NL2027551B1

NL2027551B1 - 1immunogenic polypeptides and pharmaceutical compositions

Info

Publication number: NL2027551B1
Application number: NL2027551A
Authority: NL
Inventors: J Cruz Luis; Kit Chung Chih; Eich Christina; G Da Silva Cândido
Original assignee: Academisch Ziekenhuis Leiden
Priority date: 2021-02-12
Filing date: 2021-02-12
Publication date: 2022-09-12
Also published as: WO2022173302A1; NL2027551A

Abstract

The invention relates to immunogenic polypeptides, particularly polypeptides comprising an amino acid sequence selected from the group set out in SEQ ID NOs: 1 to 26 of Table 1, or variant of such sequences that incorporate an amino acid sequence that differs from one of SEQ ID NOs: 1 to 26 by alteration of up to 2 amino acid residues within the coronavirus-derived portion of the sequence set out in Table 1. The polypeptides may be linear, or branched, and may be subject to modification, such as by addition of palmitic acid, at the N or C terminal. There are also provided nucleic acid sequences encoding the polypeptides, pharmaceutical compositions comprising such polypeptides or nucleic acid sequences, and cells expressing the polypeptides or comprising the nucleic acid sequences. These agents are useful for the prevention or treatment of coronavirus-based infections, such as COVlD-19 caused by SARS—CoV-2. Accordingly, the invention also provided medical uses and methods of treatment directed to the prevention or treatment of such infections. Certain formulations using the polypeptides in combination with adjuvants and/or carriers are demonstrated to offer particular advantages.

Description

IMMUNOGENIC POLYPEPTIDES AND PHARMACEUTICAL COMPOSITIONS

FIELD OF THE INVENTION The present invention relates to immunogenic polypeptides comprising amino acid sequences derived from coronaviruses. The invention also relates to the nucleic acid sequences encoding such polypeptides, as well as to pharmaceutical compositions that comprise the polypeptides and/or nucleic acid sequences. The pharmaceutical compositions, polypeptides or nucleic acid sequences are suitable for use in methods of treatment or medical uses for the prevention or treatment of coronavirus infections. The invention further relates to cells that express polypeptides of the invention, and to methods of producing polypeptides of the invention.

BACKGROUND Coronaviruses are positive-sense single-stranded RNA viruses belonging to the family Corona viridae. This family has a size ranging from 60 nm to 140 nm in diameter with spike like projections on its surface giving it a crown like appearance under the electron microscope; hence the name of coronavirus. It is subdivided into the genera Alpha, Beta, Gamma and Delta coronavirus. These viruses mostly infect animals, including birds and mammals. In humans, they generally cause mild respiratory infections, such as those observed in the common cold and the majority of the population has usually been exposed at some point throughout their lives. However, there have been two events in the past two decades where the crossover from animal beta coronaviruses to humans has resulted in severe disease. The first was in 2002-2003 in the Guangdong province of China when a new coronavirus of the B genera and with origin in bats, via the intermediate host of palm civet cats, was transmitted to humans. This virus was designated as severe acute respiratory syndrome coronavirus (SARS-CoV) and affected 8422 people mostly in China and Hong Kong and caused 916 deaths (mortality rate 11%) before being contained. Later on in 2012, in Saudi Arabia, emerged the Middle East respiratory syndrome coronavirus (MERS-CoV), also of bat origin and with dromedary camels as the intermediate host. It affected 2494 people and caused 858 deaths (mortality rate 34%). MERS-CoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ~10% and ~35%, respectively.

In December 2019, in Wuhan, capital city of Hubei province and a major transportation hub of China, many people started presenting to local hospitals with severe pneumonia of unknown cause. The outbreak caused by this new coronavirus was extended over the world. On the 7th of January 2020 the virus was identified as a novel beta coronavirus eventually named COVID- 19, that had >95% homology with the bat coronavirus and >70% similarity with the SARS-CoV. This new coronavirus was called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS- CoV-2) by their similitude with SARS-CoV. SARS-CoV-2 has a genome size of ~30 kilobases which, like other coronaviruses, encodes for multiple structural and non-structural proteins. The structural proteins include the spike (S) protein, the envelope (E) protein, the membrane (M) protein, and the nucleocapsid (N) protein. Due to the recent discovery of SARS-CoV-2 very little information is currently available on its immunogenicity (e.g., epitopes eliciting antibody or T cell responses). Some studies have determined that SARS-CoV-2 is quite similar to SARS-CoV both in an analysis of the full-length of its genomes, as well as its cell entry mechanisms and the human cellular receptor that it uses to infect. Moreover, all coronaviruses present quite high similar protein compositions.

All coronaviruses present the envelope spike protein (S Protein). The monomer of the S protein from SARS-CoV-2 contains 1273 amino acids, with a molecular weight of about 140 kDa. Self- association naturally assembles the S protein into a homo-trimer, typically similar to the first class of membrane fusion protein (Class | viral fusion protein). The S protein contains two subunits (S1 and S2). The S1 subunit can be further defined with two domains termed the N-terminal domain (NTD) and the C-terminal domain (CTD). The receptor binding domain (RBD) is located in the CTD. The use of S proteins as antigens in vaccine development include the full-length S protein, the RBD domain, the S1 subunit, NTD, and the internal membrane fusion peptide (FP). RBD: most of SARS-CoV-2 subunit vaccines currently under development use the RBD as the antigen. Recombinant RBD consists of multiple conformational neutralizing epitopes that can induce high titer of neutralizing antibodies against SARS-CoV. The RBD domain is relatively conserved as compared with the S1 subunit. The S2 subunit contains the basic elements required for membrane fusion, including an internal membrane FP, two 7-peptide repeats (HR), a membrane proximal external region (MPER), and a trans-membrane domain (TM). The N protein is the most abundant protein in coronavirus. This protein has been reported to be highly antigenic, with 89% of patients who developed SARS producing antibodies to this antigen.

Several institutions have initiated programs on the SARS-CoV-2 subunit vaccine, and almost all of them use the S protein as antigens.

Recently, it was reported that 149 sites of mutations were identified across the genome of 103 sequenced strains of SARS-CoV-2, and the virus had evolved into two subtypes, termed L and S subtype. The study also indicated that the two subtypes showed great differences in geographical distribution, transmission ability, and severity of disease, which add more difficulties for vaccine design.

Vaccines designed for SARS-CoV have shown a protective role of both humoral and cell- mediated immune responses. Antibody responses generated in mouse models against the S protein, the most prominent protein of SARS-CoV, have been demonstrated to protect from infection. Likewise, antibodies generated against the N protein of SARS-CoV, which is profusely expressed during infection, and were particularly prevalent in SARS-CoV-infected patients have shown to be a highly immunogenic protein. However, the antibody response was shown to be short-lived in convalescent SARS-CoV patients.

SUMMARY OF THE INVENTION In a first aspect, the invention provides a polypeptide comprising an amino acid sequence selected from the group set out in SEQ ID NOs: 1 to 28 of Table 1, or a variant thereof comprising an amino acid sequence that differs from one of SEQ ID NOs: 1 to 26 by alteration of up to 2 amino acid residues within the coronavirus-derived portion of the sequence set out in Table 1. These polypeptides in accordance with the first aspect of the invention may be referred to as “polypeptides of the invention” or “peptides of the invention”. The polypeptides of the invention that differ from one of SEQ ID NOs: 1 to 26 by alteration of up to 2 amino acid residues in the coronavirus-derived portion of the sequence may be referred to as “variants” of the polypeptides that comprised one of SEQ ID NOs: 1 to 26.

In a second aspect, the invention provides a nucleic acid sequence encoding a polypeptide according to the first aspect of the invention. Such nucleic acid sequences will also be referred to as “nucleic acid sequences of the invention” or “nucleic acids of the invention”.

In a third aspect, the invention provides a pharmaceutical composition comprising a polypeptide according to the first aspect of the invention and/or a nucleic acid sequence according to the second aspect of the invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions are also referred to as “pharmaceutical compositions of the invention” or “compositions of the invention”. As discussed in more detail below, a pharmaceutical composition of the invention may comprise one or more polypeptides of the invention. Alternatively, or additionally, a pharmaceutical composition of the invention may comprise one or more nucleic acid sequences of the invention. It will be appreciated that, as described in more detail below, the polypeptides of the first aspect of the invention, the nucleic acid sequences of the second aspect of the invention, and the pharmaceutical compositions of the third aspect of the invention are all suitable for use as medicaments. These medical uses of the invention are particularly suitable for the prevention or treatment of coronavirus infections. A fourth aspect of the invention provides a method of preventing or treating a coronavirus infection, the method comprising providing to a subject in need of such prevention or treatment a therapeutically effective amount of a polypeptide in accordance with the first aspect of the invention. For the sake of brevity, such preventative or curative methods may both be referred to herein as “methods of treatment of the invention”. Suitably the therapeutically effective amount of the polypeptide in accordance with the first aspect of the invention may be provided by administration to the subject of a pharmaceutical composition according to a third aspect of the invention. In a fifth aspect, the invention provides a prokaryotic or eukaryotic cell expressing a polypeptide according to the first aspect of the invention. Cells in accordance with this embodiment of the invention may be referred to as “cells of the invention”. Suitably, a prokaryotic or eukaryotic cell according to the fifth aspect of the invention may comprise a nucleic acid sequence according to the second aspect of the invention. Cells of the invention may be used to produce polypeptides of the invention. Indeed, in a sixth aspect the invention provides a method of producing a polypeptide in accordance with the invention, the method comprising growing a cell in accordance with the fifth aspect of the invention such that the cell expresses a polypeptide of the first aspect of the invention.

FIGURE LEGENDS

Figure 1 shows results achieved in reciprocal quantification of SARS-CoV-2 specific IgG antibodies after vaccination with distinct pre-clinical and clinical adjuvants.

Four doses schedule (days 0, 14, 28 and 42). Extraction was performed on day 56. All groups were inoculated with 100pL through the sc route. A) Study of pre-clinical adjuvants. Depicted are 5 the IgG levels of naive (i.e. non-vaccinated mice) versus 1. Alu+MPLA ; 2. MPLA; 3. R848; 4.

Poly (I:C); versus Nanohydrogel(peptide only); versus the same adjuvants but encapsulated in NanoHydrogels. B) Study of clinical adjuvants. Depicted are the IgG levels of naive (i.e. non- vaccinated mice) versus 1. Alu+MPLA; 2. MPLA; 3. R848; 4. Poly (I:C); versus Nanohydrogel (peptide only); versus the same adjuvants but encapsulated in NanoHydrogels. Statistics were calculated using the unpaired Student's t test with Welch's correction. Statistical differences were considered significant at p < 0.05. * =p < 0.05; **p =<0.01; ***p < 0.001.

Note: For clarity reasons only the control groups Nanohydrogel (peptide only) and Peptide only are labeled with “peptide” although all groups were vaccinated with peptides equality, with the exception of the naive group. The NanoHydrogel concentration was 26% (W/V).

Abbreviations: Alu: Aluminum; MPLA: Monophosphoryl lipid A. Figure 2 illustrates results achieved investigating SARS-CoV-2 specific T cell responses after vaccination with distinct pre-clinical and clinical adjuvants.

Four doses schedule (days 0, 14, 28 and 42). Extraction was performed on day 56. All groups were inoculated with 100pL through the sc route. A+B) CD8 cytotoxic T cell response after ex vivo restimulation of splenocytes with dendritic cells pre-loaded with peptides. Depicted is the quantification of production of TNFa by CD8 T cells determined by flow cytometry as a measure of cognate recognition of the peptides. C+D) CD4 T helper cell response after ex vivo restimulation of splenocytes with dendritic cells pre-loaded with peptides. Depicted is the quantification of double positive CD4 T cells for the production of TNFa and IFNy determined by flow cytometry as a measure of cognate recognition of the peptides. Statistics were calculated using the unpaired Student's t test with Welch's correction. Statistical differences were considered significant at p < 0.05. * =p < 0.05; **p =<0.01; ***p < 0.001.

Note: For clarity reasons only the control groups nanohydrogel (peptide only) and peptide only are labeled with “peptide” although all groups were vaccinated with peptides equality, with the exception of the naive group. The nanohydrogel concentration was 26% (W/V). Abbreviations: Alu: Aluminum; MPLA: Monophosphoryl lipid A.

Figure 3A demonstrates that compositions of the invention are effective via intranasal administration, and shows the impact of formulation on the activity of such compositions Three doses schedule (days 0, 14 and 28). Extraction was performed on day 42. Groups 1, 2, 3, y 6 were inoculated through the nasal route with 50 HL. Group 4 was inoculated sc with 100 HL. 5 Further details of the study are set out in the Examples. Figure 3B demonstrates positive recognition of the spike protein by the IgG antibodies generated in response to polypeptides and compositions of the invention The western blot gels were loaded with denatured spike protein (r&d systems prod. # 10549-cv- 100) and then the protein was visualized at high gain (©).

Figure 4 illustrates the impact of formulation and delivery systems on effectiveness of the compositions of the invention. Two doses schedule {days O and 14). Extraction was performed on day 24. All groups were inoculated nasally with 100uL. The composition of experimental groups is shown in Table 5 to the figure. Further details of the study undertaken are set out in the Examples.

DETAILED DESCRIPTION OF THE INVENTION The inventors have devised a new approach for the design and generation of novel immunogenic polypeptides. They have used this new approach to generate a panel of immunogenic polypeptides (which they refer to as “chimeric synthetic polyvalent lipo-multiepitope peptides”) comprising the amino acid sequences set out in Table 1.

Table 1 provides 26 amino acid residue sequences (SEQ ID NOs: 1 to 26) that can be utilised in peptides of the invention able to promote an immune response that is protective or curative in respect of disorders caused by coronavirus infections. Polypeptides of the invention comprising these amino acid sequences (or variants of the sequences, as referred to herein) are suitable for medical use, for example in vaccines. Their ability to induce both humoral and cell-mediated immune responses makes them especially suited to therapeutic applications.

The methods of treatment and medical uses described herein may be employed in the prevention or treatment of infections caused by a coronavirus selected from the group consisting of: SARS- CoV-2; SARS-CoV; and MERS-CoV. In particular, the polypeptides of the invention have proven to be of benefit in promoting an immune response which is protective in respect of SARS-CoV-2, the coronavirus responsible for COVID-19. As will be recognised, the COVID-19 pandemic means that there remains a well-recognised clinical need for medicaments that can be used to prevent or treat this disease.

The polypeptides of the invention provide such medicaments, as demonstrated by the experimental results set out in the Examples. These illustrate that administration of polypeptides of the invention to a subject is able to bring about an immune response against coronavirus in the recipient. They demonstrate not only the effectiveness of pharmaceutical compositions of the invention, but also particular benefits associated with specific composition formulations.

It will be appreciated that these results illustrating the therapeutic efficacy of polypeptides of the invention also support the therapeutic use of nucleic acids of the invention (since expression of such nucleic acids by a recipient's cells will lead to the production of a therapeutically effective amount of a polypeptide of the invention).

As set out in Table 1, each of the amino acid sequences of SEQ ID NOs: 1 to 26 comprises a coronavirus-derived portion. These coronavirus-derived portions comprise one or more epitopes from the proteins of SARS-CoV-2. Details of the epitopes incorporated in the coronavirus-derived portion of each amino acid sequence are set out in Table 1.

The coronavirus-derived portions of SEQ ID NOs: 1 to 26 are taken from the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, or envelope (E) of SARS-COV-2. Sequences comprising epitopes in the S, E, M and N structural proteins were selected due to their potential to induce dominant and long-lasting immune responses. The selected sequences include regions defining linear B cell epitopes and T cell epitopes.

While the sequences incorporated are taken from SARS-CoV-2, the portions of the proteins used have been selected on the basis that they are from regions where the sequences of the SARS- CoV-2 proteins are identical with the corresponding sequences within SARs-CoV and/or MERS- CoV. Furthermore, the regions selected do not appear to be subject to mutation in the available strains of SARS-CoV-2, SARs-CoV or MERS-CoV. These epitopes have the potential to induce immune responses in respect of not only SARS-CoV-2, but also SARS-CoV, and MERS-CoV. Accordingly, polypeptides of the invention comprising or consisting of these sequences (or comprising or consisting of variants of SEQ ID NOs: 1 to 28) have the capacity to be used in medical uses or methods of treatment for the prevention or treatment of a range of coronavirus infections, including (but not limited to) infections caused by SARS-CoV-2, SARS-CoV, or MERS- CoV.

The coronavirus-derived portions of the amino acid sequences set out in SEQ ID Nos: 1 to 26 contain Bor T cell (MHC | and Il) epitopes, or a combination of such epitopes. The T cell epitopes were selected for those associated with MHC that seek to maximize the coverage of a global population. The nature of the coronavirus-derived epitope or epitopes found in each sequence is set out in Table 1.

Generally, amino acid sequences of SEQ ID NOs: 1 to 14, 16 and 23 to 26 each include coronavirus-derived B cell epitopes, while the amino acid sequences of SEG ID NOs: 15, and 17 to 22 each include coronavirus-derived T cell epitopes (MHC | and Il).

In addition to the coronavirus-derived epitopes, the amino acid sequences of SEQ ID NOs: 1 to 14 and 23 to 26 each also include a distinct universal T-helper epitope. These are derived from tetanus toxoid (noted as “TT” in Table 1), diphtheria toxoids (noted as “DDT” in Table 1), or the universal T-helper epitope PADRE (noted as “Padre” in Table 1).

In view of the above, it will be recognized that polypeptides of the invention can be selected for their suitability to induce either B cell or T cell immune responses.

A polypeptide of the invention that comprises an amino acid sequence selected from the group set out in SEQ ID NOs: 1 to 14, 16 and 23 to 26 of Table 1, or a variant thereof comprising an amino acid sequence that differs from one of SEQ ID NOs: 1 to 14, 16 and 23 to 26 by alteration of up to 2 amino acid residues within the coronavirus-derived portion of the sequence set out in Table 1, may be selected for therapeutic use when it is desired to induce a B cell immune response.

On the other hand, a polypeptide of the invention that comprises an amino acid sequence selected from the group set out in SEQ ID NOs: 15, and 17 to 22 of Table 1, or a variant thereof comprising an amino acid sequence that differs from one of SEQ ID NOs: 15, and 17 to 22 by alteration of up to 2 amino acid residues within the coronavirus-derived portion of the sequence set out in Table 1, may be selected for therapeutic use when it is desired to induce a T cell immune response.

A combination of polypeptides of the invention including at least one that comprises an amino acid sequence selected from the group set out in SEQ ID NOs: 1 to 14, 16 and 23 to 26 of Table 1, or a variant thereof comprising an amino acid sequence that differs from one of SEQ ID NOs:

1to 14, 16 and 23 to 26 by alteration of up to 2 amino acid residues within the coronavirus-derived portion of the sequence set out in Table 1, and at least one that comprises an amino acid sequence selected from the group set out in SEQ ID NOs: 15, and 17 to 22 of Table 1, or a variant thereof comprising an amino acid sequence that differs from one of SEQ ID NOs: 15, and 17 to 22 by alteration of up to 2 amino acid residues within the coronavirus-derived portion of the sequence set out in Table 1 may be used when it is desired to induce both a B cell and T cell based immune response in a subject. Comparing homology of the spike protein sequence between SARS-CoV-1, SARS-CoV-2 and MERS virus results in a sequence identity of 76% and 35% for SARS-CoV-1, SARS-CoV-2 and MERS, respectively (the low homology of MERS being expected, since it binds to DPP-IV in human cells, instead of ACE2). Polypeptides of the invention may comprise spike B-epitopes from regions of the sequence that share a high degree of homology between these species were selected (SEQ ID NO: 14 and SEQ ID NO: 16).

Polypeptides of the invention may comprise linear MERS B-epitopes, for example the B-epitope comprising residues 736-761 of MERS. This epitope is found in SEQ ID NO: 16. Polypeptides of the invention may comprise MHC Class | and/or MHC Class II T-cell epitopes. Examples of such epitopes providing MHC-peptide binding affinity may be identified using computing approaches, for example with the NetMHCpan-4.0 and NetMHCII servers to identify potentially immunogenic peptides derived from M, N, E and S viral proteins. In the present case, two spike-protein MHC-I epitopes, highly conserved across SARS-CoV-1, SARS-CoV-2, and MERS (LITGRLQSL and NLNESLIDL), have been incorporated in certain polypeptides of the invention to promote their generation of a cross-protective vaccine against these related viral infections. These sequences are found in SEQ ID NOs: 17 to 22. In addition to designing the immunogenic polypeptides of the invention, the inventors have also developed pharmaceutical compositions comprising these agents that remarkably enhance the effects of the polypeptides when used in vaccine formulations. These pharmaceutical compositions comprise the polypeptides of the invention in combination with selected adjuvants and/or delivery systems. The compositions, their constituents, and the advantages that they offer are discussed in more detail elsewhere in the present disclosure. The efficacy of the polypeptides of the invention and pharmaceutical composition of the invention is demonstrated by the results set out in the Examples.

The results that the inventors have achieved using the polypeptides and pharmaceutical compositions of the invention indicated that they offer a number of notable advantages as compared to previously available vaccines.

Medical use of the polypeptides and pharmaceutical compositions of the invention allows a wider spectrum of immune responses to coronaviruses to be generated due to the presence of epitopes from many different sites within proteins of SARS-CoV-2. Furthermore, the IgG serum levels of anti-SARS-COV-2 antibodies directed to epitopes from spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) achieved using the compositions of the invention (comprising suitable adjuvants and delivery systems) via systemic and mucosal inoculation is as intense as the response obtained with the systemic inoculation in alum per se. Furthermore, the impact of the adjuvants on efficacy of the compositions of the invention means that these compositions do not require the same high quantity of antigen (i.e. of the polypeptides of the invention) has been required by previous approaches. The simplicity of the production process for polypeptides of the invention also makes manufacture of these vaccines very cheap as compared to other vaccine types. Both of these factors provide marked advantages in assisting the production of large quantities of the compositions of the invention in an timely, cost- effective, and efficient manner. The invention will now be further described with reference to the following definitions and Examples.

DEFINITIONS Polypeptides of the invention The first aspect of the invention provides polypeptides defined with respect to the amino acid sequences set out in SEQ ID NOs: 1 to 26 of Table 1. The polypeptides of the invention either comprise an amino acid sequence set out in SEQ ID NOs: 1 to 26, or comprise an amino acid sequence that differs from one of SEQ ID NOs: 1 to 26 by alteration of up to 2 amino acid residues within the coronavirus-derive part of these sequences. The polypeptides of the invention comprising an amino acid sequence selected from the group set out in SEQ ID NOs: 1 to 22 of Table 1, or a variant thereof comprising an amino acid sequence that differs from one of SEQ ID NOs: 1 to 22 by alteration of up to 2 amino acid residues within the coronavirus-derived portion of the sequence set out in Table 1, represent a useful sub-set of the polypeptides of the invention. As referred to above, each of the amino acid sequences set out in SEQ ID NOs: 1 to 26 comprises a portion that is derived from the coronavirus SARS-CoV-2. These portions of SEQ ID NOs: 1 to 26 are taken from the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, or envelope (E) of SARS-COV-2 and comprise one or more B cell or T cell epitopes. The coronavirus-derived portions of SEQ ID NOs: 1 to 26 are defined in Table 1, along with details of the B or T cell epitopes incorporated. The amino acid sequences of SEQ ID NOs: 1 to 26 each also comprise further amino acid residues that are not derived from coronavirus sources. These include T-helper cell epitopes, (derived from tetanus toxin, diphtheria toxin, or the universal T-helper epitope PADRE) and linkers between the epitopes. In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 1. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 1.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 1 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 1. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 1 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 1. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 1 is set out in Table 1. In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 2. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 2. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 2 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 2. In a suitable embodiment the invention provides a polypeptide comprising an amine acid sequence that varies from SEQ ID NO: 2 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 2. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 2 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 3. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 3.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 3 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 3. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 3 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 3. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 3 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 4. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 4.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 4 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 4. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 4 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 4. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 4 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 5. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 5.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 5 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 5. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 5 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO5 1. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 5 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 6. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 6.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 6 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 8. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 6 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 6. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 6 is set out in Table 1. In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 7. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 7.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 7 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 7. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 7 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 7. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 7 is set out in Table 1. In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 8. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 8. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 8 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 8. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 8 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 8. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 8 is set out in Table 1. In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 9. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 9.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 9 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 9. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 9 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO9 1. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 9 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 10. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 10.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 10 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 10. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 10 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 10. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 10 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 11. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 11.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 11 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 11. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 11 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 11. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 11 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 12. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 12.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 12 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 12. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 12 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 12. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 12 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 13. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 13.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 13 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 13. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 13 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 13. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 13 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 14. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 14.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 14 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 14. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 14 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 14. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 14 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 15. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 15.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 15 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 15. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 15 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 15. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 15 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 16. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 16.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 16 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 16. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 16 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 16. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 16 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 17. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 17.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 17 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 17. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 17 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 17. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 17 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 18. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 18.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 18 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 18. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 18 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 18. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 18 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 19. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 19.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 19 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 19. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 19 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 19. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 19 is set out in Table 1. In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 20. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 20.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 20 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 20. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 20 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 20. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 20 is set out in Table 1. In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 21. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 21. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 21 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 21. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 21 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 21. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 21 is set out in Table 1. In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 22. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 22.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 22 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 22. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 22 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 22. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 22 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 23. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 23.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 23 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 23. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 23 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 23. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 23 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 24. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 24.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 24 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 24. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 24 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 24. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 24 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 25. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 25.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 25 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 25. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 25 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 25. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 25 is set out in Table 1.

In a suitable embodiment the invention provides a polypeptide that comprises the amino acid sequence set out in SEQ ID NO: 26. In a suitable embodiment the invention provides a polypeptide that consists of the amino acid sequence set out in SEQ ID NO: 26.

In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 26 by alteration of a single amino acid residue within the coronavirus-derived portion of SEQ ID NO: 26. In a suitable embodiment the invention provides a polypeptide comprising an amino acid sequence that varies from SEQ ID NO: 26 by alteration of two amino acid residues within the coronavirus-derived portion of SEQ ID NO: 26. For the avoidance of doubt, the coronavirus-derive portion of SEQ ID NO: 26 is set out in Table 1.

In the context of the preceding paragraphs, references to sequences that vary from one of the sequences set out in SEQ ID NOs: 1 to 26 by “alteration of a single amino acid residue” should be taken as requiring that a total of one residue is altered as compared to the reference sequence in Table 1 (i.e. not “one or more” residues). Similarly, references to sequences that vary from one of the sequences set out in SEQ ID NOs: 1 to 26 by “alteration of two amino acid residues” should be taken as requiring that a total of two residues are altered as compared to the reference sequence in Table 1 (i.e. not “two or more” residues).

As noted above, polypeptides of the invention incorporating alteration of a single amino acid residue, or alteration of two amino acid residues, in the coronavirus-derived portions of SEQ ID NOs: 1 to 26 may be referred to as variants of SEQ ID NOs: 1 to 26.

Polypeptides variants of SEQ ID NOs: 1 to 26 may additionally or alternatively contain alterations of amino acid residues in the non-coronavirus-derived portions of the sequences set out in Table

1. As appropriate, a variant in accordance with this embodiment of the invention may comprise a single alteration in the non-coronavirus-derived portion of SEQ ID NOs: 1 to 28, or up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, upto 8, up to 9, or up to 10 alterations in the non-coronavirus- derived portion of SEQ ID NOs: 1 to 26.

Suitably, alterations of the amino acid sequence in a variant of any of SEQ ID NOs: 1 to 26 may be selected such that they do not disrupt epitopes within the coronavirus-derived and/or non- coronavirus-derived portions of the sequence.

A variant comprising an alteration in the non-coronavirus-derived portion of SEQ ID NOs: 1 to 26 may comprise 2 such alterations, 3 such alterations, 4 such alterations, 5 such alterations, 6 such alterations, 7 such alterations, 8 such alterations, 9 such alterations, or 10 such alterations. Indeed, a variant comprising an alteration in the non-coronavirus-derived portion of SEQ ID NOs: 1 to 26 may comprise 2 or more such alterations, 3 or more such alterations, 4 or more such alterations, 5 or more such alterations, 6 or more such alterations, 7 or more such alterations, 8 or more such alterations, 9 or more such alterations, or 10 or more such alterations.

The alterations that may be incorporated in the non-coronavirus-derived sequences of such variants will be subject to the same considerations applicable to the alterations in the coronavirus- derived portions of the sequences, as set out below.

Each alteration of an amino acid residue present in a variant of SEQ ID NOs: 1 to 26 may be independently selected from the group consisting of: substitution of an amino acid residue; addition of an amino acid residue; and deletion of an amino acid residue.

A number of different substitutions of amino acid residues are suitable for incorporation in variants of SEQ ID NOs: 1 to 26.

Merely by way of example, amino acids maybe substituted for one another within similar classes or subclasses. Three examples of such classes are: hydrophilic amino acids, hydrophobic amino acids and cysteine-like amino acids. A polypeptide of the invention comprising a substitution within the coronavirus-derived amino acid sequence of any of SEQ ID NOs: 1 to 26 may incorporate one or two substitutions of hydrophilic amino acid residues with hydrophilic amino acid residues, one or two substitutions of hydrophobic amino acid residues with hydrophobic amino acid residues, or one or two substitutions of cysteine-like amino acid residues with cysteine-like amino acid residues.

Hydrophilic amino acids include amino acids having acidic, basic or polar side chains.

"Hydrophilic amino acid" refers to an amino acid having a side chain that is attracted by aqueous solution. Examples of genetically encoded hydrophilic amino acids are Ser and Lys. Examples of non-genetically-encoded hydrophilic amino acids are Cit and hCys.

"Acidic amino acid" refers to a hydrophilic amino acid having a side chain pK value of less than

7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Examples of genetically encoded acidic amino acids include aspartic acid (aspartate) and glutamic acid (glutamate).

"Basic amino acid" refers to a hydrophilic amino acid having a side chain pK value of greater than

7. Basic amino acids typically have positively charged side chains at physiological pH due to association with a hydronium ion. Examples of genetically encoded basic amino acids include arginine, lysine and histidine. Examples of non-genetically-encoded basic amino acids are the acyclic amino acids omithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine.

"Polar amino acid" refers to a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but which has a bond in which the pair of electrons shared by two atoms is held more closely by one of the atoms. Examples of genetically encoded polar amino acids are asparagine and glutamine. Examples of non-genetically-encoded polar amino acids are citrulline, N-acetyl lysine and methionine sulfoxide.

"Hydrophobic amino acid" refers to an amino acid having a side chain that is uncharged at physiological pH and that is repelled by aqueous solution. Examples of genetically encoded hydrophobic amino acids are lle, Leu and Val. An example of a non-genetically-encoded hydrophobic amino acid is t-BuA. Hydrophobic amino acids include amino acids having aromatic or apolar side chains.

"Aromatic amino acid" refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated TT-electron system (aromatic group). The aromatic group may be further substituted with groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl, nitro and amino groups, among others. Examples of genetically encoded aromatic amino acids are phenylalanine, tyrosine and tryptophan. Commonly encountered non-genetically-encoded aromatic amino acids include phenylglycine, 2-naphthylalanine, beta.-2-thienylalanine, 1,2,3,4-tetrahydroisoquinoline- 3-carboxylic acid, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine and 4- fluorophenylalanine.

"Apolar amino acid" refers to a hydrophobic amino acid having a side chain that is generally uncharged at physiological pH and that is not polar. Examples of genetically encoded apolar amino acids are glycine, proline and methionine. An example of a non-genetically-encoded apolar amino acid is Cha. Apolar amino acids may be further subdivided to include, among others, aliphatic amino acids. The definitions of the classes of amino acids as used herein are as follows: "Aliphatic amino acid" refers to an apolar amino acid having a saturated or unsaturated straight, branched or cyclic hydrocarbon side chain. Examples of genetically encoded aliphatic amino acids are Ala, Leu, Val and lle. An example of a non-genetically-encoded aliphatic amino acid is Nie. "Cysteine-like amino acid" refers to an amino acid having a side chain capable of forming a covalent linkage, such as a disulfide linkage, with a side chain of another amino acid. Cysteine- like amino acids generally have a side chain containing at least one thiol (SH) group. An example of a genetically encoded cysteine-like amino acid is cysteine. Examples of non-genetically- encoded cysteine-like amino acids are homocysteine and penicillamine. One of skill in the art will appreciate that the aforementioned classifications are not absolute. Several amino acids can be included in more than one category. For example, tyrosine has both an aromatic ring and a polar hydroxyl group, and thus can be included in both the aromatic and polar categories. Similarly, cysteine can form disulfide linkages but is also apolar. Thus, while not strictly classified as a hydrophobic or apolar amino acid, cysteine can often be used to confer hydrophobicity to a peptide.

Certain commonly encountered amino acids which are not genetically encoded and which can be present, or substituted for an amino acid, in the peptides and peptide analogues of the present invention include, but are not limited to, B-alanine (b-Ala) and other omega-amino acids such as 3-aminopropionic acid (Dap), 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; 30 .alpha.-aminoisobutyric acid (Aib); .epsilon.-aminohexanoic acid (Aha); .delta.-aminovaleric acid (Ava); methylglycine (MeGly); omithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); 2-naphthylalanine (2-Nal); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)}; 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), .beta.-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (hArg); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid (Dab); 2,3-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe(pNH.sub.2)); N-methyl valine (MeVal); homocysteine (hCys) and homoserine (hSer). These amino acids also fall into the categories defined above.

Further examples of substitutions of amino acid residues that may be incorporated in the polypeptides of the invention comprising or consisting of variants of SEQ ID NOs: 1 to 26 are set out below.

In a suitable embodiment, substitution of an amino acid residue is selected from the group consisting of. substitution of a lysine residue with an arginine residue or a histidine residue; substitution of an arginine residue with a lysine residue or a histidine residue; and substitution of a histidine residue with a lysine residue or an arginine residue. In another suitable embodiment, substitution of an amino acid residue is selected from the group consisting of: substitution of a leucine residue with an isoleucine residue or a norleucine residue; substitution of an isoleucine residue with a leucine residue or a norleucine residue; and substitution of a norleucine residue with a leucine residue or an isoleucine residue. In a suitable embodiment a substitution of an amino acid residue is selected from the group consisting of: substitution of a serine or threonine residue with a cysteine residue; and substitution of a cysteine residue with a serine or threonine residue. A substitution of an amino acid residue may be selected from the group consisting of: substitution of a glutamine residue with an asparagine residue; and substitution of an asparagine residue with a glutamine residue. A substitution of an amino acid residue may be selected from the group consisting of: substitution of a glutamic acid residue with an aspartic acid residue; and substitution of an aspartic acid residue with a glutamic acid residue.

Suitably, substitution of an amino acid residue is selected from the group consisting of: substitution of a glycine residue with an alanine residue or a beta alanine residue; substitution of an alanine residue with a glycine residue or a beta alanine residue; and substitution of a beta alanine residue with a glycine residue or an alanine residue.

In a suitable embodiment, substitution of an amino acid residue is selected from the group consisting of: substitution of a phenylalanine residue with a tyrosine or a tryptophan residue; substitution of a tryptophan residue with a phenylalanine residue or a tyrosine residue; and substitution of a tyrosine residue with a tryptophan residue or a phenylalanine residue.

An appropriate substitution of an amino acid residue may be substitution of a L-amino acid with a D-amino acid, or with other analogous building blocks. It will be appreciated that appropriate substitutions from the group set out above may be selected with reference to the amino acid residues present in the coronavirus-derived portion of any of SEQ ID NOs: 1 to 26. It will also be appreciated that, in the case of variants of SEQ ID NOs: 1 to 26 incorporating two substitutions of amino acid residues present in the coronavirus-derived portion of the reference sequence, each substitution may be independently selected from the various options set out above. The polypeptides of the invention may have many different forms. In a suitable embodiment, a polypeptide of the invention has the form of a linear polypeptide.

The polypeptides of the invention may comprise a single polypeptide chain. Alternatively, a polypeptide of the invention may comprise two polypeptide chains. A suitable embodiment of such a polypeptide of the invention may comprise two polypeptide chains each comprising the amino acid sequence of SEQ ID NO: 5. A suitable embodiment of such a polypeptide of the invention may consist of two polypeptide chains each consisting of the amino acid sequence of SEQ ID NO: 5. In a further embodiment, a polypeptide of the invention may comprise four polypeptide chains. Suitably such a polypeptide of the invention may comprise four polypeptide chains each comprising the amino acid sequence of SEQ ID NO: 6. Suitably such a polypeptide of the invention may consist of four polypeptide chains each consisting of the amino acid sequence of SEQ ID NO: 6. The present invention also encompasses the modification of peptides of the invention to confer stability, to facilitate uptake and absorption, and/or to improve any other characteristic or property of the peptides that is known to one of skill in the art. For example, the active peptides described in this invention can be cyclized, and charges on the peptides can be neutralized, and the peptides can be linked to other chemical moieties.

Accordingly, in a suitable embodiment, a polypeptide of the invention may have the form of a cyclic polypeptide. Peptides of the invention can be cyclized by any suitable method known to those skilled in the art. For example, the N-terminal and C-terminal ends of a peptide (such as the N-terminal and C- terminal ends of a sequence set out in one of SEQ ID NOs: 1 to 26) can be condensed to form a peptide bond by known procedures.

In an alternative approach, functional groups present on the side chains of amino acid residues within the peptide sequence can also be joined to cyclize the peptides of the invention. Pairs of functional groups that can form covalent bonds include --COOH and --OH; --COOH and --NH; and --COOH and --SH. Pairs of amino acids that can be used to cyclize a peptide include Asp and Lys; Glu and Lys; Asp and Arg; Glu and Arg; Asp and Ser; Glu and Ser; Asp and Thr; Glu and Thr; Asp and Cys; and Glu and Cys.

Other examples of amino acid residues that are capable of forming covalent linkages with one another include cysteine-like amino acids such as Cys, hCys, B-methyl-Cys and Pen, which can form disulfide bridges with one another. An example of a pair of cysteine-like amino acid residues that can form a covalent interlinkage is Cys and Pen. Other pairs of amino acids that can be used for cyclization of the peptide will be apparent to those skilled in the art. Methods for preparing cyclic peptides and for otherwise modifying peptides are well-known in the art, such as those described in the chapter "Peptide Backbone Modifications" in the book Chemistry and Biochemistry of Amino Acids Peptides and Proteins.

The polypeptides of the invention comprise both coronavirus-derived and non-coronavirus- derived amino acid sequences. The coronavirus-derived portions of the polypeptides of the invention may be unmodified as compared to the coronavirus from which they are derived. Alternatively, the coronavirus-derived portions and/or the non-coronavirus-derived portions of the amino acid sequences of the polypeptides of the invention may comprise alterations that modify the naturally occurring sequences in a manner that influences the biological, structural, or other properties of the polypeptides of the invention.

The design of sequences used in the polypeptides of the invention provides freedom to generate new epitopes that recapitulate the three-dimensional structure of solvent-exposed natural antigens that generate a strong and specific immune response. Polypeptides of the invention may incorporate new B-cell SARS-CoV-2 antigens designed based on the structure of the spike (S) protein of the virus, which plays a key role in ACE2 receptor recognition and cell membrane fusion processes. To design such polypeptides, open and close conformations of the protein (PDB ID: 6MOJ, 6VXX and 6VYB) may be inspected and solvent-exposed domains identified. In some cases, cysteine residues may be added in strategic positions of the peptide epitopes for disulphide bridge formation. Alterations to add cysteine residues can serve to rigidify the peptide backbone in a more bioactive-like conformation. Such alteration can increase resistance of the polypeptides of the invention to exopeptidase degradation. Examples of such alterations are found in SEQ ID NOs: 1- 15 and SEQ ID NOs: 23- 26.

The S protein of coronavirus must be activated by target cell proteases, which cleave the S protein into S1 and S2 subunits: The S protein sequence comprising two cleavage sites (site 1 and site 2). Polypeptides of the invention may comprise alterations that rigidify cleavage site 2. Such alterations may enable disulphide-bridged cyclisation in order to stabilise the native B-hairpin conformation. Examples of such alteration are found in SEQ ID NO: 7.

The D814G mutation of the spike protein has rapidly become one of the dominant forms worldwide. Polypeptides of the invention may incorporate this mutated epitope. The presence of this sequence alteration serves as a potentially protective epitope against mutated form of the virus comprising this change. SEQ ID NO: 13 comprises this alteration.

The polypeptides of the invention may also be subject to one or more covalent modifications. In a suitable example, at least one of the N-terminus and the C-terminus of a polypeptide of the invention may be independently modified by a fatty acid.

In a suitable embodiment, the fatty acid is a (2-28C) fatty acid. For example, the fatty acid may be a (8-20C) fatty acid.

In a suitable embodiment, the fatty acid is palmitic acid, oleic acid, lauric acid, capric acid, myristic acid, stearic acid, linoleic acid or linolenic acid. Specifically, there is disclosed a polypeptide of the invention modified by addition of palmitic acid at the N-terminus or C-terminus.

The invention discloses a polypeptide (in accordance with any of the embodiments described herein), wherein at least one of the N-terminus and the C-terminus is independently modified by a polymer. The invention also discloses a polypeptide as herein described with a reactive or free N-terminus and/or C-terminus.

Suitably, in the case of a polypeptide of the invention modified by a polymer, the polymer is selected from polyethylene glycol, polypropylene glycol, polytrimethylene glycol, polyoxyethylene, polylactic acid, polyacrylic acid and polyacrylamide. In a particularly suitable embodiment of such a polypeptide of the invention, the polymer is polyethylene glycol. There is also disclosed a polypeptide according to any embodiment described herein, wherein at least one of the N-terminus and the C-terminus is independently modified by a polysaccharide.

In a suitable embodiment of such a polypeptide, the polysaccharide is dextran. In a suitable embodiment of a polypeptide of the invention {according to any of the embodiments described herein), at least one of the N-terminus and the C-terminus is independently modified by a hydrocarbyl group selected from (1-24C)alkyl, (1-24C)alkoxy, (2-24C)alkenyl, (2- 24C)alkynyl, aryl, aryl(1-4C)alkyl, heteroaryl, heteroaryl(1-4C)alkyl, carbocyclyl, carbocyclyl(1- 4C)alkyl, heterocyclyl and heterocyclyl(1-4C)alkyl. In a suitable embodiment, the invention provides a polypeptide according to any of the embodiments herein described, wherein any one or more of the fatty acid, polymer, polysaccharide and hydrocarbyl groups is independently optionally substituted with one or more substituents independently selected from hydroxy, cyano, oxo, halogen, (1-6C)alkyl, (1- 6C)alkoxy, phenyl, benzyl, -NR'R?, -C(O)-R', -C(0)-OR?, -O-C(O)-Rt, -C(O)-NR'R?, -N(R")C(O)- Rt, -S(O}o2R', -S(0)NR'R', and -N(R")-S(O):R', wherein R' and R? are each independently hydrogen or (1-3C)alkyl. Table 1 sets out details of a number of exemplary modifications that may be made in respect of the polypeptides of the invention. In a suitable embodiment, the invention provides a polypeptide comprising the amino acid sequence of one of SEQ ID NOs: 1 to 28, or a variant of such a sequence, and further comprising the exemplary modification set out in respect of the relevant amino acid sequence in Table 1.

A polypeptide of the invention may comprise a reversible modification. Suitably, such a reversible modification may be selected such that the modification increases the bioavailability of the polypeptide of the invention. Alternatively, or additionally, the reversible modification may be selected such that the it increases the ability of the polypeptide of the invention to induce an antibody response and/or induce a T cell immune response in a subject to whom the polypeptide is provided. The ability of reversible modifications to achieve such changes in the properties of polypeptides of the invention may be investigated by comparing polypeptides comprising a putative modification of interest with suitable control polypeptides. Nucleic acid sequences of the invention As set out above, the second aspect of the invention provides a nucleic acid sequence encoding a polypeptide according to the first aspect of the invention. In a suitable embodiment the nucleic acid sequence is a DNA sequence. In a suitable embodiment the nucleic acid sequence is an RNA sequence.

A nucleic acid sequence of the invention may be provided in the form of a vector comprising the nucleic acid sequence. As set out in more detail elsewhere in the specification, the nucleic acid sequences of the invention are suitable for applications in medical uses or methods of treatment of the invention. When used in such applications, the nucleic acids sequences of the invention may be incorporated in pharmaceutical compositions of the invention. Pharmaceutical compositions of the invention In a third aspect, the invention provides a pharmaceutical composition comprising a polypeptide according to the first aspect of the invention, and/or a nucleic acid sequence according to the second aspect of the invention, and a pharmaceutically acceptable carrier. In a suitable embodiment, a pharmaceutical composition of the invention comprises a polypeptide according to the first aspect of the invention.

In a suitable embodiment, a pharmaceutical composition of the invention comprises a mixture of polypeptides according to the first aspect of the invention.

A suitable embodiment, a pharmaceutical composition of the invention comprises a mixture of polypeptides including 2, 3, 4 or 5 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of polypeptides including 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of polypeptides including 21, 22, 23, 24, 25 or 26 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. In suitable embodiment, a pharmaceutical composition of the invention comprises a mixture of polypeptides including up to 2, up to 3, up to 4 or up to 5 of the amino acid sequences set out in SEQ ID NOs: 1to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of polypeptides including up to 6, up to 7, up to 8, up to ©, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19 or up to 20 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of polypeptides including up to 21, up to 22, up to 23, up to 24, or up to 25 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment, a pharmaceutical composition of the invention comprises a mixture of polypeptides including at least 2, at least 3, at least 4 or at least 5 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of polypeptides including at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of polypeptides including at least 21, at least 22, at least 23, at least 24 or at least 25 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of polypeptides that includes polypeptides comprising each of the amino sequences set out in SEQ ID NOs: 1 to 22. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of polypeptides that includes polypeptides comprising each of the amino sequences set out in SEQ ID NOs: 1 to 26. Suitably pharmaceutical composition may comprise a substantially equal mixture of polypeptides of the invention incorporated in the pharmaceutical composition, which is to say that each of the polypeptides of the invention incorporated in the pharmaceutical composition may be present in an amount substantially equal to that of each other polypeptide of the invention.

In addition, or as an alternative, to the embodiments set out above, a pharmaceutical composition of the invention may comprise a nucleic acid sequence according to the second aspect of the invention.

In a suitable embodiment, a pharmaceutical composition of the invention comprises nucleic acid sequences of the invention encoding a mixture of polypeptides according to the first aspect of the invention.

A suitable embodiment, a pharmaceutical composition of the invention comprises nucleic acid sequences of the invention encoding a mixture of polypeptides including 2, 3, 4 or 5 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of nucleic acid sequences of the invention encoding polypeptides including 8, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of nucleic acid sequences of the invention encoding polypeptides including 21, 22, 23, 24, 25 or 26 of the amino acid sequences set out in SEQ ID NOs: 1to 26. In suitable embodiment, a pharmaceutical composition of the invention comprises a mixture of nucleic acid sequences of the invention encoding polypeptides including up to 2, up to 3, up to 4 or up to 5 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of nucleic acid sequences of the invention encoding polypeptides including up to 6, upto 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19 or up to 20 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of nucleic acid sequences of the invention encoding polypeptides including up to 21, up to 22, up to 23, up to 24, up to 25 or up to 26 of the amino acid sequences set out in SEQ ID NOs: 1 to 26.

A suitable embodiment, a pharmaceutical composition of the invention comprises a mixture of nucleic acid sequences of the invention encoding polypeptides including at least 2, at least 3, at least 4 or at least 5 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of nucleic acid sequences of the invention encoding polypeptides including at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of nucleic acid sequences of the invention encoding polypeptides including at least 21, at least 22, at least 23, at least 24 or at least 25 of the amino acid sequences set out in SEQ ID NOs: 1 to 26. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of nucleic acid sequences of the invention encoding polypeptides that includes polypeptides comprising each of the amino sequences set out in SEQ ID NOs: 1 to 22. A suitable embodiment of a pharmaceutical composition of the invention comprises a mixture of nucleic acid sequences of the invention encoding polypeptides that includes polypeptides comprising each of the amino sequences set out in SEQ ID NOs: 1 to 26.

Suitably pharmaceutical composition may comprise nucleic acid sequences of the invention encoding a substantially equal mixture of polypeptides of the invention incorporated in the pharmaceutical composition, which is to say that each of the polypeptides of the invention encoded by the nucleic acid sequences incorporated in the pharmaceutical composition may be present in an amount substantially equal to that of each other polypeptide of the invention. Pharmaceutical compositions of the invention may suitably be compositions suitable for use to generate a protective immune response. Suitably pharmaceutical compositions of the invention may be vaccine formulations. Such vaccines may be used to prevent coronavirus infections, such as infection with SARS-CoV-2 responsible for COVID-19. A pharmaceutical composition of the invention, such as a pharmaceutical composition suitable for use as a vaccine, may comprise an adjuvant in addition to a polypeptide or nucleic acid of the invention. Suitably, an adjuvant may be any agent capable of stimulating an immune response to a polypeptide or nucleic acid of the invention. Illustrative examples of adjuvants suitable for use in pharmaceutical compositions of the invention are set out below.

Merely by way of example, a pharmaceutical composition may comprise an adjuvant selected from the group consisting of: AS03; AddaS03; AS04; MF59; AddaVax; an aluminium salt adjuvant; Poly I:C; R848; Cpg; virus-like particles; virosomes; MPL; and flagellin protein. Adjuvants suitable for use in the pharmaceutical compositions of the invention are considered in more detail below. A suitable adjuvant may be an agonist of Toll-like receptors (TLRs). For example, a suitable adjuvant may be an agonist of a TLR selected from the group consisting of: TLR3; TLR4; TLR7; TLRS; and TLR9.

Polyinosic:polycytidylic acid (also referred to as Poly I:C) is suitable example of an adjuvant of this sort that is an agonist of TLR3. MLP is an example of an adjuvant that is an agonist of TLR4 suitable for use in such an embodiment. R848 (an imidazoquinoline) is an example of an adjuvant that is an agonist of TLR7 or TLR8 suitable for use in such embodiments. CpG oligodeoxynucleotides (also referred to as CpG, or CpG ODNs) constitute an example of an adjuvant that is an agonist of TLR9 suitable for use in such embodiments. Thus a pharmaceutical composition of the invention may comprise an adjuvant that is a TLR agonist selected from the group consisting of: Poly I:C; MLP; R848; and CpG ODNSs.

Suitable adjuvants that may be used in the compositions of the invention include those derived from bacterial sources. Examples of such adjuvants suitable for use in the pharmaceutical composition of the invention include MPL (also referred to as monophosphoryl lipid A) and flagellin protein.

Adjuvant system 03 (AS03) and AddaS03 AS03 adjuvant, is a combination of an emulsion oil in water with squalene, vitamin E (DL-a- tocopherol) and polysorbate. The addition of vitamin E helps improve cell-mediated immunity in the elderly. The combination presented by AS03 induces the activation of cells of the innate immune system. Macrophages are the probable initiators of the response of cytokines such as IL-6 and chemokines, enhancing the recruitment of DCs and increasing the uptake of antigens (such as the polypeptides of the invention). A study in humans showed that AS03 increases the humoral response and the proliferation of CD4 T lymphocytes against different strains of influenza A/H1IN1 and A/H5N1. The use of this adjuvant has decreased the amount of antigen necessary to induce response, in addition to a high level of immunity, generating a cross reaction against heterologous strains of H5N1. To date this adjuvant is used in the vaccine against pandemic influenza H5N1 in the United States, which it has a reserve in case of a pandemic, but it is not for sale to the general public.

AddaSO03 is an oil-in-water nano-emulsion adjuvant available commercially from Invivogen. The formulation of AddaS03 is highly similar to that of the adjuvant system AS03. It is composed of the same percentage of two biodegradable oils, squalene and DL-a-tocopherol, and the surfactant, polysorbate 80 (Tween® 80), for increased stability AddaSO03 represents an alternative adjuvant to ASO3 that can be substituted for AS03 in any situation calling for use of this adjuvant (including in any of the compositions of the invention described herein). Either AS03 or AddaS03 may be employed as an adjuvant in the pharmaceutical compositions of the invention.

As demonstrated in the Examples, AS03 (or the closely related adjuvant AddaS03) constitutes a particularly effective adjuvant for use in the pharmaceutical compositions of the invention. Pharmaceutical compositions comprising AddaS03 are able to elicit highly effective immune responses.

Adjuvant system 04 (AS04) AS04 consists of the combination of two adjuvants, MPL and aluminium salts (both considered individually below). In AS04, the effects on the innate immunity generated by the MPL and the effect of the aluminium salts are added to induce a response with a high long-lasting antibody production. Aluminium salts have the function of stabilizing the presence of MPL and of the antigen (for example a polypeptide of the invention), to subsequently modulate and prolong the cytokine response, generated by MPL at the administration site. The joint action of these adjuvants, improves the response of the vaccine against a given antigen. The adjuvant has been considered to have an adequate safety profile, so it has been approved for use in humans by the FDA in 2005 and by the EMA in 2007.

In AS04's mechanism of action, MPL plays an important role in activating both adaptive and innate immune system; direct stimulation on the TLR-4 receptor, leads to the activation and maturation of APCs that migrate to lymph nodes, likewise induces the production of proinflammatory cytokines such as TNF-q, activation of the transcription factor NF-kB and induction of an adaptive immune response, both of T and B lymphocytes. AS04 has been shown to generate a high level of memory B cells and a high production of antibodies, and is used in the formulation of the vaccine against the Hepatitis B52 virus and in the HPV vaccine. These properties also make AS04 suitable for use as an adjuvant in the pharmaceutical compositions of the invention. MF59 and AddaVax MF59 is a nanoemulsion composed of biodegradable squalene oil, which does not contain additional immunostimulants and is stabilized by two nonionic surfactants: polysorbate 80 and Span 85 (sorbitan trioleate 85), with a low ionic strength citrate buffer phase. Squalene is biodegradable and biocompatible. MF59 has powerful adjuvant activity with an acceptable profile of safety. It is used as a delivery system for immunostimulatory adjuvants such as MLP and QS21. This emulsion stimulates the antibody response against a wide range of antigens. MF59 allows the use of a lower number of doses and stimulates the production of immunological memory of populations with a Th1 and Th2 cell phenotype in murine models.

Among the most studied mechanisms of action of MF59 are: the ability to stimulate recruitment of macrophages and dendritic cells (DCs) at the site of inoculation and local lymphoid nodules. MF59 directly activates muscle fibres at sites where it is administered, and this causes the production of immune mediators, which in turn activate local DCs, thus increasing uptake and efficiency of antigenic presentation. Clinical studies have shown that these effects of MF59 make it possible to significantly reduce the amount of antigen (such as a polypeptide of the invention) necessary to trigger a satisfactory immune response. MF59 was determined to be safe and well tolerated for use in older adults, children, and newborns. The effects of MF59 have been proven in studies of cytomegalovirus, Herpes Simplex Virus (HSV), Human Immunodeficiency Virus (HIV) and seasonal influenza and H1N1. MF59 is present in seasonal and pandemic influenza vaccines, in H5N1 and H5N3 influenza vaccines. Furthermore, it was shown that when used in vaccines against H5N1 virus, in patients previously vaccinated for H5N3 (vaccinated 6 years previously), the antibody response was greater than in patients without prior immunization, showing that the use of influenza vaccines with this adjuvant, generate long-term memory B cells, with the potential to respond against variants of the virus. AddaVax is a squalene-based oil-in-water nano-emulsion commercially available from Invivogen. It has a formulation similar to that of MF59 that has been licensed in Europe for adjuvanted flu vaccines. Squalene is an oil more readily metabolized than the paraffin oil used in Freund's adjuvants. This class of adjuvants is believed to act through recruitment and activation of APC and stimulation of cytokines and chemokines production by macrophages and granulocytes.

Squalene oil-in-water emulsions, such as MF59 and AddaVax elicit both cellular (Th1) and humoral (Th2) immune responses. AddaVax is represents an alternative adjuvant to MF59 that can be substituted for MF59 in any situation calling for use of this adjuvant (including in any of the compositions of the invention described herein). The properties of MF59 and AddaVax make either of these agents suitable for use as an adjuvant in the pharmaceutical compositions of the invention. Aluminium salts Also known as alum salts, suitable aluminium salt adjuvants include those selected from: aluminium hydroxide Al (OH)3, aluminium phosphate AIPO4 and aluminium phosphate sulfate (AIOHPO4S0.). These components have been shown to have ample ability to be combined with a wide variety of antigens. Despite advances in the generation of new adjuvants, phosphate and aluminium hydroxide continue to be the adjuvants most used in humans worldwide, due to their high degree of safety and low cost. The proper selection of aluminium salts enables increased immunogenicity of the antigen. To be effective, the antigen (such as a polypeptide of the invention) should be adsorbed onto charged aluminium particles. The hydrophobic interactions that are generated with the antigen allow the maximum adjuvant effect. Despite a lot of studies and being the adjuvant most used for 100 years, the mechanism of action of the aluminium compounds is still not entirely described. Administration of antigens in aluminium salts has shown an increase in Th2 cells and cytokines IL-4, IL-5 and IL-10 in mice, as well as an increase in humoral response. The present invention shows enhanced humoral immune response by co-administration of aluminium salt adjuvnants with the polypeptides of the invention, indicating the suitability of these adjuvants for use in the pharmaceutical compositions of the invention.

MPL MPL is obtained by hydrolysis and purification of LPS, derived from the bacterium Salmonella Minnesota RC-595. The exact mechanism of action of MPL is still under investigation. The APC recipient who recognizes this adjuvant is TLR-4 (Toll-like receptor 4); when MPL binds to TLR-4, it induces a TRIF-mediated signaling cascade, subsequently activates the transcription factor NF- kB, and induces the expression of proinflammatory cytokines such as TNF-a, IFN-y, IL-12 , IL-6, IL-1B, IFN-a and GM-CSF24 and the production of mediators such as GM-CSF, IP-10, CCL2 and

CCL5. The humoral response produced by this adjuvant is characterized by generating 1gG1 isotypes and IgG4 in humans. In a study using a murine model, MPL has shown that it can induce a high production of Th1-type antibodies such as IgG2a and IgG1. It is suggested that the TLR- 2 activation may also be involved in the activation of APCs by MPL. MPL is currently in clinical trials for use in HPV, HSV, Malaria and Tuberculosis (TB) vaccine. It is part of the adjuvant system AS01, AS02 AS04, and synthetically in the adjuvant RC-529. MPL is suitable for use as an adjuvant in the pharmaceutical compositions of the present invention.

Formulation of pharmaceutical compositions of the invention The pharmaceutical compositions of the invention may comprise an antigen delivery system. Any delivery system that can serve as a transport vehicle for polypeptides of the invention may be used. Examples of suitable antigen delivery systems include liposomes, virosomes, and virus- like particles. Their tissue compatibility and biodegradability make these a valuable system for releasing synthetic antigens.

Suitably, a pharmaceutical composition of the invention may comprise one or more nanoparticles. Nanoparticles are support delivery systems which can be used as antigen delivery systems. They are normally prepared with synthetic polymers. The tissue compatibility of such nanoparticles makes them a valuable system for slowly release of peptides of the invention. A suitable nanoparticle to be employed in a pharmaceutical composition of the invention may be a biodegradable and/or biocompatible polyester nanoparticles. Such a suitable nanoparticle may be of a material selected from the group consisting of: polylactic-co-glycolic (PGL), poly (DL-lactic- coglycolic acid) (PLGA), poly (DL-lactic) (PLA) and poly (ortho-esters) (POE). The use of PGL nanoparticles is advantageous for their ability to release the polypeptides of the invention in a controlled way. Thus, a single administration of a vaccine pharmaceutical composition of the invention comprising PGL nanoparticles to generate effects similar to the vaccines that are administered in various doses, or in multiple incidences of administration.

Suitable particles may differ in electrical charge and hydrophobicity. This allows antigens, such as the polypeptides of the invention, to be adsorbed on biodegradable and/or non-biodegradable particles. In addition, such compositions allow several peptide antigens or nucleic acid sequences to be released simultaneously. This can considerably increase serum antibody levels and the response mediated by cytotoxic T Iymphocytes, thus improving therapeutic efficacy.

Nanoparticles are able to be captured by APCs. This can be advantageous in triggering an immune response mediated by cytotoxic T lymphocytes, in addition to inducing the production of high specific antibody titers. PGL nanoparticles for therapeutic purposes are commercially available.

A suitable pharmaceutical composition of the invention may comprise PLGA. This biodegradable polymer is hydrolyzed into its monomeric components, lactic and glycolic acid, which are natural metabolites of the human body. PLGA is particularly suitable for antigen delivery (such as in pharmaceutical compositions of the invention) because of its slow-release characteristics. Compositions of the invention may employ encapsulation of important components, such as the polypeptides of the invention, in order to activate immune cells. Compositions of the invention comprising PLGA may prevent unwanted body dispersion and degradation of the vaccine components (such as the polypeptides of the invention, or suitable adjuvants) during the administration at the injection site and thereby increase half-life of the vaccine.

A particularly suitable form of compositions of the invention provides polypeptides of the invention within nanoparticles of a biodegradable, biocompatible PLGA polymer. Such a composition may facilitate the delivery of the peptides of the invention to immune cells. This may be achieved by targeting specific dendritic cell receptors via markers such as CD40, DEC-205, CD11c or Fc receptors. Suitably the nanoparticles may be targeted by the addition of antibodies directed to the markers (e.g. by the addition of antibodies directed to a marker selected from the group consisting of: CD40, DEC-205, CD11c, and Fc receptors) to the surface of the nanoparticles. The remarkable effectiveness of such compositions of the invention is demonstrated in the Examples. Formulating the compositions of the invention with nanoparticles, such as PLGA-nanoparticles, also serves to protect the polypeptides of the invention. Suitably the polypeptides of the invention may be co-encapsulated with selected adjuvants (such as R848, Poly I.C, MPL, CpG, etc.). Compositions in accordance with this embodiment of the invention are well suited to activate the immune system via TLR ligands and provide the pro- inflammatory context for antigen recognition. The inventors have also found that compositions of the invention comprising the polypeptides of the invention formulated with thermosensitive Poloxamer 407 hydrogels are very well suited to vaccine use.

Poloxamer 407 (PF-127) is a nonionic surfactant composed of polyoxyethylene-polyoxypropylene copolymers in a concentration ranging from 20 to 30%. The unique thermoreversible and promising drug release characteristics of PF-127 render it an attractive candidate as a pharmaceutical vaccine vehicle for the delivery of polypeptides of the invention through many different routes of administration. Compositions of the invention comprising PF127 may be suitable for mucosal administration. Such composition may include other agents, such as tetanus toxoid (TT) or chitosan, in the presence of a non-ionic block copolymer, which are well suited to the generation of systemic and mucosal immune responses. Accordingly, compositions of the invention comprising both PF-127 and chitosan or tetanus toxoid, in addition to the polypeptides of the invention, represent embodiments particularly suitable to be employed for mucosal vaccine delivery, and appear to exert an additive or synergistic effect on the immune response.

Compositions of the invention comprising PF-127 gels in combination with PLGA nanoparticles are well suited to for the combinatorial and parenteral delivery of polypeptides of the invention having short half-lives. Compositions of the invention comprising PF-127 gel formulations containing polypeptides of the invention (optionally encapsulated in suitable nanoparticles) may be used when it is desired to provide a controlled delivery system for polypeptides of the invention.

In a suitable embodiment of a pharmaceutical composition of the invention, a polypeptide of the invention may be encapsulated or selectively bound to delivery system selected from the group consisting of: biodegradable biopolymers (such as PLGA, PLA, or pluronic F127 hydrogels); liposomes; dextran; and alginate. Such compositions are suitable for the controlled release of the polypeptides of the invention, as well as protecting them from enzymatic degradation and increasing their stability within compositions.

A combination of both adjuvant and delivery system as a vehicle (for example, PLGA, PLA, pluronic PF127 gels, liposomes, dextran, alginate or others) may be used for the controlled release of polypeptides of the invention, while also serving to protect the polypeptides from enzymatic degradation, increase their metabolic stability, and to release the polypeptides of the invention in a controlled manner). Such formulations of the compositions of the invention allow smaller doses of the polypeptides of the invention to be used to achieve a therapeutic effect. Other stabilizers and preservatives can also be incorporated in such compositions of the invention.

Pharmaceutical compositions of the invention can be provided in a form selected from the group consisting of emulsions, hydrogels, hydrocolloids, mix-formulations, liposomes, micro/nanoparticles, creams, sprays, foams, and gels.

In suitable embodiment, the pharmaceutical compositions of the invention can be formulated for provision by any desired route of administration, including (but not limited to) those selected from the group consisting of: intravenous administration; subcutaneous administration; intramuscular administration; intradermal administration; sublingual administration; and intranasal administration.

In a suitable embodiment, a pharmaceutical composition of the invention comprised a nanoparticle formulation, in which polypeptides or nucleic acids of the invention are provided in combination with microparticles or nanoparticles. The polypeptides of the invention may be provided on the surface of the microparticles or nanoparticles or may be encapsulated within the microparticles or nanoparticles (to yield microcapsules or nanocapsules). As discussed elsewhere, such microparticles or nanoparticles may also enclose other components of the composition, such as adjuvants or the like.

In a suitable embodiment, such compositions of the invention comprising microparticles or nanoparticles may further comprise a targeting agent attached to the surface of the microparticles or nanoparticles. The incorporation of such an agent on the surface of the microparticles or nanoparticles allows their targeting (and so targeting of the polypeptides of the invention) to cell types or tissues of interest. Suitable targeting agents are able to bind to cell types or tissues of interest. Many examples of such targeting agents will be known to those skilled in the art. Of particular utility in such embodiments are antibodies that bind to a target molecule of interest. For example, antibodies to CD40 may be incorporated in a pharmaceutical composition of the invention (suitably on the surface of microparticles or nanoparticles within the composition), as targeting agents.

In an embodiment of the present invention, a composition comprises nano/microparticles that have at least one shell comprising a polymeric material. In a suitable embodiment the micro/nanoparticles are bilayered polymeric particles, i.e. the nano/microcapsules comprises an inner nano/microcapsule comprising a core and a first polymeric shell which is coated with a second polymeric shell. Suitably the first and/or second the polymeric shells may comprise one or more biodegradable polymers.

In addition, the targeted nano/microcapsules to DCs of the invention have the advantage that they show to induce potent humoral and CD8 T cell responses. As demonstrated in the Examples, targeted nano/microcapsules against CD40 were more efficiently targeted to DCs in vivo upon injection than were non-targeted control formulations.

Use of targeted nano/microcapsules in the compositions of the present invention allows a decreased quantity of polypeptides of the invention to be employed. As a consequence, the potential for development of side-effects is reduced. Furthermore, targeted nano/microcapsules also have the advantage that they are capable of penetrating inside immune cells, such as in DCs, more efficiently. In a suitable embodiment, the carrier may be provided in the form of nanoparticles or microparticles. Suitably may the carrier may be a polymer. In a suitable embodiment, the polymer may be biodegradable. In an embodiment suitable polymer carriers for use in the pharmaceutical compositions of the invention (which may be incorporated in the form of nano/microcapsules) are selected from the group consisting of poly(D,L-lactide-co-glycolide), polylactic acids, poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)] block copolymer, poly-anhydride poly(fumaric-co-sebacic) anhydride, poly (ethylene oxide)-poly(lactide/glycolide), poly{amino acid), polyvinyl alcohol, alginate, dextran, chitosan, hydroxyapatite, collagen, fibrin, hyaluronic acid, carbomers, poly(amino acid) and poly(ethylene glycol). In a suitable embodiment, the nano/microcapsules of a composition of the invention are bilayered polymeric nano/microcapsules which comprise an inner layer polymer, and an outer layer polymer in the outer surface of the microcapsule. The polymers of the inner layer and the outer shell layer may be independently selected from the group consisting of: poly(D,L-lactide-co-glycolide), polylactic acids, poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)] block copolymer, poly-anhydride poly(fumaric-co-sebacic) anhydride, poly (ethylene oxide)—poly(lactide/glycolide), poly(amino acid), polyvinyl alcohol, alginate, dextran, chitosan, hydroxyapatite, collagen, fibrin, hyaluronic acid, carbomers, poly(amino acid) and poly(ethylene glycol).

In a suitable embodiment the inner layer polymer and the outer layer polymer are different. In a suitable embodiment, the inner layer polymer is poly (D,L lactide-co-glycolide) (PLGA) and the outer layer polymer is polyvinyl alcohol (PVA). In a suitable embodiment, at least some of the functional groups of the inner layer polymer, preferably when the functional groups are carboxyl groups of the inner layer polymer, are present in the outer surface of the nano/microcapsule, together with the polymer in the outer surface of the nano/microcapsule. In a suitable embodiment when the inner layer polymer is poly (D,L lactide-co-glycolide) (PLGA) and the outer layer polymer is polyvinyl alcohol (PVA), the carboxyl groups from the PLGA polymer are present on the outside surface of the microcapsule. Suitably, in such an embodiment, PLGA has a lactide/glycolide molar ratio from 40:60 to 60:40, for example 50:50.

The inventors’ studies set out in the Examples have led them to identify two formulations of compositions of the invention that are of particular interest. In a suitable embodiment, a pharmaceutical composition of the invention may comprise a polypeptide or nucleic acid sequence of the invention in combination with nanoparticles of poly (DL-lactic-coglycolic acid) (PLGA) coated with chitosan. Additionally, or alternatively, a pharmaceutical composition of the invention may comprise a polypeptide or nucleic acid sequence of the invention in combination with Aluminium salts and MPLA. Many of the embodiments, above have been described with reference to compositions of the invention comprising polypeptides of the invention. Each of these embodiments described herein should also be taken as suitable for, and disclosed in respect of, pharmaceutical compositions comprising nucleic acids of the invention (which may be encapsulated in or otherwise associated with nanoparticles within a formulation of the invention). Routes of administration The pharmaceutical compositions, methods of treatment, and medical uses of the invention may provide polypeptides or nucleic acids of the invention to a recipient via any suitable route of administration. Merely by way of illustration, the results set out in the Examples demonstrate that the compositions of the invention are effective when provided by mucosal routes of administration (e.g. by nasal administration) or by systemic routes of administration (e.g. by subcutaneous administration). The formulations considered above provide examples that can be administered via any desired route of administration. Compositions of the invention, or medical uses of the invention, may make use of a route of administration selected from the group consisting of: intravenous (iv) administration; subcutaneous (sc) administration; intramuscular {im} administration; intradermal (id) administration; sublingual (sl) administration; and intranasal administration. The skilled person will be able to determine suitable forms of the compositions of the invention for use with the desired route of administration.

As noted above, a PF-127 based composition comprising peptides of the invention with chitosan and cholera B toxin adjuvants, has proved particularly effective in stimulating a systemic and mucosal immune response. This allows smaller doses of the peptides of the invention to be used in such vaccine formulations. Compositions of this sort, being for use in mucosal delivery, are suitable for nasal administration.

Nasal administration of the polypeptides of the invention (in which the polypeptides are directed to the nasal associated lymphoid tissue — NALT) is advantageous, since it allows access to specialized mechanisms by which the polypeptides of the invention may cross epithelial barriers, including antigen uptake by M cells. M cells facilitate the interaction of polypeptides with cells of the immune system, like Langerhans DCs and macrophages, in a compartment protected from the modulatory effect of systemic immunity.

Another potentially advantageous route of mucosal immunization is through interaction of polypeptides of the invention with the dense populations of DCs in the tonsils.

Therapeutically effective amounts of polypeptides or nucleic acids of the invention The pharmaceutical compositions of the invention will comprise the polypeptides or nucleic acids of the invention in an amount sufficient to provide a therapeutically effective amount of the active agent. This therapeutically effective amount may be provided by one or more incidences of administration of the pharmaceutical composition.

A pharmaceutical composition of the invention may comprise a polypeptide or nucleic acid sequence of the invention in an amount of between about 0.1 and 5000 ppm, as characterised as proportion of the total weight of the polypeptide (or polypeptides) or nucleic acid sequence (or sequences) of the invention as compared to the total weight of the composition of the invention. As set out below, a composition of the invention may suitably comprise the polypeptide(s) or nucleic acid sequence(s) of the invention in an amount of between 1 ug and 1000 ug.

The pharmaceutical compositions of the present invention may be formulated to provide a therapeutically effective amount of a polypeptide or nucleic acid sequence of the invention in a volume of about 0.01 mL to 10 mL. A preferred volume within this range may be selected based upon factors including the inoculation route; the species to be immunized; and the concentration of polypeptides or nucleic acid sequences of the invention within the composition.

In a suitable example, a pharmaceutical composition of the invention may be formulated to provide a dose of between 1g and 1000ug of the polypeptides or nucleic acid sequences of the invention in each incidence of administration. Suitably, a pharmaceutical composition of the invention may be formulated to provide a dose of between 50kg and 750ug of the polypeptides or nucleic acid sequences of the invention in each incidence of administration. For example, a pharmaceutical composition of the invention may be formulated to provide a dose of between 100ug and 500g of the polypeptides or nucleic acid sequences of the invention in each incidence of administration.

In an embodiment, a pharmaceutical composition of the invention may be formulated to provide a dose of approximately 260g of the polypeptides or nucleic acid sequences of the invention in each incidence of administration.

It will be appreciated that, in the case of pharmaceutical compositions of the invention comprising a mixture of polypeptides or nucleic acid sequences of the invention, the doses considered above are suitable as examples of the total amount of the polypeptides or nucleic acid sequences of the invention to be provided in an incident of administration (that is to say that the total amount of the different polypeptides or nucleic acid sequences making up the mixture will correspond to the selected dose).

The skilled person will be aware of means by which the appropriate dose of a polypeptide of the invention or nucleic acid sequence of the invention may be modified from the suggestions set out above, in order to take into account variables such as the formulation of the composition, the size, weight or age of the subject requiring treatment, and the extent of disease to be addressed. Methods of treatment and medical uses of the invention A fourth aspect of the invention provides a method of preventing or treating a coronavirus infection, the method comprising providing to a subject in need of such prevention or treatment a therapeutically effective amount of a polypeptide in accordance with the first aspect of the invention.

It will be appreciated that the polypeptides of the first aspect of the invention, the nucleic acid sequences of the second aspect of the invention, and the pharmaceutical compositions of the third aspect of the invention are all suitable for use as medicaments. Thus, the invention also provides a polypeptide of the invention for use as a medicament, a nucleic acid of the invention for use as a medicament, and a pharmaceutical composition of the invention for use as a medicament.

The methods of treatment and medical uses described herein may be employed in the prevention or treatment of infections caused by a coronavirus selected from the group consisting of: SARS- CoV-2; SARS-CoV; and MERS-CoV. In particular, the polypeptides of the invention have proven to be of benefit in promoting an immune response which is protective in respect of SARS-CoV-2, the coronavirus responsible for COVID-19. As will be recognised, the continuing COVID-19 pandemic means that there remains a well-recognised clinical need for medicaments that can be used to prevent or treat this disease. However, the methods of treatment and/or medical uses of the invention are more broadly applicable than therapeutic use only in respect of SARS-CoV-2. They may be used in the prevention or treatment of infections caused by a coronavirus selected from the group consisting of: SARS-CoV-2; SARS-CoV; and MERS-CoV. As set out in Table 1, each of the amino acid sequences of SEQ ID NOs: 1 to 26 comprises a coronavirus-derived portion. These portions are derived from the proteins of SARS-CoV-2, however, the sequences used have been selected for their potential to induce immune responses in respect of more than one of SARS-CoV-2, SARS- CoV, and MERS-CoV. Thus, polypeptides of the invention comprising or consisting of these sequences (or comprising or consisting of variants of SEQ ID NOs: 1 to 26) have the capacity to be used therapeutically in a range of coronavirus infections. Suitably the therapeutically effective amount of the polypeptide in accordance with the first aspect of the invention used in a method of treatment of the invention may be provided by administration to the subject of a pharmaceutical composition according to a third aspect of the invention, or of a nucleic acid sequence according to the second aspect of the invention. Administration of a nucleic acid of the invention to a subject requiring treatment enables the subjects own cells to express polypeptides of the invention, encoded by the nucleic acid sequence of the invention, in a therapeutically effective amount. The methods of treatment or medical uses of the fourth aspect of the invention may employ a pharmaceutical composition of the invention in accordance with any of the embodiments of such compositions described herein.

The methods of treatment or medical uses of the fourth aspect of the invention may employ a single polypeptide of the invention. Alternatively the methods of treatment or medical uses of the fourth aspect of the invention may employ 2 or more independently selected polypeptides of the invention, 3 or more independently selected polypeptides of the invention, 4 or more independently selected polypeptides of the invention, 5 or more independently selected polypeptides of the invention, 6 or more independently selected polypeptides of the invention, 7 or more independently selected polypeptides of the invention, 8 or more independently selected polypeptides of the invention, 9 or more independently selected polypeptides of the invention, 10 or more independently selected polypeptides of the invention, 11 or more independently selected polypeptides of the invention, 12 or more independently selected polypeptides of the invention, 13 or more independently selected polypeptides of the invention, 14 or more independently selected polypeptides of the invention, 15 or more independently selected polypeptides of the invention, 16 or more independently selected polypeptides of the invention, 17 or more independently selected polypeptides of the invention, 18 or more independently selected polypeptides of the invention, 19 or more independently selected polypeptides of the invention, or more independently selected polypeptides of the invention, 21 or more independently selected polypeptides of the invention, 22 or more independently selected polypeptides of the invention, 23 or more independently selected polypeptides of the invention, 24 or more independently selected polypeptides of the invention, 25 or more independently selected 20 polypeptides of the invention, or even 26 or more independently selected polypeptides of the invention each comprising a different amino acid sequence comprising or consisting of one of SEQ ID NOs: 1 to 26, or a variant thereof. Cells of the invention In a fifth aspect, the invention provides a prokaryotic or eukaryotic cell expressing a polypeptide according to the first aspect of the invention. Suitably, a prokaryotic or eukaryotic cell according to the fifth aspect of the invention may comprise a nucleic acid sequence according to the second aspect of the invention.

It will be appreciated that the polypeptides and nucleic acids of the invention are artificial. As a result, cells in accordance with this fifth aspect of the invention may readily be distinguished from those found in nature, or the prior art.

Suitable examples of the cells of the invention include plant cells, animal cells, yeast cells, and bacterial cells.

Methods of producing polypeptides of the invention The sixth aspect of the invention provides methods of producing polypeptides of the invention.

In such method, cells in accordance with the fifth aspect of the invention are grown in conditions such that the cell produces a polypeptide of the first aspect of the invention.

For the purposes of the present invention, references to “growing” cells should be taken as encompassing techniques in which cells are maintained, or in which cells are divide or proliferate.

A method in accordance with the sixth aspect of the invention may further comprise isolating the polypeptide of the invention produced by the cell of the invention.

Suitably, the method of the sixth aspect of the invention may comprise fermentation to yield the polypeptide of the invention.

In such an embodiment, the cell of the invention may be selected from the group consisting of: a plant cell; a bacterial cell; and a yeast cell.

A method in accordance with the sixth aspect of the invention may comprise a further step of post-translationally modifying the polypeptide produced.

Any of the modifications considered elsewhere in this disclosure may constitute suitable examples of modifications that may be used in such embodiments.

As set out in the Examples, the polypeptides of the invention are also capable of synthesis by methods well known to those skilled in the art.

EXAMPLES 1 EXAMPLE 1

SYNTHESIS OF THE PEPTIDES OF THE INVENTION Peptides of the invention comprising each of SEQ ID NOs: 1 to 26, set out in Table 1, were synthesized using the solid-phase method and Fmoc/Bu strategy. Linear peptide synthesis Linear peptides of the invention (comprising the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4,7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 to 26 set out in Table 1) were synthesised on Fmoc-Rink-MBHA (1.08 mmol, 0.72 mmol/g) or Aminomethyl CM (ChemMatrix) Resin (0.1 mmol, 0.4 mmol/g). In the Aminomethyl CM Resin. The linker was coupled manually using Fmoc-Rink linker (3 equiv), PyBOP (3 equiv), HOAt (3 equiv), and DIEA (9 equiv) in DMF. Before Fmoc removal, an acetylation step [Ac20-DIEA-DMF (10:5:85)] was performed to block any possible free amino groups. In all cases, the Fmoc group was removed using 2 treatments of 10 min each with 5mL/g resin of 20% Piperidine in DMF). After that, the all aminoacids were coupled using Fmoc/Bu standard coupling conditions (i.e. Fmoc-Aa/DIPCDI/HOBt 3:3:3 in DMF during 1.5h). Elongation of the peptide was achieved by repeating coupling/deprotection steps. After introduction of the last aminoacid, the peptide was acylated either using acetic acid, palmitic acid or other aliphatic chain acid in the presence of DIPCDI and HOB (3:3:3 in DMF/DCM during 2h). In all cases, coupling reaction was monitored either by ninhidrin (primary amines) or cloranil assays {secondary amines).

All polyvalent lipo-multiepitope peptidil-resin cleavage were performed using 95% TFA, 2.5% TIS and 2.5% water in all cases (10mL/g resin). Lipo-multiepitope peptides were precipitated with cold MTBE after TFA removal under a Nz stream. The polyvalent lipo-multiepitope were dissolved in water and lyophilized to obtain the final product. After that each lipo-multiepitope peptide were further purified using HPLC-preparative. Branched peptide synthesis. The 2 and 3 branched version of the polyvalent multiepitope peptides (i.e. peptides of the invention comprising SEQ ID NOs: 5 or 6) were assembled by stepwise Fmoc solid-phase methods, using the protection scheme described for Fmoc strategy. The Lys residues responsible for branching (one in the divalent, the another one in the tetravalent versions of the branched, respectively) were incorporated as Fmoc-Lys(Fmoc)OH and coupled with TBTU/DIEA activation. Double couplings with 10-fold excess of Fmoc-amino acids and DIEA/HOBt were used for most residues of the distinct epitope. When required by the Kaiser test, capping steps were performed with 4% acetic anhydride and 1% DIEA in DMF. All branched polyvalent multiepitope peptidil- resin cleavage were performed using 95% TFA, 2.5% TIS and 2.5% water in all cases (10mL/g resin). Branched polyvalent multiepitope peptides were precipitated with cold MTBE after TFA removal under a Nz stream. The branched polyvalent multiepitope peptides were dissolved in water and lyophilized to obtain the final product. After that each branched polyvalent multiepitope peptides were further purified using HPLC-preparative. 2 EXAMPLE 2

CHARACTERISATION OF THE PEPTIDES OF THE INVENTION All peptides of the invention manufactured above were analyzed by RP-HPLC and HPLC-MS or MALDI-TOF. Table 2 shows the characterization of each polypeptide obtained in this patent using the methods described in Example 1. The analysis by RP-HPLC allowed determination of the purity and retention time. An HPLC-MS or a MALDI-TOF analysis was used for unambiguous characterization.

Characterization is showed in Table 2. EM corresponds to HPLC-MS or MALDI-TOF and the exact mass was calculated using Chemdraw Ultra. 2 EXAMPLE 3

PHARMACEUTICAL COMPOSITIONS OF THE INVENTION INDUCE AN IMMUNE

RESPONSE Immunogenicity of an experimental pharmaceutical composition of the invention (comprising polypeptides of the invention) was investigated in a mouse model.

13 groups of 7 female BALB/c mice were inoculated subcutaneously with a dose of an experimental composition comprising a mixture of polypeptides of the invention with amino acid sequences consisting of each of SEQ. ID. No. 1, SEQ. ID. No. 2, SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID. No. 5, SEQ. ID. No. 8, SEQ. ID. No. 7, SEQ. ID. No. 8, SEQ. ID. No. 9, SEQ. ID. No.

10, SEQ. ID. No. 11, SEQ. ID. No. 12, SEQ. ID. No. 13, SEQ. ID. No. 14, SEQ. ID. No. 15, SEQ. ID. No. 16, SEQ. ID. No. 17, SEQ. ID. No. 18, SEQ. ID. No. 19, SEQ. ID. No. 20, SEQ. ID. No.

21, SEQ. ID. No. 22. Each of these polypeptides of the invention was provided in an amount of 260 pg. Each of the 13 groups was inoculated with distinct adjuvants described in Table 3. The group 13 was inoculated with the polyvalent lipo-multiepitope in phosphate-saline (PBS) buffer. Group 14 was used as the untreated control group (naive). The inoculation schedule was as follows: day 0, 14, 28, 42 and the extraction was done on day 56. The antibody response was quantified by immunoenzymatic assay (ELISA) to determine the IgG antibodies against the spike SARS-CoV- 2 protein in sera.

Unpaired Student's t test with Welch's correction was performed to analyze statistically the results, p<0.05 was considered a significant difference. The results of this study demonstrated that the pharmaceutical compositions of the invention, comprising polypeptides of the invention, are able to stimulate an immunogenic response leading to the generation of antibodies reactive to SARS-CoV-2 in subjects to whom they are administered. Furthermore, the results demonstrated that by modifying the specific adjuvants provided in the pharmaceutical compositions of the invention it is possible to markedly increase the spike SARS- CoV-2 antibody immune response. Turning to the results set out in Figure 1, it can be seen that when the polypeptides are provided without an adjuvant (i.e. with the polypeptides in PBS alone), only a low immune response is generated. The responses after subcutaneous inoculation, using either aluminium or AddaVax {an MF59 equivalent} as adjuvant were increased and approximately similar to one another. The immune response was found to be increased in the groups treated with PF127 Nanohydrogel and combined adjuvants (Aluminium+MPLA) compared to single adjuvant respectively. The results set out in Figure 2 illustrate that the corresponding CD4 and CD8 T cell responses followed a similar pattern in terms of their magnitude. The highest induction of SARS-CoV-2 specific IgG antibodies and of SARS-CoV-2 specific T cells were observed in the mice vaccinated with the adjuvant AddaS03 (AS-03 like).

In conclusion, the polypeptides of the invention, when administered via the subcutaneous route, are able to achieve high immunogenicity when formulated using distinct clinical approved adjuvants (as experimental pharmaceutical compositions of the invention). We observed an improvement in the immune response in the experimental group in which the agents of the invention were provided in the nanohydrogel combination with MPL + aluminum as compared to single adjuvants. 4 EXAMPLE 4

PHARMACEUTICAL COMPOSITIONS OF THE INVENTION ARE EFFECTIVE VIA INTRANASAL ADMINISTRATION, AND IMPACT OF FORMULATIONS ON ACTIVITY This study investigated the ability to enhancing the effects of the polypeptides of the invention (when administered intranasally) by using a nanohydrogel formulation in combination with recombinant cholera Toxin B subunit and/or chitosan.

For this experiment 6 groups of 8 female C57BL/6J mice were selected. The inoculation schedule was as follows: day 0, 14 and 28 and extraction on day 42. The assayed groups are summarized in Table 4. All groups except 4 were inoculated intranasally. Group 4 was inoculated subcutaneously for comparison.

The Mann Whitney test was used to analyze statistically the results against PBS control, p<0.05 was considered a significant difference. The results of this study are set out in Figure 3A. They indicate that it is possible to induce systemic immune responses to the polypeptides of the invention after intranasal administration. The serum IgG response for the two immunized groups (1-2) provided with compositions of the invention comprising adjuvants was high and it was significantly higher to that obtained by inoculation of polyvalent lipo-multiepitope in PF127 nanoHydrogel and similar to that attained by the group inoculated in sc. Titers obtained by intranasal administration inoculation in PF127 NanoHydrogel + 1% Chitosan alum did not differ significantly from that of PF127 NanoHydrogel plus cholera Toxin B subunit recombinant group by the nasal route. In Figure 3B, a pool of the generated anti-Covid-19 IgG antibodies from Group 1 were validated on a western blot to test the recognition of the recombinant spike protein. Using a commercially availabe anti-spike antibody (1:500, first lane), with negative control serum 1:250 (lane 2), with positive control serum 1:250 (lane 3), and with neg. and pos. serum 1:2000. The tested 1:2000 dilution gave a high signal indicating high recognition of the spike protein, even at a low dilution. EXAMPLE 5 5 IMPACT OF FORMULATION AND DELIVERY SYSTEMS ON EFFECTIVENESS OF

COMPOSITIONS OF THE INVENTION Different target delivery systems and immune adjuvants were employed with the aim of studying the controlled release effect and impact of different delivery systems on the compositions of the invention.

8 experimental formulations of compositions of the invention were prepared using different targeted delivery systems and adjuvants, in order to investigate the impact of controlled release and delivery systems on agents of the invention. As with the preceding studies, each composition comprised an equal mixture of polypeptides of the invention comprising the amino acids set out in SEQ ID NOs: 1 to 22.

Each experimental formulation (and one control group of PBS alone) was provided by subcutaneous injection to a separate group of 8 female BALB/c mice. Mice were were immunized on days 0, 14 and extraction took place 10 days after second inoculation.

The formulation provided to Group 1 comprised the mixture of polypeptides of the invention in a PF127 nanohydrogel, with MPL and aluminium salt as adjuvants.

The formulations provided to Groups 2 to 4 each comprise a backbone of polymer poly(lactic-co- glycolic acid) nanoparticles. PLGA is a carrier that is biodegradable, biocompatible, and FDA approved. These PLGA-based formulations of compositions of the invention facilitate targeting of the nanovaccine to specific DC receptors via specific surface receptors as DEC-205. The PLGA-nanoparticles also confer protection and controlled release of encapsulated compounds.

The interior of the nanoparticles co-encapsulated the mixture of polypeptides of the invention either alone (in Group 2) or in tandem with adjuvants R848 and Poly I:C (in both Groups 3 and 4). These adjuvants are able to activate the immune system via TLR ligands and to provide the pro-inflammatory context for antigen recognition.

The compositions provided to Groups 3 and 4 differed from one another in that the nanoparticles in the formulation of Group 3 were targeted via CD40 antibodies, while the nanoparticles in Group 4 used an isotype control antibody.

Groups 5 and 6 respectively received compositions of the invention in which the polypeptides were provided in liposomes and in chitosan nanoparticles. The compositions provided to Group 7 comprised the polypeptides of the invention with aluminium salts, while the compositions provided to Group 8 comprised the polypeptides in a PBS control carrier.

The results of this study are set out in Figure 4. Here it can be seen that Groups 1, 2, 5 and 6 all exhibit approximately the same high level of the antibody titers against SARS-CoV. In contrast, Groups 7 and 8 (formulations without carriers, and in the case of Group 8 without adjuvants) yielded much lower antibody titers.

Formulations of compositions of the invention comprising PLGA nanoparticles and the adjuvants R848 and Poly I:C achieved the highest titers of antibodies against SARS-CoV. Of these, the greatest response was produced by Group 3, in which the PLGA nanoparticles were targeted with CD40 antibodies.

Taken as a whole, the results demonstrated that all compositions of the invention are able to achieve a potentially therapeutic immune response, but that incorporation of an appropriate delivery system is able to improve the immune responses obtained. Compositions of the invention in which the polypeptides of the invention are encapsulated in nanoparticles (such as PLGA nanoparticles) with adjuvants (such as R848 and Poly |:C) are particularly effective, and this efficacy can be increased by appropriate targeting of the nanoparticles (for example, by coating with antibodies such as anti-CD40 antibodies).

The results also showed that the compositions of the invention incorporating the polypeptides of the invention in PF127 NanoHydrogel, with MPL and aluminium hydroxide adjuvants, enhanced antibody responses against SARS-CoV, (as demonstrated for the results of Group 1 represented in Figure 4). Statistical analysis using Student's t test (p<0.05 was considered a significant difference) did not show any difference between Groups 1 to 6.

Table 1 / / . / Design and linear Exemplary modification - | Protein region . structure {1) or Sequence (coronavirus- and position of Type B/ T-cell : ‚ / branched structure Reference derived portion coronavirus- {MHC tand 1) . | . {2 = 2 branches) or underlined) derived portion (3 = 4 branches) SEQ Palm- ID QYIKANSKFIGITELKKIT | Spike RBD TT{830-844)-B( NO: 1 | LOFTLERGVOYQFYRY | 481 RBD,481) and 1 TheiperandB | Unpublished data SEQ Paim- Spike TT(830-844)-8 T hel J ID QYIKANSKFIGITELKKY | RBD (830-844) 8( elperandB | published data NO: 2 | EYRSMSSTELKPEERD | 411 RBD,411) and1 | epitope SEQ Palm- Spike iD VHHNTEEIVAQSIALSSL RBD DDT(331-350)-8( T helper and B Unpublished dat td Sg tert Fre . npublis a NO:3 | MVKKEISDYNYKLPDDE | © RBD,412) and1 | epitope punished ce SEQ Paim- Spike ID AKFVAAWTLKAAAKKG! RED Padre-B(RBD,351) T helper and 8 Unpublished dat NO: 4 | YQISNERVEEVREENK | and 1 epitope PIPER SEQ | Paim3CSK4K-- Spike dre (RED | ID | AKEVAAWTLKAAAKKES | RBD Padre-B(RBD,351} | ThelperandB | published data NO: 5 | IEKGYGY 351 an 2 epitope SEQ | Palm3CSK4K,- Spike 4 el ID AKFVAAWTLKAAAKKES | RBD Padre-B[RBD,351) T © per and B Unpublished data NO:6 | TEKGYGV 351 and 3 epitope SEQ Palm- . Spike DDT{331-350)-B T helper and ID VHHNTEEIVAQSIALSSL | oF ja p NO 7 | MVPRRARSVASGAACE Cleavage site 1 682-690}-B{804- twoB Unpublished data | ee | and2 818) and 1 epitopes SEQ | Paim- Spike TT{830-844)-B( - T helper and B . ID QYIKANSKFIGITELKKS | RBD RBD,477-490) and . Unpublished data NO: 8 | TPONGVEGENCYE (477-450) 1 epitope SEQ Palm- T epitope { MHCH, | T cell{ MHCH, 692)- Tand & an ID HAYTMSLGAENSVAYK | 692) from B Spike | B{ RBD,494-509) ) Unpublished data NO: 9 | KSYGUPTNOVEYRYR | RBD (494-509) | and 1 epitope SEQ Palm- spike ID VHHNTEEIVAQSIALSSL | <1 DDT(331-350)- ThelperandB | Lo NO: | MVKKSGECRDATID B(563-578)and1 | epitope nptblished cota me (563-578)

LVER SEQ Palm- Spike ID AKFVAAWTLKAAAKKG en Padre-B(RBD,336- | T helper and 8 | NO: | EEGEVENATREASVYA | 100 361) and 1 epitope | nPUIEed aie SEQ Paim- ID NNFTVSFWLRVPKVSA | Spike S2 DDT(271-290)- Thelperand® | oypliched dat NO: | SHLETKKMSLGAENSY | (697-708 B(697-708)and 1 | epitope published data SEQ | Paim- Spike TT{580-599)-B(604- Doi ID NSVDDALINSTKIYSYFP | (604-626) 626) T helper and B 10.1 TL /2000.04.29.0 NO: SVKKTSNEVAVLYRBY | prevalent D614G | D614G mutation epitope 69054 RE 13 NGIEVSMAN mutation and 1

SEQ Palm- Spike RBD Padre-B(481)- iD AKFVAAWTLKAAAFTLK | (481) T helper and NO: ROG YORYRVALSER B{898-907)-8 / . IEMA INA IRN YVR | (898-907) (815-826) and 1 three B Unpublished data 14 LAHARAAFSMQUAYREN (815-826) epitopes AARSFIEDLLFNKYK Homology MERS SEQ Palm- Spike protein T evi MICH ID NLLLQYGSFSTQLNRKL | epitope ( MHCH, epitope | : NO: | ITGRLOSLKKNLNESLID | 738) T epitope | [oor epitope | Three T cell I DEN STE MHC, 972)-T- epitopes and . LHKEAVYDOALDELBETR | MHC, 972)-T | ) prop Unpublished data OTLESE / epitope MHC}, one B cell LRRD epitope MHC, . . / 1143)-B epitope epitope 1143)-B epitope {287-306} and 1 (287-306) SEQ | Palm- . - Spike protein ID QYIKANSKFIGITELKKA MERS 8 eitee TT(830-844)- B{737- | T helper and NO: | LEDIPSTLIPRSVRSYP POP | 761)B{541:555) | twoB Unpublished data MVR AEST ADA A ELON en {737-761)-B | 18 GEMRLASAACREQLER | sapsss) | 3991 epitopes LESGGMLVE epitope (341-553) SEQ Paim- Spike protein T epit (MHC ID YLOQPRTFLLKKFIAGLIAI | T epitope { MHC, 68) eee . CIENET ONAN WE SR -T epitope NO: | VKKEGECNDDELKKVVL | 268)-T epitope pitop Four T cell 17 SEELLNAPATVCG MHC}, 1219)-T- SSS] MHC}, 1219)-T- . epitopes Unpublished data . epitope MHC, epitope MHCI, . {MHC! and 11) . 132)-T epitope 132)-T epitope MHCH, 512) and 1 MHCH, 512) SEQ | Palm- Spike protein T anit NIH iD SUAYTMSLKKIADYNYE | T epitope ( MHC], coon one! ' 3 SONI oD REIT . “18 to e NO: | LKKVLNDILSRLKKINITE | 690)-T epitope JT epitap Four T cell 18 FOTLLALMREY LTS MHC, 416)-T- | | MIST ISD AE MHC, 416)-T- . epitopes Unpublished data . epitope MHCI, epitope MHC, / {MHCl and 1) / 975)-T epitope 975)-T epitope MHCH, 233) and 1 MHC, 233) SEQ Paim- Spike protein T enit MHC! ID | LLENKWILAKRLGSLOT | Tepitope (MHC, on one! . of Oi ie . -T epitope NO: FVKKVELEVIYVKKQE | 820)-T epitope )-T epitop Four T cell 19 SNOUTVLRPELLTD MHC, 999)-T- . . DSLR Ei ik MHC, 999)-T- . epitopes Unpublished data ‚ epitope MHCI, epitope MHC, . {MHC and 1) . 1059)-T epitope 1059)-T epitope MHC, 853) and 1 MHC, 853) SEQ Paim- N protein T epit MHC ID | LLLDRINGUKKGMSRIS | Teptope (me, | | cPiope MAC, NO: | MEVKKRWYEYYLSTSP | 221)Tepitope | 2) cPiope | Three Tcell warns MHC}, 315)-T epitopes Unpublished data Lh MHC], 315)- T ‚ . epitope MHCIL, (MHC! and Hf) epitope MHCH, 107) and 1 107) SEQ | Palm- E protein T enit MHC] ID | ELAEVVELLKKSEVKESE | Tepitope (MHC, | se "| tree cel . AAC ATE AN TAL TA . -T epitope ree 1 ce NO: YNKKLMTLAI TALBLCA | 19)-T epitope )-T epitop / / 21 i MHCL, 49)-T epitopes Unpublished data X MHCI, 49)-T / . epitope.

MHCII, 28) | {MHCl and 1) epitope MHC, and 1 28)

SEQ Palm- M protein Tenis (MHC! ID | GLMMMLSYEIKKLLEGW | T epitope ( MHC, ee . iY ENG A ANSI Ti . -1 epitope NO: NLVKKEVEAAVYRIKRTL | g8)-T epitope )-T epitop Four T cell 22 SYYHLOASORVASDS MHC, 14)-T- / . REESE MHC, 14)-T- . epitopes Unpublished data epitope. MHC, 64)- epitope MHC, . (MHC! and tl) . T epitope MHCIL, 64)-T epitope 175) and 1 MHCI, 175) NO: Palm- . . Spike protein B (RBD, 477-490)- B epit Ee | BREE | 20 een | unpublished data QYIKANSKFIGITEL (RED, 477-490) | TT{830-844) and 1 | and T helper NO: Paim- . . / / . Spike protein B (RBD,494-509)- B epitopes 2¢ | SYGCOPTYGUGYGOYR | (oH \ | eens | unpublished data KQYIKANSKFIGITEL | (RED: TT(830-844) and 1 | and Thelper NO: Palm . . am ee Spike protein B (RBD 442-451)- B epitopes 25 | CAKVOONYNCKKQYIK ke 0 451) | ) oop Unpublished data ANSKFIGITEL (RBD, 442- TT{830-844) and1 | an elper NO: Palm- 26 OLSVYAYHENNKSVMES | Spike protein ( B{ NTD,140-158)- B epitopes ; rr LEI DAN ZED A RIGS Ef re Un pu blished data EFCKKQYIKANSKFIGIT | NTD,140-158) TT{830-844) and 1 | and T helper

EL

Table 2 Exemplary Purity Foun Co Exact SEQ modification and (RP- d mass ID NO | Sequence HPLC) (g/mol (m+H 0, mo (%) £ /2) 1 Palm- 92.00 QYIKANSKFIGITELKKIT 80 5521.7 5522. LCFTLKRGVGYQPYRVV | 9 43

VLSFELLHAP 2 Palm- 6.7 | 94.63 4431.3 | 4432. QYIKANSKFIGITELKKY 1 29

FYRSHRSTKLKPFERD 3 Palm- 8.5 | 89.17 4490.3 | 4491. VHHNTEEIVAQSIALSSL 2 21

MVKKCIADYNYKLPDDF

TGC 4 Palm- 8.8 [92.70 4144.0 | 4145. AKFVAAWTLKAAAKKGI 4 07

YQTSNFRVPPVRFPNITN Palm 3CSK4KAAKo- 10. | 90.96 7234.2 AKFVAAWTLKAAAKKFS | 4 6 TFKCYGV-Acetyl Palm3CSK4KAAK,. 12. | 92.56 12976. AKFVAAWTLKAAAKKFS | 9 24

TFKCYGV 7 Palm- 10. [92.36 5327.3 | 5328. VHHNTEEIVAQSIALSSL | 1 5 45

MVPRRARSVASQAACIL PDPSKPSKRSFC

Palm- 96.43 3736.4 | 3738. QYIKANSKFIGITELKKS 9 49

TPCNGVEGFNCYF Palm- 8.1 [95.30 3529.9 | 3530. ITAYTMSLGAENSVAYKK 8 67

SYGQPTNGVGYQYR Palm- 92.09 4512.4 | 4514. VHHNTEEIVAQSIALSSL 4 30

MVKKCQFCRDIADTTD

CVRD 11 Palm- 94.90 AKFVAAWTLKAAAKKCP 78 4858.8 | 4850. FGEVFNATRFASVYAW 8 76

NRKRISNC 12 Palm- 92.90 NNFTVSFWLRVPKVSAS 81 4136.8 | 4137. HLETKKMSLGAENSVA | 9 09

YS 13 Palm- 91.98 NSVDDALINSTKIYSYFP 78 5052.8 | 5054. SVKKTSNEVAVLYQGVN 8 01

CTEVPVAIH 14 Palm- 95.68 AKFVAAWTLKAAAFTLK 25070 | 7508. RGVGYQPYRVVVLSFEL | 6.8 LHAPAAFcMQCAYRFNA 0 07

ARSFIEDLLFNKVK Palm- 96.60 NLLLQYGSFSTQLNRKLI 67650 | 6766. TGRLQSLKKNLNESLID |7.5 LKKDAVDCALDPLSETK © 27

CTLKSF

16 Palm- 95.80 QYIKANSKFIGITELKKA 6710.0 6711. LPDTPSTLTPRSVRSVP 7.8 GEMRLASAACRKQLSP 6 87

LEGGGWLVC 17 Palm- 95.09 YLQPRTFLLKKFIAGLIAI 83 5803.2 5803. VKKFQFCNDPFLKKVVL 3 23

SFELLHAPATVCG 18 Palm- 91.90 SIAYTMSLKKIADYNYK 9.7 6249.6 6260. LKKVLNDILSRLKKINIT 9 08

RFQTLLALHRSYLTP 19 Palm- 90.67 LLENKVTLAKRLQOSLOT 5682.0 5682. YVKKVVFLHVTYVKKQK 9:5 7 98

FNGLTVLPPLLTD Palm- 92.68 LLLDRLNQLKKGMSRIG 4583.6 4584. MEVKKRWYFYYLGTGP 4 78

EAGL 21 Palm- 94.59 FLAFVVELLKKSLVKPSF 4455.7 4456. YVKKLVTLAILTALRLCA 8 92

Y 22 Palm- 94.78 GLMWLSYFIKKLLEQW 5991.3 5992. NLVKKFVLAAVYRIKRTL 3 45

SYYKLGASQRVAGDS palm 97.8 3735.5 | 3737.

23 STPCNGVKGFNCYFKKQ | 7.8

Palm: am 96.4 3935.6 | 3936. 24 SYGCQPTYGVGYQCYR |8.3 j u

KQYIKANSKFIGITEL Pal am 95.2 3242.0 | 3244. CSKVGGNYNCKKQYIKA | 7.2 j 1

NSKFIGITEL Palm- 96.7% ; CLGVYYHKNNKSWMES 4793.4 | 4794.

EFCKKQYIKANSKFIGITE 4 89

L

Table 3. Details for study of Example 3, investigating impact of different adjuvants.

Summary of immunization schedules for the vaccine composition of the invention. Vaccine (distinct 4x 260 ug Aluminum salts 7 Polyvalent lipo- multiepitope) Vaccine (distinct 4x 260 ug AddaVax (MF-59 7 Polyvalent lipo- like) multiepitope) 3 Vaccine (distinct 4x 260 ug AddaS03 (AS-03 7 Polyvalent lipo- like) multiepitope) Vaccine (distinct 4x 260 ug MPLA 7 Polyvalent lipo- multiepitope) Vaccine (distinct 4x 260 ug Aluminum salts + 7 sc Polyvalent lipo- MPLA multiepitope) Vaccine (distinct 4x 260 ug R848 7 Polyvalent lipo- multiepitope) 7 Vaccine (distinct 4x 260 ug Poly (I:C) 7 Polyvalent lipo- multiepitope) Vaccine (distinct 4x 260 ug PF127 7 Polyvalent lipo- NanoHydrogel multiepitope) Vaccine (distinct 4x 260 ug PF127 7 sc Polyvalent lipo- NanoHydrogel + multiepitope) MPLA Vaccine (distinct 4x 260 ug PF127 7 Polyvalent lipo- NanoHydrogel + multiepitope) R848 Vaccine (distinct 4x 260 ug PF127 7 Polyvalent lipo- NanoHydrogel + multiepitope) Poly (I:C) Vaccine (distinct 4x 260 ug PF127 7 Polyvalent lipo- NanoHydrogel multiepitope) (MPLA + Aluminum hydroxide

Polyvalent lipo- multiepitope) Table 4. Details for study set out in Example 4, investigating intranasal route of administration using PF127 NanoHydrogel as a vehicle.

Summary of immunization schedules Groups Doses Adjuvant Mice route (ug) Vaccine (distinct | 400 ug PF127 NanoHydrogel+ 10 ug Polyvalent lipo- Cholera Toxin B subunit multiepitope) recombinant} Vaccine (distinct | 400 ug PF127 NanoHydrogel + 1% Polyvalent lipo- Chitosan) multiepitope) 3 | Vaccine (distinct | 400 ug PF127 NanoHydrogel Polyvalent lipo- multiepitope) Vaccine (distinct | 400 ug Aluminum salts control sc Polyvalent lipo- multiepitope) Vaccine (distinct | 400 ug PBS control Polyvalent lipo- multiepitope) © [Cool [ous |Nawecomwol |e nm

Table 5. Experiment set out in Example 5 investigating use of distinct delivery systems as a carriers in the pharmaceutical compositions of the invention.

Summary of immunization schedules

Groups Doses Adjuvant/carrier Mice route

(ng)

Vaccine (distinct 400 ug PF127 NanoHydrogel

Polyvalent lipo- (MPL + Aluminum multiepitope) hydroxide

Vaccine (distinct 400 ug PLGA nanoparticles

Polyvalent lipo-

multiepitope)

Vaccine (distinct 400 ug PLGA nanoparticles

Polyvalent lipo- (R848 + Poly I:C)

multiepitope) Target with CD40 antibodies

Vaccine (distinct 400 ug PLGA nanoparticles

Polyvalent lipo- ((R848 + Poly I:C)

multiepitope) Target with Isotypes control

Vaccine (distinct 400 ug Liposomes

Polyvalent lipo-

multiepitope)

Vaccine (distinct 400 ug Chitosan nanoparticles

Polyvalent lipo-

multiepitope)

7 Vaccine (distinct 400 ug Aluminum salts

Polyvalent lipo-

multiepitope)

Vaccine (distinct 400 ug PBS control

Polyvalent lipo-

multiepitope) I on Je

SEQLTXT

SEQUENCE LISTING <110> Academisch Ziekenhuis Leiden <120> Immunogenic polypeptides and pharmaceutical compositions <130> P308047NL <160> 26 <170> PatentIn version 3.5 <210> 1 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Chimeric immunogenic polypeptide <400> 1 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys 1 5 10 15 Lys Ile Thr Leu Cys Phe Thr Leu Lys Arg Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 40 45 <210> 2 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 2 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys 1 5 10 15 Lys Tyr Phe Tyr Arg Ser His Arg Ser Thr Lys Leu Lys Pro Phe Glu 20 25 30 Pagina 1

SEQLTXT Arg Asp <210> 3 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 3 Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser 1 5 10 15 Ser Leu Met Val Lys Lys Cys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro

Asp Asp Phe Thr Gly Cys

<210> 4 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 4 Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Lys Lys Gly 1 5 10 15 Ile Tyr Gln Thr Ser Asn Phe Arg Val Pro Pro Val Arg Phe Pro Asn

20 25 30

Ile Thr Asn

35 <210> 5 <211> 24 <212> PRT <213> Artificial Sequence

Pagina 2

SEQLTXT

<220> <223> Artificial immunogenic polypeptide <400> 5 Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Lys Lys Phe 1 5 10 15 Ser Thr Phe Lys Cys Tyr Gly Val

<210> 6 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 6 Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Lys Lys Phe 1 5 10 15 Ser Thr Phe Lys Cys Tyr Gly Val

20 <210> 7 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 7 Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser 1 5 10 15 Ser Leu Met Val Pro Arg Arg Ala Arg Ser Val Ala Ser Gln Ala Ala

20 25 30 Cys Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Cys

40 45 Pagina 3

SEQLTXT <210> 8 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 8 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys 1 5 10 15 Lys Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe

<210> 9 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 9 Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr 1 5 10 15 Lys Lys Ser Tyr Gly Gln Pro Thr Asn Gly Val Gly Tyr Gln Tyr Arg 20 25 30 <210> 10 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 10 Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser 1 5 10 15 Pagina 4

SEQLTXT Ser Leu Met Val Lys Lys Cys Gln Phe Cys Arg Asp Ile Ala Asp Thr

Thr Asp Cys Val Arg Asp

<210> 11 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 11 Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Lys Lys Cys 1 5 10 15 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 20 25 30 Trp Asn Arg Lys Arg Ile Ser Asn Cys 35 40 <210> 12 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 12 Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala 1 5 10 15 Ser His Leu Glu Thr Lys Lys Met Ser Leu Gly Ala Glu Asn Ser Val 20 25 30 Ala Tyr Ser 35 Pagina 5

SEQLTXT <210> 13 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 13 Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr 1 5 10 15 Phe Pro Ser Val Lys Lys Thr Ser Asn Glu Val Ala Val Leu Tyr Gln

Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile His 40 <210> 14 <211> 65 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 14 Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Phe Thr Leu 1 5 10 15 Lys Arg Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe 20 25 30 Glu Leu Leu His Ala Pro Ala Ala Phe Cys Met Gln Cys Ala Tyr Arg 35 40 45 Phe Asn Ala Ala Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val 50 55 60 Lys 65 Pagina 6

SEQLTXT <210> 15 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 15 Asn Leu Leu Leu Gln Tyr Gly Ser Phe Ser Thr Gln Leu Asn Arg Lys 1 5 10 15 Leu Ile Thr Gly Arg Leu Gln Ser Leu Lys Lys Asn Leu Asn Glu Ser

Leu Ile Asp Leu Lys Lys Asp Ala Val Asp Cys Ala Leu Asp Pro Leu 40 45

Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe

50 55 <210> 16 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 16 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys 1 5 10 15 Lys Ala Leu Pro Asp Thr Pro Ser Thr Leu Thr Pro Arg Ser Val Arg

20 25 30 Ser Val Pro Gly Glu Met Arg Leu Ala Ser Ala Ala Cys Arg Lys Gln 35 40 45

Leu Ser Pro Leu Glu Gly Gly Gly Trp Leu Val Cys

50 55 60

Pagina 7

SEQLTXT <210> 17 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400e> 17 Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Lys Phe Ile Ala Gly Leu 1 5 10 15 Ile Ala Ile Val Lys Lys Phe Gln Phe Cys Asn Asp Pro Phe Leu Lys

Lys Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 40 45 Gly <210> 18 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 18 Ser Ile Ile Ala Tyr Thr Met Ser Leu Lys Lys Ile Ala Asp Tyr Asn 1 5 10 15 Tyr Lys Leu Lys Lys Val Leu Asn Asp Ile Leu Ser Arg Leu Lys Lys 20 25 30 Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr 35 40 45 Leu Thr Pro 50 Pagina 8

SEQLTXT <210> 19 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 19 Leu Leu Phe Asn Lys Val Thr Leu Ala Lys Arg Leu Gln Ser Leu Gln 1 5 10 15 Thr Tyr Val Lys Lys Val Val Phe Leu His Val Thr Tyr Val Lys Lys

Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp

40 45 <210> 20 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 20 Leu Leu Leu Asp Arg Leu Asn Gln Leu Lys Lys Gly Met Ser Arg Ile 1 5 10 15 Gly Met Glu Val Lys Lys Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly

20 25 30

Pro Glu Ala Gly Leu

35 <210> 21 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide

Pagina 9

SEQLTXT <400> 21 Phe Leu Ala Phe Val Val Phe Leu Leu Lys Lys Ser Leu Val Lys Pro 1 5 10 15 Ser Phe Tyr Val Lys Lys Leu Val Thr Leu Ala Ile Leu Thr Ala Leu

Arg Leu Cys Ala Tyr

<210> 22 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 22 Gly Leu Met Trp Leu Ser Tyr Phe Ile Lys Lys Leu Leu Glu Gln Trp 1 5 10 15 Asn Leu Val Lys Lys Phe Val Leu Ala Ala Val Tyr Arg Ile Lys Arg

20 25 30

Thr Leu Ser Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Ala Gly Asp

35 40 45 Ser <210> 23 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 23 Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Lys Lys 1 5 10 15

Pagina 10

SEQLTXT Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu

<219> 24 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 24 Ser Tyr Gly Cys Gln Pro Thr Tyr Gly Val Gly Tyr Gln Cys Tyr Arg 1 5 10 15 Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu 20 25 30 <210> 25 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 25 Cys Ser Lys Val Gly Gly Asn Tyr Asn Cys Lys Lys Gln Tyr Ile Lys 1 5 10 15 Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu 20 25 <210> 26 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Artificial immunogenic polypeptide <400> 26 Pagina 11

SEQLTXT Cys Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser 1 5 10 15 Glu Phe Cys Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly

Ile Thr Glu Leu

Pagina 12

Claims

CONCLUSIONS

A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26 of Table 1, or a variant thereof comprising an amino acid sequence different from any of SEQ ID NOs: 1 to and 26, by an alteration of up to two amino acid residues within the coronavirus-derived portion of the sequence, listed in Table 1.

The polypeptide of claim 1, wherein at least one of the N-terminus and the C-terminus is independently modified by a fatty acid.

The polypeptide of claim 2, wherein the fatty acid is a (2-28C) fatty acid.

The polypeptide of claim 3, wherein the fatty acid is an (8-20C) fatty acid.

The polypeptide of any one of claims 1 to 4, wherein the fatty acid is palmitic acid, oleic acid, lauric acid, capric acid, myristic acid, stearic acid, linoleic acid, or linolenic acid.

A polypeptide according to any one of the preceding claims, wherein the fatty acid is palmitic acid.

A polypeptide according to any one of the preceding claims, comprising a reactive or a free N-terminus or C-terminus.

A polypeptide according to any one of the preceding claims, wherein at least one of the N-terminus and the C-terminus is modified by a polymer.

The polypeptide of claim 8, wherein the polymer is selected from polyethylene glycol, polypropylene glycol, polytrimethylene glycol, polyoxyethylene, polylactic acid, polyacrylic acid, and polyacrylamide.

The polypeptide of claim 8 or 9, wherein the polymer is polyethylene glycol.

A polypeptide according to any one of the preceding claims, wherein at least one of the N-terminus and the C-terminus is independently modified by a polysaccharide.

The polypeptide of claim 11, wherein the polysaccharide is dextran.

The polypeptide of any preceding claim, wherein at least one of the N-terminus and the C-terminus is independently modified by a hydrocarbyl group selected from (1-24C)alkyl, {1-24C)alkoxy, (2- 24C)alkenyl, (2-24C)alkynyl, aryl, aryl(1-4C)alkyl, heteroaryl, heteroaryl(1-4C)alkyl, carbocyclyl, carbocyclyl(1-4C)alkyl, heterocyclyl, and heterocyclyl(1-4C) alkyl.

The polypeptide of any preceding claim, wherein any or more of the fatty acid, polymer, polysaccharide, and hydrocarbyl groups are independently optional or substituted with one or more substituents independently selected from hydroxy, cyano, oxo, halogen, ( 1-6C)alkyl, (1-6C)alkoxy, phenyl, benzyl, -NR'R?, -C(O)-R?, -C(O)-OR*, -O-C(O)-Rt, - C(0)-NR'R2, -N(R")C(O)-R", -S(O)0-2R', -S(O)2NR'R7, and -N(R") -S(O) 2 R 7 , where R 1 and R 7 are each independently hydrogen or (1-3C)alkyl.

A polypeptide according to any one of the preceding claims, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26 of Table 1.

The polypeptide of claim 15, wherein the amino acid sequence of the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26 of Table 1.

The polypeptide of claim 15 or 18, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 26, further comprising the exemplary modification listed for the amino acid sequence in Table 1.

A polypeptide according to any one of claims 1 to 14, comprising an amino acid sequence which differs from any of the group of SEQ ID NOs: 1 to 26 by an alteration of a single amino acid residue within the coronavirus-derived portion of the sequence, can be found in Table 1.

A polypeptide according to any one of claims 1 to 17, comprising an amino acid sequence which differs from any of the group of SEQ ID NOs: 1 to 26 by an alteration of two amino acid residues within the coronavirus-derived portion of the sequence, back found in Table 1.

The polypeptide of claim 18 or 19, wherein each alteration is independently selected from the group consisting of: substitution of an amino acid residue; addition of an amino acid residue; and deletion of an amino acid residue.

The polypeptide of claim 20, wherein substitution of an amino acid residue is selected from the group consisting of: substitution of a lysine residue with an arginine residue or a histidine residue; substitution of an arginine residue with a lysine residue or a histidine residue; and substitution of a histidine residue with a lysine residue or an arginine + residue.

The polypeptide of any one of claims 20 to 21, wherein substitution of an amino acid residue is selected from the group consisting of: substitution of a leucine residue with an isoleucine residue or a norleucine residue; substitution of an isoleucine residue with a leucine residue or a norleucine residue; and substitution of a norleucine residue with a leucine residue or an isoleucine residue.

The polypeptide of any one of claims 20 to 22, wherein an amino acid residue substitution is selected from the group consisting of: substitution of a serine or threonine residue with an α cysteine residue; and substitution of a cysteine residue with a serine residue.

The polypeptide of any one of claims 20 to 23, wherein an amino acid residue substitution is selected from the group consisting of: substitution of a glutamine residue for an asparagine residue; and substitution of an asparagine residue for a glutamine residue.

The polypeptide of any one of claims 20 to 24, wherein a substitution of a residue is selected from the group consisting of: substitution of a glutamic acid residue with an aspartic acid residue; and substitution of an aspartic acid residue for a glutamic acid residue.

The polypeptide of any one of claims 20 to 25, wherein an amino acid residue substitution is selected from the group consisting of: substitution of a glycine residue for an alanine residue or a beta-alanine residue; substitution of an alanine residue with a glycine residue or a beta-alanine residue; and substitution of a beta-alanine residue with a glycine residue or an alanine residue.

The polypeptide of any one of claims 20 to 26, wherein an amino acid residue substitution is selected from the group consisting of: substitution of a phenylalanine residue with a tyrosine residue or a tryptophan residue; substitution of a tryptophan residue with a phenylalanine residue or a tyrosine residue; and substitution of a tyrosine residue with a tryptophan residue or a phenylalanine residue.

The polypeptide of any one of claims 20 to 27, wherein an amino acid residue substitution is selected from the group consisting of: substitution of an L-amino acid for a D-amino acid.

The polypeptide of any one of claims 20 to 28, wherein an amino acid residue substitution is selected from a similar molecular building block to a non-natural amino acid.

The polypeptide of any one of claims 18 to 29, comprising an amino acid sequence variant selected from the group consisting of SEQ ID NOs: 1 to 26, further comprising the exemplary modification found for the reference / basic amino acid sequence in Table 1.

A polypeptide according to any one of claims 1 to 30 comprising a single polypeptide chain.

A polypeptide according to any one of claims 1 to 31 comprising two polypeptide chains.

The polypeptide of claim 32, comprising two polypeptide chains each comprising the amino acid sequence of SEQ ID NO: 5.

A polypeptide according to any one of claims 1 to 30 comprising four polypeptide chains.

The polypeptide of claim 34 comprising four polypeptide chains each comprising the amino acid sequence of SEQ ID NO: 6.

A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 35 and a pharmaceutically acceptable carrier.

A pharmaceutical composition according to claim 37, wherein the composition comprises a mixture of polypeptides according to any one of claims 1 to

35.

The pharmaceutical composition of claim 37, wherein the composition comprises a mixture of polypeptides comprising any of the amino acid sequences listed in SEQ ID NOs: 1 to 22.

A pharmaceutical composition according to claim 37 or claim 38, wherein the polypeptides are present in equal amounts.

A pharmaceutical composition according to any one of claims 36 to 39 comprising an adjuvant.

The pharmaceutical composition of claim 40, wherein the adjuvant is selected from the group consisting of: AS03; AddaSO3; AS04; MPL; MF59; AddaVax; Poly I:C; R848; CpG; flagellin protein; and an aluminum salt adjuvant.

The pharmaceutical composition of any one of claims 36 to 41, wherein the carrier is selected from the group consisting of: a polymer; a liposome; dextran; and alginate.

The composition of claim 42, wherein the polymeric carrier is selected from the group consisting of: poly(D,L-lactide-co-glycolide), polylactic acids, poly(propylene fumarate-co-ethylene glycol) [P(PF-co- EG)] block copolymer, polyanhydride poly(fumaric acid-co-sebacic acid anhydride, poly(ethylene oxide)-poly(lactide/glycolide), poly(amino acid), polyvinyl alcohol, alginate, dextran, chitosan, hydroxyapatite, collagen, fibrin, hyaluronic acid, carbomers, poly(amino acid) poly(ethylene glycol), and PF127 pluronic gel.

A pharmaceutical composition according to any one of claims 36 to 43, wherein the carrier is provided as microspheres or nanospheres.

A pharmaceutical composition according to any one of claims 36 to 44 comprising a mixture of polypeptides of the invention, a PF127 carrier, chitosan, and cholera B toxin.

A pharmaceutical composition according to any one of claims 36 to 44 comprising a PF127 gel and PLGA nanoparticles as carriers.

A pharmaceutical composition according to any one of claims 36 to 46, for use as a medicament for mucosal administration.

A polypeptide or pharmaceutical composition according to any one of claims 1 to 47, for use as a medicament.

A polypeptide or pharmaceutical composition according to claim 48, for use as a vaccine.

The polypeptide or pharmaceutical composition for use according to claim 49, for use as a vaccine for the prevention and/or treatment of a coronavirus infection.

The polypeptide or pharmaceutical composition for use according to claim 50, for use as a vaccine for preventing and/or treating an infection caused by a coronavirus selected from the group consisting of: SARS-CoV-2; SARS CoV; and MERS-CoV.

A polypeptide or pharmaceutical composition for use according to any one of claims 48 to 51, wherein the medical use comprises providing the polypeptide with one or more additional peptides according to any one of claims 1 to 35.

A nucleic acid sequence encoding a polypeptide according to any one of claims 1 to 35.

A prokaryotic or eukaryotic cell expressing protein according to any one of claims 1 to 35.

A prokaryotic or eukaryotic cell comprising a nucleic acid sequence according to claim 53.

A method of producing a polypeptide according to any one of claims 1 to 35, the method comprising growing a cell according to claim

54 or 55, in such a way that the cell expresses a polypeptide according to the first aspect of the invention.