expected.

Table E

In summary, dendroaspin stimulated DC- T cell co-culture induced T cell proliferation and cytokine production in v/tro.The cytokines induced by dendroaspin were anti-inflammatory (IL10 and TGFP) and were significantly higher (p=0.04 and 0.02) than control in the culture supernatants stimulated with dendroaspin which leads to the conclusion that dendroaspin can act to induce an anti-inflammatory immune response.

4.3 EXAMPLE C

Figures 62A-F displays FACS data showing that in vitro culture with dendroaspin activates T reg cells. Figure 62A shows T reg cells day 0 1.2%; Figure 62B shows control at 1.5%, Figure 62C shows PHA 14.2%, Figure 62D shows 2F1 1.9%, Figure 62E shows HSP65 1.1% and Figure 62F shows HSP65 + 2F1 2.2%. These results indicated that dendroaspin can be a suitable adjuvant. 4.4 EXAMPLE D

Further in vitro co-culturing experiments were undertaken to assess the effects of dendrtic cells activated with dendroaspin and HSP65 on regulatory T cells (T reg cells). Table F (below) shows the increase in the T reg cells compared to day 0 after co-culture with dendrtic cells activated with dendroaspin and HSP65. The number of regulatory T cells was found to increase in the DC T cell co culture experiments upon stimulation with dendroaspin

Table F

4.5 EXAMPLE E

The effect of pre-treatment of monocytes and macrophages with dendroaspin on the inflammatory response was investigated. Monocyte derived dendritic cells were cultured as described earlier. Mouse macrophage cell line J774 was procured from National Center for Cell Sciences (NCCS, Pune). Lipopolysaccharide (LPS) present in all gram-negative organisms is a potent inducer of tumor necrosis factor (TNF)-a. We studied the effect of pre- treatment with dendroaspin on inflammatory cytokine secretion. MDDC or J774 cells were cultured at 1x10 ⁶ cells/well in serum free X-vivo 20 media. Dendroaspin 10ug/ml was added to the cells for 24 hours, washed and treated with two different concentrations of LPS for another 24 hrs. Cytokines were estimated in the culture supernatant by CBA. Figure 63 shows a bar chart of the effect of dendroaspin pretreatment on TNF- a concentration in culture supernatant of macrophages activated with LPS in MDDC cells (Figure 63A) and J774 cells (Figure 63B). Significant reduction in TNF- a secretion was observed upon pre treatment of MDDC and mouse macrophages with dendroaspin, pre-treatment of MDDC and macrophages with dendroaspin reduced the LPS induced inflammatory response.

4.6 EXAMPLE F

To study in vivo polarization of immune response groups of mice were immunized with glutathione synthetase (GST) and dendroaspin conjugated GST (DSP-GST). Mice were given primary immunization with 50 pg of the proteins and 2 boosters with 25 pg of the protein with alum as an adjuvant. Three days after the last dose, serum was collected from the animals and GST specific antibody response was studied by ELISA. Figure 64 shows a bar chart of the effect of dendroaspin on IgG subtypes I gG 1 , lgG2a, lgG2b and lgG3 response to GST. Results show that dendroaspin is an immunogenic protein and induces the Th2 response.

4.7 EXAMPLE G

Experiments were conducted to investigate dendroaspin as a mucosal adjuvant. Groups of ApoB ^tm2S9y /Ldlr ^{tm1 Her/J} mice (5-6 weeks) were orally dosed five times on alternate days with dendroaspin-GST or GST alone, 20 pg per animal, per dose. Induction of tolerance was studied 6 days after the last dose. Mice were fed on a diet high in fat and cholesterol (Harlan, TD 96121 Indianapolis, USA (21 % fat and 1.25% cholesterol), for 10 weeks to develop atherosclerosis. Quantification of atherosclerotic lesions was carried out as per the protocol approved by the Animal Models of Diabetic Complications Consortium (http://www.diacomp.org). Flow cytometry was carried out to assess the CD4 ⁺ CD25 ⁺ FOXP3 ⁺ regulatory T cells in lymphoid organs. Figure 65 shows a bar chart of the increases in the percentage of FOXP3 positive T reg cells in lymphoid organs (lymph nodes and spleen) following oral dosing. Mucosal administration of dendroaspin induced an increase in foxp3 cells in the lymphoid organ of mice and the percentage of T reg cells increased in lymph nodes and spleen (30% and 26% respectively) in dendroaspin treated mice compared to GST alone, though the increase was not statistically significant. Oral treatment with dendroaspin resulted in an 8.9% reduction in lesion in the aortic sinus (Figure 66A), while macrophage infiltration into the sinus was reduced by 37.5 % in ApoB48/LDLr mice (Figure 66B). In a mucosal tolerance rabbit model, oral dosing of dendroaspin was found to increase the mean percentage of FOXP3 positive cells in the spleen compared to GST treated animals (3.46±0.99 vs 11.38±2.14 , p=0.008) (Figure 67). Oral treatment with dendroaspin reduced the lesion development by 24.4 % compared to GST control in the aortic sinus (P=0.03). Figure 68 shows a bar chart of the reduction in lesion area in total surface area in the aortic sinus of dendroaspin treated rabbits. In rabbits treated orally with dendroaspin, lipid accumulation as observed by oil red O (ORO) staining in the aortic sinus was reduced by 14.3% in dendroaspin treated rabbits in comparison to GST (P=NS). In addition, macrophage infiltration in aorta was significantly reduced in dendroaspin treated rabbits compared to GST ( P=0.03) . Figure 69 shows a bar chart of the reduction in macrophage infiltration as a percentage of CD68 positive area in lesions of DSP treated rabbits. From the data it can be concluded that dendroaspin stimulates anti inflammatory immune response and activates T regulatory cells. Oral administration of dendroaspin reduces increases the T reg cells in lymphoid organs and can moderately reduce early atherosclerosis in mice and rabbit models.

4.8 EXAMPLE G

The effect of carrier proteins on the ApoB-specific Ig subclass and IgG subtype composition in the sera (BALB/C) was studied using 5-7-week old male KO (BQ,'\29S-Apobtm2Sgy Ldlrtm1Her/S) mice, sera were collected 1 week after immunization with either (i) a dendroaspin-GST construct or (ii) KLH-conjugate or (iii) dendroaspin. Table G shows the constructs used for immunization and the effect on IgG and subtypes IgG 1 and lgG2a and Ig subclasses IgM and IgA. As can be seen from Table 7 the dendroaspin-GST construct was the only construct able to illicit an immune response against lgG2a and that the dendroaspin-GST treated mice produced a high level of IgG specific responses. able G

5-7-week old male KO (BQ,'\29S-Apobtm2Sgy Ldlrtml Her/ 3) mice were immunized with 20 pg of either GST-2f1 or GST at days 0, 2, 5, 7 and 9 in combination with Alum adjuvant (50 pl/mouse) in a volume of 240 pl and injected subcutaneously into 8 sites. Serum samples were collected 2 weeks after the third inoculation and tested against four peptide antigens ApoB, PDGF, HSP60 and Cpn. Table H shows the results. able H

Table I shows the proteins used for immunization against the dendroaspin-GST antigen construct.

Table I

Results showed protein 191 induced ApoB and C. pn. peptide-specific IgG response; Protein N8154 induced ApoB peptide-specific IgG response; Protein 679 induced ApoB peptide- specific IgG response and inconsistent PDGF-specific IgG response; Protein N103 induced inconsistent ApoB peptide-specific IgG response; Protein H29 induced inconsistent MicroHSP-specific IgG response; Protein H13 induced inconsistent MicroHSP-specific IgG response.

In conclusion, the results showed that different combinations of epitopes result in different immune responses.

Tabte 1: Prop® rites of oonstrocte

Table 2. Construction of TRI88-Vx using a Dendroaspin (Den) scaffold.

The data was produced using an on-line programm https://www.ebi.ac.uk/Tools/psa/emboss needle/

• Software calculation.

Table 3. Epitopes derived from SARS-CoV-2 and IL-6

Table 4. Vaccine constructs

NTD* denotes N-terminal domain; M-RBM** denotes modified receptor binding motif AA ^{436- 510} , modified to include additional disulfide bonds.

Table 5. Epitopes derived from SARS-CoV2 S protein

RBM modified denotes modified receptor binding motif AA ^436-510 , modified to include additional disulfide bonds.

Table 6: S protein mutations

Gene code Other location TRI88-V7 TRI88-V11 TRI88-V12 mutations Country

21656- F32I China

21658

21707- H49Y China

21709

21765- Danish mink,

HV 69-70 HV 69-70

21770 UK del del deletion

21991- UK. European

21993 Y144 del Y144 del countries (EU) deletion

21995- Y145H

21997

22226- A222V UK, EU

A222V A222V

22228

22277- Q239K

22279

22301- S247R Australia

22303

22622- N354D China

22624

22652- D364Y China

22654

22661- V367F France

22623

22811- K417N S. Africa

22813 22877- Scotland, EU

N439K* N439K*

22879

22919- Y453F Danish mink

Y453F Y453F

22921

22988- G476S

22990

22991- S477N

22993

22991- S477I

22993

23009- V483A

23011

23012- E484K S. African

23014 Brazil

N501Y N501Y UK, EU. S.

A23063T

(TAT) (TAT) African

C23271A A570D A570D UK, EU

23402- D614G Danish mink,

D614G D614G

23404 Germany, UK

Nigeria, UK,

C23604A P681 H P681 H

EU

23635- I692V Danish mink 23638

C23709T T716I T716I UK

23948- D796H UK, EU

23950

24053- A831V

24055

24077- D839Y/N/E

24079 24389- S943P Belgium

24391

T24506G S982A S982A UK, EU

G24914C D1118H D1118H UK, EU

24989- P1143L Australia

24911

25349- P1263L

25351

25247- Danish mink

M 12291 M 12291

25249

Table 7. Epitopes of TRI88-V11 and V12 derived from SARS-CoV-2 S protein

Modified RBM denotes modified receptor binding motif AA ^436-510 and AA ^319-541 , modified to include additional disulfide bonds.

Table 8. The properties of the TRI vaccine constructs

Table 9: list of CoVs and their features

Table 10

Epitopes of TRI88-V14 and V15 derived from N terminus of N protein of MERS, C terminus of N protein and S protein of Cov2

Modified RBM denotes modified receptor binding motif AA ^436-510 and AA ^319-541 , modified to include additional disulfide bonds.

Table 11 : Prediction of antigenic epitopes of TRI88-V14 Table 12: Prediction of antigenic epitopes of TRI88-V15 Table 13. Zika Vaccine properties

Table 14. Construction of TRI88-Vx using a Dendroaspin (Den) scaffold.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. Sequences