WO2021260176A1

WO2021260176A1 - Synthetic epitopes of betacoronaviruses

Info

Publication number: WO2021260176A1
Application number: PCT/EP2021/067487
Authority: WO
Inventors: Anna Kathleen SUMERAY; John Anthony Robinson; Kerstin MÖHLE
Original assignee: Virometix Ag
Priority date: 2020-06-25
Filing date: 2021-06-25
Publication date: 2021-12-30

Abstract

The present invention relates to fields of epitope vaccine design. In particular, it relates to a polypeptide comprising an amino acid sequence of SEQ ID NO: 1: X₁SNNLDSKVGGNYNYX₂YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQX₃YGFQPTNGVGYQPX₄, wherein each of X1 to X4 is independently at least one amino acid, or a variant of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 or a fragment of the variant of SEQ ID NO: 1, and a nucleic acid molecule encoding the polypeptide of the invention. The invention further relates to a conjugate comprising (i) the polypeptide of the invention, (ii) a peptide moiety comprising at least one coiled coil peptide chain segment, and (iii) a lipid moiety. The invention further relates to a synthetic virus-like particle (sVLP) consisting of helical lipopeptide bundles comprising the conjugates of the invention, and its use as a vaccine against SARS-CoV and SARS-CoV-2 diseases and in preventing or treating SARS-CoV and SARS-CoV-2 diseases.

Description

SYNTHETIC EPITOPES OF BETACORONAVIRUSES

The present invention relates to fields of epitope vaccine design. In particular, it relates to a polypeptide comprising an amino acid sequence of SEQ ID NO: 1:

X_ISNNLDSKVGGNYNYX₂YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQ X3YGFQPTNGVGYQPX4, wherein each of XI to X4 is independently at least one amino acid, or a variant of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 or a fragment of the variant of SEQ ID NO: 1, and a nucleic acid molecule encoding the polypeptide of the invention. The invention further relates to a conjugate comprising (i) the polypeptide of the invention, (ii) a peptide moiety comprising at least one coiled coil peptide chain segment, and (iii) a lipid moiety. The invention further relates to a synthetic virus-like particle (sVLP) consisting of helical lipopeptide bundles comprising the conjugates of the invention, and its use as a vaccine against SARS-CoV and SARS-CoV-2 diseases and in preventing or treating SARS-CoV and SARS-CoV-2 diseases.

RELATED ART

A previously unknown coronavirus, named SARS-CoV-2, was discovered in December 2019 in Wuhan, Hubei province of China and was sequenced and isolated by January 2020. Like SARS- CoV and MERS-CoV, SARS-CoV-2 also belongs to the Beta-coronavirus genus and is associated with an ongoing outbreak of atypical pneumonia (Covid-2019) and the World Health Organization declared the SARS-CoV-2 epidemic a public health emergency of international concern. Coronavirus entry into host cells is mediated by the transmembrane spike (S) glycoprotein that forms homotrimers protruding from the viral surface. S comprises two functional subunits responsible for binding to the host cell receptor (SI subunit) and fusion of the viral and cellular membranes (S2 subunit). The distal SI subunit comprises the receptor-binding domain(s) (RBD) and contributes to stabilization of the prefusion state of the membrane -anchored S2 subunit that contains the fusion machinery. ACE2 could mediate SARS- CoV-2 S-mediated entry into cells, establishing it as a functional receptor for this newly emerged coronavirus (Walls et ah, Structure, Function, and Antigenicity ofthe SARS-CoV-2 Spike Glycoprotein, Cell 180, 1-12, 2020).

Both S glycoprotein and the RBD are highly immunogenic and both have been found to elicit neutralizing antibodies. Pak et al. reported the RBD in complex with the Fab of a neutralizing mouse monoclonal antibody, F26G19, elicited by immunization with chemically inactivated SARS-CoV. The structure reveals that the RBD surface recognized by F26G19 overlaps with the surface recognized by ACE2 and, as such, suggests that F26G19 likely neutralizes SARS-CoV by blocking the virus-host cell interaction (Pak et al., Structural Insights into Immune Recognition of the Severe Acute Respiratory Syndrome Coronavirus S Protein Receptor Binding Domain, J. Mol. Biol. 388, 815-823, 2009).

Wrap et al. tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to SARS-CoV-2 S protein, suggesting that antibody cross reactivity may be limited between the two RBDs (Wrapp et al., Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation, Science 367, 1260-1263, 2020).

Marasco and co-workers identified a neutralizing human monoclonal antibody against the SI RBD, designated “80R,” from two non-immune human antibody libraries. 80R binds SI with nanomolar affinity, blocks the binding of SI to ACE2, and prevents the formation of syncytia in vitro. Mapping of the 80R epitope showed it is located within the N-terminal 261-672 amino acids of S protein and is not glycosylation-dependent (Sui et al., Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S 1 protein that blocks receptor association, Proc. Natl. Acad. Sci. USA 101, 2536-2541, 2004).

Wu et al., isolated a neutralizing monoclonal antibodies called B38 and H4 block binding between the spike glycoprotein receptor binding domain (RBD) of the virus and the cellular receptor angiotensin converting enzyme 2 (ACE2) (Wu et al., A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2, Science, 2020, 368, 1274-1278). The RBD-B38 complex structure revealed that most residues on the epitope overlap with the RBD-ACE2 binding interface, explaining the blocking effect and neutralizing capacity.

The emergence of the coronavirus pandemic demands the development of medical countermeasures to address this public health crisis. Thus, there is a high need of new vaccines that provide protection against members of the Betacoronavirus genus such as SARS-CoV and SARS-CoV - 2 in order to control its spread and to prevent rapid progress of disease and mortalities.

SUMMARY OF THE INVENTION

The inventors identified a SARS-CoV and SARS-CoV-2 epitope derived from a loop structure on the surface of the RBD. It is defined by a relatively small and well-defined sequence centered on residues 437-508 of receptor binding domain of a SARS-CoV-2 spike protein. To have the correct folded structure of the epitope is likely critical for recognition by neutralizing antibodies, and for eliciting efficiently potent neutralizing antibodies against SARS-CoV and SARS-CoV-2.

Beside identification of the epitope of residues 437-508, the inventors developed artificial peptidomimetics, which comprise inter alia circulating cysteine residues as conformational constraints to stabilize the biologically relevant conformation of the peptidomimetics.

Combined with the lipopeptide building block of the invention, the inventors produced conjugates and sVLP in order to activate efficiently B and T cells in a host organism to produce exclusively neutralizing antibodies against the target viruses SARS-CoV and SARS-CoV-2 and to avoid inducing virus-related TH2 T-cell responses.

Thus, in a first aspect, the invention relates to (i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 : Xi SNNLD SK V GGNYN YX₂ YRLFRK SNLKPFERDIS TEI Y Q AGS TPCN GVEGFNC YFPLQ X₃YGFQPTNGVGYQPX₄, wherein each of XI to X4 is independently at least one amino acid; or (ii) a variant of SEQ ID NO: 1, wherein in said variant at most 33 amino acids are exchanged as compared to SEQ ID NO: 1; or (iii) a fragment of SEQ ID NO: 1 or a fragment of the variant of SEQ ID NO: 1.

In a further aspect, the invention relates to a nucleic acid molecule encoding the polypeptide of the invention.

In a further aspect, the invention relates to a conjugate comprising

(i) the polypeptide of the invention,

(ii) a peptide moiety comprising at least one coiled coil peptide chain segment, and

(iii) a lipid moiety comprising two or three, preferably two hydrocarbyl chains, wherein the peptide moiety is covalently linked at one end to the polypeptide of the invention and at the other end to the lipid moiety, either directly or through a coupling moiety.

In another aspect, the present invention provides for a bundle of conjugates comprising 2, 3, 4, 5, 6 or 7, preferably 2, 3, 4 or 5, more preferably 3, of the inventive conjugate.

In a further aspect, the invention relates to a synthetic virus-like particle consisting of helical lipopeptide bundles comprising two, three, four, five, six or seven conjugates of the invention.

In again another aspect, the present invention provides for the conjugate of the present invention or the synthetic virus like particle of the present invention for use as a medicament.

In a further aspect, the invention relates to the polypeptide, the conjugate, the synthetic virus like particle of the invention for use as a vaccine against SARS-CoV or SARS-CoV-2 diseases and in preventing or treating SARS-CoV or SARS-CoV-2 diseases.

In another aspect, the present invention provides for a pharmaceutical composition comprising an immunologically effective amount of the conjugate of the present invention or the synthetic virus like particle of the present invention, together with a pharmaceutically acceptable diluent, carrier or excipient, wherein preferably said pharmaceutical composition is a vaccine.

Further aspects and embodiments of the present invention will become apparent as this description continues.

DESCRIPTION OF THE FIGURES

FIG. 1: Protein sequence alignment of the receptor binding domain (RBD) of the SARS-CoV (top) and SARS-CoV-2 (bottom) spike proteins.

FIG. 2: The RBD from the SARS-CoV-2 spike protein, from residue 331 to 527, taken from protein data base PDB, file 6VW1. The stretch including residues 436 to 508 is shown in dark grey.

FIG. 3: Mimetic- 1, mimetic-2, and mimetic-3 are derived from the regions of the complete RBD boxed by dotted lines. Non-native disulfide bonds and a D-Arg-Pro dipeptide unit are introduced as conformational restraints: In mimetic- 1, a disulfide between two non native Cys residues is introduced at the N- and C-termini (452-494). In mimetic-2, a disulfide bond between two non-native Cys residues (437-508) is introduced and two amino acid stretches are newly combined and fused by a D-Arg-Pro dipeptide backbone cross-link between 454 and 492 replacing L-Arg454 to Leu492. In mimetic 3, a disulfide between two non-native Cys residues is introduced at the N- and C-termini (437-508), with the native sequence in- between.

FIG. 4: ELISA measurements of IgG responses - Graphs show averaged OD450 values from mice sera at a dilution of 1:400 and reflect levels of IgG antibodies.

FIG. 5: ELISA measurements of IgG responses - Graphs show individual OD450 values from mice sera (4 mice per group, per dose) at a dilution of 1:400 and reflect levels of IgG antibodies.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Throughout this specification and the claims, which follow, unless the context requires otherwise, the term “comprise” and its variations such as “comprises” and “comprising” etc., are to be understood as a non-exhaustive wording and imply the inclusion of a stated feature or element but not the exclusion of any other feature or element. The term “comprise” and its variations cover the term “consisting of’. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents, unless the content clearly dictates otherwise. The terms “reduce”, “inhibit” or “decrease”, as used herein, include a just detectable reduction but also a reduction down to zero (reduction by 100%). The term “between” includes the indicated limit value. A peptide or peptide moiety, as defined herein, is any peptide-bond-linked chain of amino acids, regardless of length, secondary and tertiary structure, number of subunits or post- translational and artificial modification. Thus, the term “peptide” of “peptide moiety” is to be understood as covering the terms “polypeptide”, “protein”, “amino acid chain” and “polypeptide chain”. Amino acids included in the peptide of the invention are proteinogenic, non-proteinogenic and synthetic amino acids. Peptides can be an open linear peptide chain or cyclic peptides; and may include at least one chemical modification, such as lipidation, glycosylation and phosphorylation. Peptides can be produced by chemical synthesis, RNA translation and/or recombinant processes.

The term “amino acid”, as used herein, refers to organic compounds containing the functional groups amine (-NH2) and carboxylic acid (-COOH) and its zwitterions, typically and preferably, along with a side chain specific to each amino acid. The term “amino acid” typically and preferably includes amino acids that occur naturally, such as proteinogenic amino acids (produced by RNA-translation), non-proteinogenic amino acids (produced by other metabolic mechanisms, e.g. posttranslational modification), standard or canonical amino acids (that are directly encoded by the codons of the genetic code) and non-standard or non-canonical amino acids (not directly encoded by the genetic code). Naturally occurring amino acids include non- eukaryotic and eukaryotic amino acids.

The term “amino acid”, as used herein, includes natural proteinogenic amino acids, non- proteinogenic amino acids and chemically synthetic unnatural amino acids; alpha- (a-), beta- (b-), gamma- (g-) and delta- (d-) etc. amino acids as well as mixtures thereof in any ratio; and, if applicable, any isomeric form of an amino acid, i.e. its D-stereoi somers (labelled with a lower-case initial letter) and L-stereoi somers (labelled with a capital initial letter) (alternatively addressed by the (R) and (S) nomenclature) as well as mixtures thereof in any ratio, preferably in a racemic ratio of 1 : 1. Amino acids in this invention are preferably in L-configuration, unless mentioned specifically as D-configuration. The term “D-stereoisomer”, “L-stereoisomer”, “D- amino acid” or “L-amino acid” refers to the chiral alpha carbon of the amino acids. Amino acid can include one or more modifications and/or attached groups, for example protecting groups used for peptide synthesis, such as Boc, Fmoc or both. The term “deletion” refers herein to a position in an amino acid sequence that is not occupied by an amino acid. In a preferred embodiment of the present invention, an amino acid is a naturally occurring amino acid, wherein said naturally occurring amino acid is in its L-configuration, in its D-configuration, or in a mixture of any ratio of said L-configuration and D-configuration. If not indicated to the contrary, an amino acid is preferably a naturally occurring amino acid, wherein said naturally occurring amino acid is preferably in its L-configuration.

The antigen may comprise a peptide, a protein or an epitope mimetic having one or more B-cell epitopes that are to be used to elicit an antigen-specific humoral immune response in an animal. The term “mimetic” or “epitope mimetic” as used herein is a molecule mimicking a natural proteinogenic, peptidic or carbohydrate epitope, including peptidic compounds containing one or more non-natural amino acids, e.g. D-amino acids, b-amino acids, g-amino acids, d-amino acids, or e-amino acids, and other replacements known in the art of epitope mimics. Preferred are conformationally constrained peptidomimetics, which are fixed in a protein-like conformation.

The term “N-terminus” as used herein relates to the beginning of an amino acid sequence or polypeptide starting with the free amine group (-NEh), modified amino or modified amine group or relates to the corresponding first amino acid comprising this free amine group or modified amino or amine group. Preferred N-terminal modifications are those that protect the N-terminus from proteolytic degradation. The term “C-terminus” as used herein relates to the end of an amino acid sequence or polypeptide terminated by a free carboxyl group (-COOH) or a modified carboxyl group, such as an amide (CONH2), or relates to the corresponding amino acid comprising this free carboxyl group. By convention and as used herein, peptide sequences are written from N-terminus to C-terminus, left to right.

As used herein, the term “coiled coil peptide chain segment” is a sequence of a peptide chain capable of forming a coiled coil with at least one other coiled coil peptide chain segment. A coiled coil is a peptide structure in which at least two coiled coil peptide chain segments, each having preferably an alpha helical secondary structure, are associated into a bundle. Coiled coil peptide chain segments of the invention contain multiple repeat units, typically and preferably consecutively linked to each other. The repeat units of the coiled coil peptide chain segment may be identical or may be different, e.g. may contain at least one discontinuity, such as an insertion, deletion or exchange of at least one, preferably exactly 1, 2, 3 or 4 amino acids within the repeat unit.

In a first aspect, the invention relates to a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 :

Xi SNNLD SK V GGNYN YX₂ YRLFRK SNLKPFERDIS TEI Y Q AGS TPCN GVEGFN C

YFPLQX3YGFQPTNGVGYQPX4, wherein each of XI (or Xi) to X4 (or X4) is independently at least one amino acid, or a variant of SEQ ID NO: 1, wherein in said variant at most 33 amino acids are exchanged as compared to SEQ ID NO: 1, or a fragment of SEQ ID NO: 1 or a fragment of the variant of SEQ ID NO: 1. In a further aspect, the invention relates to a polypeptide comprising an amino acid sequence of said SEQ ID NO: 1, wherein each of XI or X4 is independently at least one amino acid or a fragment of SEQ ID NO: 1.

The term “variant of SEQ ID NO: or “variant of sequence as used herein relates preferably to a variant amino acid sequence in which a defined number of amino acids are exchanged compared to the non-variant parent sequence. In a preferred embodiment, the term “exchanged” also includes deletion of an amino acid, i.e. exchange of an amino acid by a deletion. Preferably, the number of amino acids in the variant is not amended as compared to the non-variant sequence. If XI, X2 X3 and X4 or other amino acid positions are explicitly defined, e.g., as cysteines, they are not variable in the variant. Preferably, cysteines of (i) XI and X4 and/or (ii) X2 and X3 form disulfide bond(s).

The term “amino acids at positions... are conserved in said variant” as used herein means that amino acids at these positions are not modified as compared to the parent non-modified sequence. The nomination and numbering of the conserved amino acid positions in the variants and fragments refer to the non-fragmented SEQ ID NO: 1 which starts with amino acid 1 at the N-terminus.

In a preferred embodiment, amino acids at positions 15, 17, 25, 43, 44, 56, 60 and 66 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included. In a more preferred embodiment, amino acids at positions 4, 14, 15, 17, 25, 29, 31, 32, 43, 44, 56, 60, 61, 64 and 66 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included. In another even more preferred embodiment, amino acids at positions 4, 6, 11-15, 17, 18, 21, 25, 27-33, 43, 44, 51-53, 55, 56, 59, 60, 61, 64, 66 and 68-71 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included.

In a preferred embodiment, amino acids at positions 15, 17, 25, 43, 44, 56, 60 and 66 are conserved in said variant or in the fragment of said variant, if included, and each of X1-X4 is one cysteine. In another preferred embodiment, amino acids at positions 4, 14, 15, 17, 25, 29, 31, 32, 43, 44, 56, 60, 61, 64 and 66 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included, and each of X1-X4 is one cysteine. In another preferred embodiment, amino acids at positions 4, 6, 11-15, 17, 18, 21, 25, 27-33, 43, 44, 51-53, 55, 56, 59, 60, 61, 64, 66 and 68-71 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included, and each of X1-X4 is one cysteine.

In a preferred embodiment, amino acids at positions 15, 17, 25, 43, 44, 56, 60 and 66 are conserved in said variant or in the fragment of said variant, if included, and each of XI and X4 is one cysteine. In another preferred embodiment, amino acids at positions 4, 14, 15, 17, 25, 29, 31, 32, 43, 44, 56, 60, 61, 64 and 66 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included, and each of XI and X4 is one cysteine. In another preferred embodiment, amino acids at positions 4, 6, 11-15, 17, 18, 21, 25, 27-33, 43, 44, 51- 53, 55, 56, 59, 60, 61, 64, 66 and 68-71 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included, and each of XI and X4 is one cysteine.

In a preferred embodiment, amino acids at positions 15, 17, 25, 43, 44, 56, 60 and 66 are conserved in said variant or in the fragment of said variant, if included, and each of X2 and X3 is one cysteine. In another preferred embodiment, amino acids at positions 4, 14, 15, 17, 25, 29, 31, 32, 43, 44, 56, 60, 61, 64 and 66 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included, and each of X2 and X3 is one cysteine. In another preferred embodiment, amino acids at positions 4, 6, 11-15, 17, 18, 21, 25, 27-33, 43, 44, 51- 53, 55, 56, 59, 60, 61, 64, 66 and 68-71 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included, and each of X2 and X3 is one cysteine.

In another preferred embodiment, amino acids at positions 2, 3, 5, 7-10, 19, 20, 22-24, 26, 34-42, 45-50, 54, 57, 62, 63, 65 and 67 of SEQ ID NO: 1 are variable in said variant or in the fragment of said variant, if included. Preferably, the remaining amino acid positions are conserved. In another preferred embodiment, amino acids at positions 2, 3, 5, 7-10, 19, 20, 22- 24, 26, 34-42, 45-50, 54, 57, 58, 62, 63, 65 and 67 of SEQ ID NO: 1 are variable in said variant or in the fragment of said variant, if included, and each of XI -X4 is one cysteine. Preferably, the remaining amino acid positions are conserved. In another preferred embodiment, amino acids at positions 2, 3, 5, 7-10, 19, 20, 22-24, 26, 34-42, 45-50, 54, 57, 58, 62, 63, 65 and 67 of SEQ ID NO: 1 are variable in said variant or in the fragment of said variant, if included, and each of XI and X4 is one cysteine. In another preferred embodiment, amino acids at positions 2, 3, 5, 7-10, 19, 20, 22-24, 26, 34-42, 45-50, 54, 57, 58, 62, 63, 65 and 67 of SEQ ID NO: 1 are variable in said variant or in the fragment of said variant, if included, and each of X2 and X3 is one cysteine.

In a preferred embodiment, amino acids at positions 15, 25, 43, 44, 56, 60 and 66 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included. In a more preferred embodiment, amino acids at positions 4, 14, 15, 25, 29, 31, 32, 43, 44, 56, 60, 61, 64 and 66 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included. In another even more preferred embodiment, amino acids at positions 4, 6, 11-15, 18, 21, 25, 27-33, 43, 44, 51-53, 55, 56, 59, 60, 61, 64, 66 and 68-71 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included. In a preferred embodiment, amino acids at positions 15, 25, 43, 44, 56, 60 and 66 are conserved in said variant or in the fragment of said variant, if included, and each of X1-X4 is one cysteine. In another preferred embodiment, amino acids at positions 4, 14, 15, 25, 29, 31, 32, 43, 44, 56, 60, 61, 64 and 66 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included, and each of X1-X4 is one cysteine. In another preferred embodiment, amino acids at positions 4, 6, 11-15, 18, 21, 25, 27-33, 43, 44, 51-53, 55, 56, 59, 60, 61, 64, 66 and 68-71 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included, and each of X1-X4 is one cysteine.

Preferably, cysteines of (i) XI and X4 and/or (ii) X2 and X3 form disulfide bond(s).

In a preferred embodiment, amino acids at positions 15, 25, 43, 44, 56, 60 and 66 are conserved in said variant or in the fragment of said variant, if included, and each of XI and X4 is one cysteine. In another preferred embodiment, amino acids at positions 4, 14, 15, 25, 29, 31, 32, 43, 44, 56, 60, 61, 64 and 66 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included, and each of XI and X4 is one cysteine. In another preferred embodiment, amino acids at positions 4, 6, 11-15, 18, 21, 25, 27-33, 43, 44, 51-53, 55, 56, 59, 60, 61, 64, 66 and 68-71 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included, and each of XI and X4 is one cysteine. Preferably, cysteines of XI and X4 form a disulfide bond.

In a preferred embodiment, amino acids at positions 15, 25, 43, 44, 56, 60 and 66 are conserved in said variant or in the fragment of said variant, if included, and each of X2 and X3 is one cysteine. In another preferred embodiment, amino acids at positions 4, 14, 15, 25, 29, 31, 32, 43, 44, 56, 60, 61, 64 and 66 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included, and each of X2 and X3 is one cysteine. In another preferred embodiment, amino acids at positions 4, 6, 11-15, 18, 21, 25, 27-33, 43, 44, 51-53, 55, 56, 59, 60, 61, 64, 66 and 68-71 of SEQ ID NO: 1 are conserved in said variant or in the fragment of said variant, if included, and each of X2 and X3 is one cysteine. Preferably, cysteines of X2 and X3 form a disulfide bond. In a preferred embodiment, amino acid at position 17 of SEQ ID NO: 1 is variable in said variant or in the fragment of said variant, if included. Preferably, Y at position 17 is exchanged by a polar amino acid or an amino acid that is negatively charged at physiological pH range (about 7). Preferably, Y at position 17 is exchanged by an amino acid selected from the group consisting of phenylalanine, glutamic acid, glutamine, aspartic acid aspartate or glycine.

In a preferred embodiment, SEQ ID NO: 1, said variant of SEQ ID NO: 1 or said fragment of SEQ ID NO: 1 or said fragment of said variant of SEQ ID NO: 1 comprises 2 to 8 cysteines, more preferably 2 to 6 cysteines, again more preferably 2, 3 or 4 cysteines. In a preferred embodiment, SEQ ID NO: 1, said variant of SEQ ID NO: 1 or said fragment of SEQ ID NO: 1 or said fragment of said variant of SEQ ID NO: 1 comprises at least two cysteines. In another preferred embodiment, SEQ ID NO: 1, said variant of SEQ ID NO: 1 or said fragment of SEQ ID NO: 1 or said fragment of said variant of SEQ ID NO: 1 comprises 2 or 4 cysteines. In another preferred embodiment, SEQ ID NO: 1, said variant of SEQ ID NO: 1 or said fragment of SEQ ID NO: 1 or said fragment of said variant of SEQ ID NO: 1 comprises 4 cysteines. In a further preferred embodiment, SEQ ID NO: 1, said variant of SEQ ID NO: 1, said fragment of SEQ ID NO: 1 or said fragment of said variant of SEQ ID NO: 1 comprises 2 cysteines. Preferably, pairs of said cysteines form disulfide bonds. In case of three or four cysteines, the pair of cysteines proximal to the N and C terminus form a disulfide bond and/or the pair of cysteines distal to the N and C terminus form a disulfide bond.

Preferably, a cysteine is each located at the N and C-terminus of SEQ ID NO: 1, said variant of SEQ ID NO: 1, or said fragment of SEQ ID NO: 1 or of the variant of SEQ ID NO: 1

Each of XI to X4 is independently of each other a moiety of one or more consecutively linked amino acids. In a preferred embodiment, each of XI to X4 is independently of each other a moiety of 1 to 10 consecutively linked amino acids. In a preferred embodiment, each of XI to X4 is independently a moiety of 1 to 5, more preferably 1 to 4, again more preferably 1 to 3 consecutively linked amino acids. In another very preferred embodiment, each of XI to X4 is independently of each other one amino acid or 2 consecutively linked amino acids. Most preferably, each of XI to X4 is independently of each other one amino acid.

In a preferred embodiment, in the polypeptide of the invention comprising SEQ ID NO: 1, a variant thereof or a fragment of SEQ ID NO: 1 or a fragment of said variant, (a) each of XI and X4 and (b) each of X2 and X3 is one cysteine. In a preferred embodiment, SEQ ID NO: 1, the variant thereof or the fragment of SEQ ID NO: 1 or of said variant, each of XI, X2, X3 and X4 is independently one cysteine. In a more preferred embodiment, SEQ ID NO: 1, the variant thereof or the fragment of SEQ ID NO: 1 or of said variant, (a) each of XI and X4 or (b) each of X2 and X3 is one cysteine. In a preferred embodiment, SEQ ID NO: 1, the variant thereof or the fragment of SEQ ID NO: 1 or of said variant, each of XI and X4 is one cysteine. In a preferred embodiment, SEQ ID NO: 1, the variant thereof or the fragment of SEQ ID NO: 1 or of said variant, each of X2 and X3 is one cysteine. In a preferred embodiment, the polypeptide of the invention comprises an amino acid sequence of SEQ ID NO: 1 or a fragment thereof, or optionally a variant of SEQ ID NO: 1 or said fragment, wherein (a) each of XI to X4, or (b) each of XI and X4, or (c) each of X2 and X3 is one cysteine.

In a preferred embodiment, (i) the SEQ ID NO: 1, (ii) a variant thereof or (iii) a fragment of SEQ ID NO: 1 or of said variant comprises XI and X4, wherein each of XI and X4 is one cysteine. In a preferred embodiment, (i) the SEQ ID NO: 1, (ii) a variant thereof or (iii) a fragment of SEQ ID NO: 1 or of said variant comprises X2 and X3, wherein each of X2 and X3 is one cysteine. In a preferred embodiment, (i) the SEQ ID NO: 1, (ii) a variant thereof or (iii) a fragment of SEQ ID NO: 1 or of said variant comprises XI, X2, X3 and X4, wherein each of XI, X2, X3 and X4 is one cysteine. In a preferred embodiment, (i) the SEQ ID NO: 1, (ii) a variant thereof or (iii) a fragment of SEQ ID NO: 1 or of said variant comprises XI, X2, X3 and X4, wherein each of XI, X2, X3 and X4 is one cysteine, each of XI and X4 is one cysteine, or each of X2 and X3 is one cysteine. Preferably, pairs of said cysteines form disulfide bonds. In case of three or four cysteines, preferably the pair of cysteines proximal to the N and C terminus form a disulfide bond and/or the pair of cysteines distal to the N and C terminus form a disulfide bond. Preferably, cysteines at XI and X4 form a disulfide bond and/or cysteines at X2 and X3 form a disulfide bond, if included.

In a preferred embodiment, in the polypeptide of the invention comprising SEQ ID NO: 1, a variant thereof or a fragment of SEQ ID NO: 1 or of said variant, (a) each of XI and X4 is a cysteine, and (b) each of X2 and X3 is a non-cysteine amino acid. In a preferred embodiment, in the polypeptide of the invention comprising SEQ ID NO: 1, a variant thereof or a fragment of SEQ ID NO: 1 or of said variant, (a) each of XI and X4 is a non-cysteine amino acid, and (b) each of X2 and X3 is a cysteine.

In the polypeptide of the invention comprising a variant of SEQ ID NO: 1, at most 33 amino acids are exchanged in said variant as compared to SEQ ID NO: 1. In another preferred embodiment, at most 25 amino acids are exchanged in said variant as compared to SEQ ID NO: 1. In another preferred embodiment, at most 20 amino acids are exchanged in said variant as compared to SEQ ID NO : 1. In another very preferred embodiment, at most 15 amino acids are exchanged in said variant as compared to SEQ ID NO: 1. In another more preferred embodiment, at most 10 amino acids are exchanged in said variant as compared to SEQ ID NO: 1. In another preferred embodiment, at most 5 amino acids are exchanged in said variant as compared to SEQ ID NO: 1. In another preferred embodiment, at most 4 amino acids are exchanged in said variant as compared to SEQ ID NO: 1. In another preferred embodiment, at most three amino acids are exchanged in said variant as compared to SEQ ID NO: 1. In a further preferred embodiment, at most two amino acids are exchanged in said variant as compared to SEQ ID NO: 1. In a further preferred embodiment, one amino acid is exchanged in said variant as compared to SEQ ID NO: 1.

In a preferred embodiment, in said variant of SEQ ID NO: 1 or said fragment, one or more amino acids are exchanged by a fusion residue, described herein, as compared to SEQ ID NO: 1. Preferably, said fusion residue is selected from the group consisting of D-Pro-D-Pro, L- Pro-D-Pro, D-Pro-L-Pro, L-Arg-D-Pro, D-Arg-D-Arg, D-Arg-L-Arg, L-Arg-D-Arg and D- Arg-L-Pro. In another preferred embodiment, said fusion residue is selected from the group consisting of L-Pro-D-Pro, D-Pro-L-Pro, L-Arg-D-Pro, and D-Arg-L-Pro. In a preferred embodiment, in said variant one or more amino acids are exchanged by a D-amino acid, preferably D-proline or D-arginine, as compared to SEQ ID NO: 1. Preferably, 1-5, more preferably 1-3 amino acids are exchanged by the fusion residue or the D-amino acid. Preferably, amino acids exchanged by the fusion residue or the D-amino acid are located in SEQ ID NO: 1 between amino acid position (aa) 10 and 62, preferably aa 17 and 57. In another embodiment, exchanged amino acids are located in SEQ ID NO: 1 between aa 20 and 30.

Preferably, said amino acids are exchanged with a proteinogenic, non-proteinogenic or synthetic amino acids. Preferably, said amino acid exchanges are conservative exchanges.

Preferably, said polypeptide of the invention, said amino acid sequence of SEQ ID NO: 1, said variant thereof and said fragment of SEQ ID NO: 1 or of said variant of SEQ ID NO: 1 are each capable to be recognized by an host organism’s immune system, specifically by antibodies, B cells, or T cells of said host organism. Preferably, said polypeptide of the invention, said amino acid sequence of SEQ ID NO: 1, said variant of SEQ ID NO: 1, or said fragment of SEQ ID NO: 1 or of said variant of SEQ ID NO: 1 is each capable of eliciting an antibody against residues 437-508 of RBD from SARS-CoV-2 or residues 424-494 of RBD from SARS-CoV.

In a preferred embodiment, SEQ ID NO: 1, said variant or said fragment comprises at most 4 disulfide bridges, more preferably at most 3 disulfide bridges, even more preferably one or two disulfide bridges. In another preferred embodiment, SEQ ID NO: 1, said variant or said fragment comprises two disulfide bridges. In another preferred embodiment, SEQ ID NO: 1, said variant or said fragment comprises one disulfide bridge.

The term “antibody against residues ...” relates preferably to binding to the specified residues or part thereof. It can also relate to functionally or sterically blocking or inhibiting the specified residues.

The term “fragment of SEQ ID NO: 1” as used herein relates to one or more sequence stretches extracted from SEQ ID NO: 1. In the polypeptide of the invention, said fragment of SEQ ID NO: 1 or the variant thereof is at least 20 amino acids long. In a more preferred embodiment, said fragment is at least 25 amino acids long. In an again more preferred embodiment, said fragment is at least 30 amino acids long. In an even more preferred embodiment, said fragment is at least 35 amino acids long. In a most preferred embodiment, said fragment is at least 38 amino acids long. In another preferred embodiment, said fragment is at least 45 amino acids long. In a preferred embodiment, said fragment is between 20 and 100 amino acids long. In a preferred embodiment, said fragment is between 25 and 90 amino acids long. In a preferred embodiment, said fragment is between 25 and 80 amino acids long. In a preferred embodiment, said fragment is between 25 and 60 amino acids long. In a preferred embodiment, said fragment is between 25 and 50 amino acids long. In a preferred embodiment, said fragment is between 30 and 50 amino acids long.

In a preferred embodiment, said fragment of SEQ ID NO: 1 or of a variant thereof consists of a single continuous sequence stretch of SEQ ID NO: 1, or said fragment of SEQ ID NO: 1 or of a variant thereof comprises one or more, preferably two discontinuous sequence stretches of SEQ ID NO: 1 or of a variant thereof. As used herein, the expression discontinuous sequence stretches relates to sequence stretches that are separated by at least one amino acid in SEQ ID NO: 1 or in a variant thereof, i.e. said discontinuous sequence stretches are separate portions from SEQ ID NO: 1 that are separated by at least one amino acid.

In a preferred embodiment, each of said sequence stretches is between 10 and 71 amino acids long, preferably between 15 and 60 amino acids long, more preferably between 15 and 50 amino acids long, again more preferably between 14 and 45 amino acids long.

In a preferred embodiment, said fragment of SEQ ID NO: 1 or of a variant thereof consists of a single continuous or two discontinuous sequence stretches of SEQ ID NO: 1. Preferably, said single continuous sequence stretch of SEQ ID NO: 1 or of a variant thereof has a length of 25 to 71 amino acids, more preferably 30 to 50 amino acids. Preferably, each of said two discontinuous sequence stretch of SEQ ID NO: 1 or of a variant thereof has a length of 10 to 40 amino acids, more preferably 10 to 30 amino acids, again more preferably 10 to 20 amino acids.

In a preferred embodiment, said fragment of SEQ ID NO: 1 or of a variant thereof consists of a single continuous sequence stretch of SEQ ID NO: 1. Preferably, said single continuous sequence stretch of SEQ ID NO: 1 or of a variant thereof has a length of 25 to 71 amino acids.

In a preferred embodiment, the continuous sequence stretch corresponds to a stretch which starts at amino acid position 10 or higher and ends at amino acid position 65 or lower of SEQ ID NO: 1 or the variant thereof, wherein said single continuous sequence stretch of SEQ ID NO: 1 or of a variant thereof has a length of 25 to 50 amino acids. In a preferred embodiment, the continuous sequence stretch corresponds to a stretch which starts between amino acid positions 13 and 18 and ends between amino acid positions 56 and 60 of SEQ ID NO: 1 or the variant thereof. In a more preferred embodiment, the continuous sequence stretch corresponds to a stretch which starts between amino acid positions 15 and 17 and ends between amino acid positions 57 and 59 of SEQ ID NO: 1 or the variant thereof. In an again more preferred embodiment, the continuous sequence stretch corresponds to a stretch which starts at amino acid position 16 and ends at amino acid position 58 of SEQ ID NO: 1 or the variant thereof. Preferably, in said the continuous sequence stretch, amino acids at positions 15, 17, 25, 43, 44, 56, 60 and 66 are conserved.

In a preferred embodiment, the continuous sequence stretch corresponds to a stretch which starts at amino acid position 10 or higher and ends at amino acid position 65 or lower of SEQ ID NO: 1 or the variant thereof, wherein said single continuous sequence stretch of SEQ ID NO: 1 or of a variant thereof has a length of 25 to 50 amino acids and comprises X2 and X3 which are each cysteine. In a preferred embodiment, the continuous sequence stretch corresponds to a stretch which starts between amino acid positions 13 and 18 and ends between amino acid positions 56 and 60 of SEQ ID NO: 1 or the variant thereof and comprises X2 and X3 which are each cysteine. In a more preferred embodiment, the continuous sequence stretch corresponds to a stretch which starts between amino acid positions 15 and 17 and ends between amino acid positions 57 and 59 of SEQ ID NO: 1 or the variant thereof and comprises X2 and X3 which are each cysteine. In an again more preferred embodiment, the continuous sequence stretch corresponds to a stretch which starts at amino acid position 16 and ends at amino acid position 58 of SEQ ID NO: 1 or the variant thereof, and comprises X2 and X3 which are each cysteine. Preferably, in said the continuous sequence stretch, amino acids at positions 15, 17, 25, 43, 44, 56, 60 and 66 are conserved. In a preferred embodiment, said fragment of SEQ ID NO: 1 or of the variant thereof consists of a single continuous sequence stretch of SEQ ID NO: 1 or of the variant thereof wherein said continuous sequence stretch comprises at least one disulfide bridge, preferably one or two disulfide bridges, more preferably two disulfide bridges. Preferably, one of said one or two disulfide bridges is formed by an N-terminal cysteine and a C-terminal cysteine.

In a preferred embodiment, the fragment consists of a continuous sequence stretch or two or more, preferably two discontinuous sequence stretches, wherein the discontinuous sequence stretch located closest to the N terminus of SEQ ID NO: 1 (herein also called first discontinuous sequence stretch) or the continuous sequence stretch starts in SEQ ID NO: 1 or the variant thereof at amino acid position 2, preferably 10 or higher, and the discontinuous sequence stretch located closest to the C terminus of SEQ ID NO: 1 (herein also called second discontinuous sequence stretch) or the continuous sequence stretch ends at amino acid position 71, preferably 65 or lower in SEQ ID NO: 1 or the variant thereof. Preferably, said a continuous sequence stretch or said discontinuous sequence stretches comprise X2 and X3 which are each cysteine, preferably forming a disulfide bridge. In another preferred embodiment, the fragment consists of a continuous sequence stretch or two or more, preferably two discontinuous sequence stretches, wherein the discontinuous sequence stretch located closest to the N terminus of SEQ ID NO: 1 or the variant thereof or the continuous sequence stretch starts in SEQ ID NO: 1 or the variant thereof starts between amino acids at 13 and 18, and the discontinuous sequence stretch located closest to the C terminus of SEQ ID NO: 1 or the variant thereof or the continuous sequence stretch ends between amino acid positions 56 and 60 of SEQ ID NO: 1 or the variant thereof. Preferably, said a continuous sequence stretch or said discontinuous sequence stretches comprise X2 and X3 which are each cysteine. In another preferred embodiment, the fragment consists of a continuous sequence stretch or two or more, preferably two discontinuous sequence stretches, wherein the discontinuous sequence stretch located closest to the N terminus of SEQ ID NO: 1 or the variant thereof or the continuous sequence stretch starts in SEQ ID NO: 1 or the variant thereof starts between amino acids at 15 and 17, and the discontinuous sequence stretch located closest to the C terminus of SEQ ID NO: 1 or the variant thereof or the continuous sequence stretch ends between amino acid positions 57 and 59 of SEQ ID NO: 1 or the variant thereof. Preferably, said a continuous sequence stretch or said discontinuous sequence stretches comprise X2 and X3 which are each cysteine. In another preferred embodiment, the fragment consists of a continuous sequence stretch or two or more, preferably two discontinuous sequence stretches, wherein the discontinuous sequence stretch located closest to the N terminus of SEQ ID NO: 1 or the variant thereof or the continuous sequence stretch starts in SEQ ID NO: 1 or the variant thereof starts at amino acid 16, and the discontinuous sequence stretch located closest to the C terminus of SEQ ID NO: 1 or the variant thereof or the continuous sequence stretch ends at amino acid position 58 of SEQ ID NO: 1 or the variant thereof. Preferably, said a continuous sequence stretch or said discontinuous sequence stretches comprise X2 and X3 which are each cysteine. Preferably, in said continuous and discontinuous sequence stretches, amino acids at positions 15, 25, 43, 44, 56, 60 and 66 are conserved.

Preferably, said discontinuous sequence stretch each has a length of 3 to 50, more preferably 3 to 40, again more preferably 3 to 30 amino acids.

In a preferred embodiment, said fragment of SEQ ID NO: 1 or of the variant thereof consists of a single continuous or one or more, preferably two, continuous sequence stretches of SEQ ID NO: 1 or of the variant thereof, wherein said continuous or said discontinuous sequence stretches comprise at least one disulfide bridge, preferably one or two disulfide bridges, more preferably two disulfide bridges. Preferably, one of said one or two disulfide bridges is formed by an N-terminal cysteine and a C-terminal cysteine.

In a preferred embodiment, said fragment of SEQ ID NO: 1 or of a variant thereof comprises two discontinuous sequence stretches of SEQ ID NO: 1 or of a variant thereof. In a preferred embodiment, said fragment of SEQ ID NO: 1 comprises two discontinuous sequence stretches of SEQ ID NO: 1. Preferably, said fragment comprising two discontinuous sequence stretches of SEQ ID NO: 1 or of a variant thereof has a length of 25 to 71 amino acids.

In a preferred embodiment, said fragment of SEQ ID NO: 1 or of a variant thereof comprises two discontinuous sequence stretches of SEQ ID NO: 1 or of a variant thereof, wherein each of said two discontinuous sequence stretches comprises at least one cysteine, preferably 2 cysteines. Preferably, said cysteines of said two discontinuous sequence stretches form one or two disulfide bridges, more preferably one disulfide bridge. More preferably, said cysteines of said two discontinuous sequence stretches form one or two disulfide bridges, more preferably one disulfide bridge between said two discontinuous sequence stretches.

In a preferred embodiment, the first of said two different discontinuous sequence stretches starts at the N-terminus, preferably between amino acid positions 1 and 3 of SEQ ID NO: 1 or said variant, and the second ends at the C-terminus, preferably between amino acid positions 70 and 72 of SEQ ID NO: 1 or said variant, and each of said first and second stretch has a length of 10 to 30, preferably 12 to 25 amino acids. In a preferred embodiment, the first of said two different discontinuous sequence stretches starts between amino acid positions 1 and 3 and ends between amino acid positions 16 and 20 of SEQ ID NO: 1 or said variant and the second starts between amino acid positions 55 and 59 and ends between amino acid positions 70 and 72 of SEQ ID NO: 1 or said variant. In a preferred embodiment, the first of said two different discontinuous sequence stretches starts at amino acid position 1 or 2 and ends between amino acid positions 17 and 19 of SEQ ID NO: 1 or said variant and the second starts between amino acid positions 56 and 58 and ends at amino acid position 71 or 72 of SEQ ID NO: 1 or said variant. In a preferred embodiment, the first of said two different discontinuous sequence stretches starts at amino acid position 1 and ends at amino acid position 16 of SEQ ID NO: 1 or said variant and the second starts at amino acid position 57 and ends at amino acid position 72 of SEQ ID NO: 1 or said variant. Preferably, in said dis-continuous sequence stretches, amino acids at positions 15, 17, 25, 43, 44, 56, 60 and 66, preferably 15, 25, 43, 44, 56, 60 and 66 are conserved.

In a preferred embodiment, the first of said two different discontinuous sequence stretches starts at the N-terminus, preferably between amino acid positions 1 and 3 of SEQ ID NO: 1 or said variant and comprises XI, and the second ends at the C-terminus, preferably between amino acid positions 70 and 72 of SEQ ID NO: 1 or said variant and comprises X4, and each of said first and second stretch has a length of 10 to 30, preferably 12 to 25 amino acids, wherein preferably XI and X4 are each cysteine. In a preferred embodiment, the first of said two different discontinuous sequence stretches starts between amino acid positions 1 and 3 and ends between amino acid positions 16 and 20 of SEQ ID NO: 1 or said variant and comprises XI, and the second starts between amino acid positions 55 and 59 and ends between amino acid positions 70 and 72 of SEQ ID NO: 1 or said variant and comprises X4, wherein preferably XI and X4 are each cysteine. In a preferred embodiment, the first of said two different discontinuous sequence stretches starts at amino acid position 1 or 2 and ends between amino acid positions 17 and 19 of SEQ ID NO: 1 or said variant and comprises XI, and the second starts between amino acid positions 56 and 58 and ends at amino acid position 71 or 72 of SEQ ID NO: 1 or said variant and comprises X4, wherein preferably XI and X4 are each cysteine. In a preferred embodiment, the first of said two different discontinuous sequence stretches starts at amino acid position 1 and ends at amino acid position 16 of SEQ ID NO: 1 or said variant and comprises XI, and the second starts at amino acid position 57 and ends at amino acid position 72 of SEQ ID NO: 1 or said variant and comprises X4, wherein preferably XI and X4 are each cysteine. Preferably, in said the dis-continuous sequence stretches, amino acids at positions 15, 17, 25, 43, 44, 56, 60 and 66, preferably 15, 25, 43, 44, 56, 60 and 66 are conserved.

In a preferred embodiment, said fragment of SEQ ID NO: 1 or of a variant thereof comprises two discontinuous sequence stretches of SEQ ID NO: 1 or of a variant thereof, wherein the first of said two different discontinuous sequence stretches starts between amino acid positions 1 and 3 and ends between amino acid positions 16 and 20 of SEQ ID NO: 1 or said variant, and the second starts between amino acid positions 55 and 59 and ends between amino acid positions 70 and 72 of SEQ ID NO: 1 or said variant, and wherein each of XI and X4 is cysteine.

In a preferred embodiment, said fragment of SEQ ID NO: 1 or of a variant of SEQ ID NO: 1 consists of a single continuous sequence stretch of SEQ ID NO: 1 or two discontinuous sequence stretches, wherein the continuous sequence stretch starts between amino acid positions 13 and 18 and ends between amino acid positions 56 and 60 of SEQ ID NO: 1 or the variant thereof, and the first of said two discontinuous sequence stretches starts between amino acid positions 1 and 3 and ends between amino acid positions 16 and 20 of SEQ ID NO: 1 or said variant and the second of said two discontinuous sequence stretches starts between amino acid positions 55 and 59 and ends between amino acid positions 70 and 72 of SEQ ID NO: 1 or said variant.

In a preferred embodiment, said discontinuous sequence stretches of SEQ ID NO: 1 or of a variant thereof are covalently linked, either directly or more preferably by a fusion residue. In a preferred embodiment, said fusion residue comprises one or more amino acids, more preferably one or two amino acids.

In a very preferred embodiment, said fusion residue comprises at least one D-amino acid (herein written in lower case), more preferably one or two D-amino acids, even more preferably one D-amino acid.

In another preferred embodiment, said fusion residue comprises an amino acid selected from the group consisting of arginine (L-arginine, R), D-arginine (r), proline (L-proline, P), D- proline (p) and a mixture thereof. In a preferred embodiment, said fusion residue comprises D- arginine or D-proline or a mixture thereof. In a preferred embodiment, said fusion residue comprises or preferably consists of arginine (L-arginine) and D-proline. In a more preferred embodiment, said fusion residue comprises or preferably consists of D-arginine and proline (L- proline). In another preferred embodiment, said fusion residue comprises one or more proline or on or more arginine residues or a mixture of both; preferably 1-5, more preferably 1-3, again more preferably 2 proline or arginine residues or a mixture of both. Preferably, one of said one or more proline or arginine residues, is a D-amino acid and the other is an L-amino acid. Preferably, said fusion residue comprises, preferably consists of, (i) a D-proline covalently linked to an L-arginine or L-proline, or (ii) a D-arginine covalently linked to an L-arginine or L-proline. In another preferred embodiment, said fusion residue is selected from the group consisting of D-Pro-D-Pro, L-Pro-D-Pro, D-Pro-L-Pro, L-Arg-D-Pro, D-Arg-D-Arg, D-Arg-L- Arg, L-Arg-D-Arg and D-Arg-L-Pro. In another preferred embodiment, said fusion residue is selected from the group consisting of L-Pro-D-Pro, D-Pro-L-Pro, L-Arg-D-Pro, and D-Arg-L- Pro. In a preferred embodiment of the polypeptide of the invention, an amino acid linker is located at or attached to the N-terminus of SEQ ID NO: 1, of said variant or of said fragment. In a preferred embodiment, said N terminal linker is an N terminal amino acid linker comprising or preferably consisting of one or more consecutively and covalently linked amino acids. The amino acids of said N terminal amino acid linker are selected from the group consisting of glycine, proline, amino acids with a polar uncharged side chain, amino acids with a hydrophobic side chain and mixtures thereof. In a further preferred embodiment, said N terminal amino acid linker is a glycine linker, a linker consisting of amino acids each having a hydrophobic side chain or mixtures thereof. In a preferred embodiment, said N terminal amino acid linker consists of 1 to 6 amino acids, preferably 1 to 3 amino acids, more preferably 1 to 2 amino acids. In a further preferred embodiment, said N terminal amino acid linker consists of 1 to 6, preferably 1 to 3, more preferably 1 or 2 amino acids, wherein said amino acids are selected from the group consisting of glycine, proline, amino acids with a polar uncharged side chain, amino acids with a hydrophobic side chain and mixtures thereof. In a further preferred embodiment said N terminal amino acid linker consists of 1 to 3 or 1 or 2 amino acids, wherein said amino acids are selected from the group consisting of glycine, serine, tryptophan, tyrosine and mixtures thereof.

In a preferred embodiment of the polypeptide of the invention, an amino acid linker is located at or attached to the C-terminus of SEQ ID NO: 1, of said variant or of said fragment. In another preferred embodiment, said C terminal linker is a C terminal amino acid linker consisting of one or more consecutively and covalently linked amino acids. In a further preferred embodiment, said C terminal linker comprises amino acids with a positively charged side chain. In a further preferred embodiment, said C terminal linker comprises arginine. In a preferred embodiment, said C terminal amino acid linker consists of 1 to 6 amino acids, preferably 1 to 3 amino acids, more preferably 1 to 2 amino acids. In a preferred embodiment, said C terminal amino acid linker consists of 1 to 6 amino acids, preferably 1 to 3 amino acids, more preferably 1 to 2 amino acids and comprises amino acids with a positively charged side chain, preferably arginine. In a preferred embodiment, said C terminal linker consists of 1 or 2 amino acids comprising arginine. In another preferred embodiment, said C terminal linker consists of an arginine.

In a preferred embodiment, the polypeptide of the invention has a length of at least 25 amino acids, more preferably at least 50 amino acids, again more preferably at least 75 amino acids, again more preferably at least 100 amino acids. In another preferred embodiment, the polypeptide of the invention has a length of 25 to 250 amino acids, more preferably 25 to 200 amino acids, again more preferably 25 to 150 amino acids, again more preferably 25 to 120 amino acids, again more preferably 25 to 100, again more preferably 25 to 90 amino acids, again more preferably 25 to 80 amino acids, amino acids, even more preferably 25 to 75 amino acids. In a preferred embodiment, SEQ ID NO: 1 is a derivative of RBD from SARS-CoV-2, wherein at least two non-native Cys residues forming a disulfide bridge are introduced at amino acid positions 452 and 494 and/or 437 and 508 of SARS-CoV-2. In one embodiment, two amino acid stretches are extracted from said derivative and newly combined and fused by a fusion residues, preferably a dipeptide comprising a D-amino acid.

In a preferred embodiment, SEQ ID NO: 1 or the variant of SEQ ID NO: 1 consists of a stretch from amino acid position 437 to amino acid position 508 of spike glycoprotein of SARS-CoV-2 or from amino acid position 424 to amino acid position 494 of spike glycoprotein of SARS-CoV. In a preferred embodiment, SEQ ID NO: 1 or the variant of SEQ ID NO: 1 comprises or preferably consists of a stretch from amino acid position (aa) 437 to 508 or aa 452 to 494 of spike glycoprotein of SARS-CoV-2 or from aa 424 to 494 or aa 439 to 480 of spike glycoprotein of SARS-CoV, based on the sequences as depicted in Fig. 1. Optionally, amino acids are deleted in said stretches, wherein preferably the deleted amino acids are exchanged by the fusion residue described herein. Preferably, the deleted amino acids are located between aa 452 to 494 of spike glycoprotein of SARS-CoV-2 or between aa 439 to 480 of spike glycoprotein of SARS-CoV. Preferably, the number of deleted amino acids is between 0 and 41, preferably between 10 and 41, more preferably between 20 and 41, again more preferably between 30 and 41, again more preferably between 35 and 41, most preferably 39.

In a preferred embodiment, SEQ ID NO: 1 or the variant of SEQ ID NO: 1 consists of a stretch from amino acid position 437 to amino acid position 508 of spike glycoprotein of SARS-CoV-2 or from amino acid position 424 to amino acid position 494 of spike glycoprotein of SARS-CoV, wherein in said stretch the first and last amino acid have been exchanged each by one cysteine. In a preferred embodiment, SEQ ID NO: 1 or the variant of SEQ ID NO: 1 comprises or preferably consists of a stretch from aa 437 to 508 or aa 452 to 494 of spike glycoprotein of SARS-CoV-2, based on the sequences as depicted in Fig. 1, wherein in said stretch aa 437 and 508 and/or aa 452 and 494 have been exchanged each by one cysteine. Optionally, amino acids are deleted in said stretch and preferably exchanged by the fusion residue, wherein preferably, the deleted amino acids are located between aa 452 to 494. Preferably, the number of deleted amino acids is between 0 and 41, preferably between 10 and 41, more preferably between 20 and 41, again more preferably between 30 and 41, again more preferably between 35 and 41, most preferably 39. Preferably, said two cysteines inserted at aa 437 and 508 form a disulfide bridge, and/or said two cysteines inserted at aa 452 and 494 form a disulfide bridge, if included. In a preferred embodiment, SEQ ID NO: 1 or the variant of SEQ ID NO: 1 comprises or preferably consists of a stretch from aa 424 to 494 or aa 439 to 480 of spike glycoprotein of SARS-CoV, based on the sequences as depicted in Fig. 1, wherein in said stretch aa 424 and 494 and/or aa 439 and 480 have been exchanged each by one cysteine. Preferably, said two cysteines inserted at aa 424 and 494 form a disulfide bridge and/or said two cysteines inserted at aa 439 and 480 form a disulfide bridge, if included. Optionally, amino acids are deleted in said stretches and preferably exchanged by the fusion residue, as described in the paragraph above.

In a preferred embodiment, SEQ ID NO: 1 or the variant of SEQ ID NO: 1 consists of a stretch from amino acid position 437 to amino acid position 508 of spike glycoprotein of SARS-CoV- 2 or from amino acid position 424 to amino acid position 494 of spike glycoprotein of SARS- CoV, and the fragment of said stretch SEQ ID NO: 1 or the fragment of said stretch variant of SEQ ID NO: 1 is at least 20 amino acids long, preferably between 25 and 80 amino acids long. In a preferred embodiment, SEQ ID NO: 1 or the variant of SEQ ID NO: 1 consists of a stretch from amino acid position 437 to amino acid position 508 of spike glycoprotein of SARS-CoV- 2 or from amino acid position 424 to amino acid position 494 of spike glycoprotein of SARS- CoV, wherein in said stretch the first and last amino acid have been exchanged each by one cysteine, and the fragment of said stretch SEQ ID NO: 1 or the fragment of said stretch variant of SEQ ID NO: 1 is at least 20 amino acids long, preferably between 25 and 80 amino acids long. In another preferred embodiment, SEQ ID NO: 1 or the variant of SEQ ID NO: 1 consists of a stretch from aa 437 to 508 or aa 452 to 494 of spike glycoprotein of SARS-CoV-2 or from aa 424 to 494 or aa 439 to 480 of spike glycoprotein of SARS-CoV, and the fragment of said stretch of SEQ ID NO: 1 or the fragment of said stretch of variant of SEQ ID NO: 1 is at least 20 amino acids long, preferably between 25 and 72 amino acids long. Preferably, in said stretch from SARS-CoV-2 aa 437 and 508 and/or aa 452 and 494 have been exchanged each by one cysteine. Preferably, said two cysteines inserted at aa 437 and 508 form a disulfide bridge, and/or said two cysteines inserted at aa 452 and 494 form a disulfide bridge, if included. In a preferred embodiment, SEQ ID NO: 1 or the variant of SEQ ID NO: 1 consists of a Preferably, in said SARS-CoV stretch aa 424 and 494 and/or aa 439 and 480 have been exchanged each by one cysteine. Preferably, said two cysteines inserted at aa 424 and 494 form a disulfide bridge and/or said two cysteines inserted at aa 439 and 480 form a disulfide bridge, if included. Optionally, amino acids are deleted in said stretches and preferably exchanged by the fusion residue, wherein the deleted amino acids are preferably located between aa 439 to 480 of spike glycoprotein of SARS-CoV and between 452 and 494 of SARS-CoV-2. Preferably, the number of deleted amino acids is between 0 and 41, preferably between 10 and 41, more preferably between 20 and 41, again more preferably between 30 and 41, again more preferably between 35 and 41, most preferably 39. In a preferred embodiment, the variant of SEQ ID NO: 1 consists of a stretch from amino acid position 437 to amino acid position 508 of spike glycoprotein of SARS-CoV-2 or from amino acid position 424 to amino acid position 494 of spike glycoprotein of SARS-CoV, wherein amino acids at positions 15, 17, 25, 43, 44, 56, 60 and 66, preferably 15, 25, 43, 44, 56, 60 and 66 of each of said stretches are conserved in said stretch variant. In another preferred embodiment, the variant of SEQ ID NO: 1 consists of a stretch from amino acid position 437 to amino acid position 508 of spike glycoprotein of SARS-CoV-2 or from amino acid position 424 to amino acid position 494 of spike glycoprotein of SARS-CoV, wherein amino acids at positions 4, 14, 15, 17, 25, 29, 31, 32, 43, 44, 56, 60, 61, 64 and 66, preferably 4, 14, 15, 25, 29, 31, 32, 43, 44, 56, 60, 61, 64 and 66 of each of said stretches are conserved in said variant. In a preferred embodiment, the variant of SEQ ID NO: 1 consists of a stretch from amino acid position 437 to amino acid position 508 of spike glycoprotein of SARS-CoV-2 or from amino acid position 424 to amino acid position 494 of spike glycoprotein of SARS-CoV, wherein amino acids at positions 4, 6, 11-15, 17, 18, 21, 25, 27-33, 43, 44, 51- 53, 55, 56, 59, 60, 61, 64, 66 and 68-71, preferably 4, 6, 11-15, 18, 21, 25, 27-33, 43, 44, 51- 53, 55, 56, 59, 60, 61, 64, 66 and 68-71 of each of said stretches are conserved in said variant.

In a preferred embodiment, the variant of SEQ ID NO: 1 consists of a stretch from amino acid position 437 to amino acid position 508 of spike glycoprotein of SARS-CoV-2 or from amino acid position 424 to amino acid position 494 of spike glycoprotein of SARS-CoV, wherein amino acids at positions 15, 17, 25, 43, 44, 56, 60 and 66, preferably 15, 25, 43, 44, 56, 60 and 66 of each of said stretches are conserved in said variant, wherein in said stretch the first and last amino acid have been exchanged each by one cysteine. In another preferred embodiment, the variant of SEQ ID NO: 1 consists of a stretch from amino acid position 437 to amino acid position 508 of spike glycoprotein of SARS-CoV-2 or from amino acid position 424 to amino acid position 494 of spike glycoprotein of SARS-CoV, wherein amino acids at positions 4, 14, 15, 17, 25, 29, 31, 32, 43, 44, 56, 60, 61, 64 and 66, preferably 4, 14, 15, 25, 29, 31, 32, 43, 44, 56, 60, 61, 64 and 66, of each of said stretches are conserved in said variant, wherein in said stretch the first and last amino acid have been exchanged each by one cysteine. In a preferred embodiment, the variant of SEQ ID NO: 1 consists of a stretch from amino acid position 437 to amino acid position 508 of spike glycoprotein of SARS-CoV-2 or from amino acid position 424 to amino acid position 494 of spike glycoprotein of SARS-CoV, wherein amino acids at positions 4, 6, 11-15, 17, 18, 21, 25, 27-33, 43, 44, 51-53, 55, 56, 59, 60, 61, 64, 66 and 68-71, preferably 4, 6, 11-15, 18, 21, 25, 27-33, 43, 44, 51-53, 55, 56, 59, 60, 61, 64, 66 and 68-71 of each of said stretches are conserved in said variant, wherein in said stretch the first and last amino acid have been exchanged each by one cysteine.

In a preferred embodiment, said variant of amino acid sequence of SEQ ID NO: 1 of the polypeptide of the invention consist of a sequence having a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80%, again more preferably at least 90%, even more preferably 95%, even more preferably 99% with SEQ ID NO: 1. In a preferred embodiment, said variant of amino acid sequence of SEQ ID NO: 1 of the polypeptide of the invention consist of a sequence having a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80%, again more preferably at least 90%, even more preferably 95%, even more preferably 99% with a sequence selected from the group consisting of SEQ ID NO: 2-7. In a preferred embodiment, said variant of amino acid sequence of SEQ ID NO: 1 of the polypeptide of the invention consist of a sequence having a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80%, again more preferably at least 90%, even more preferably 95%, even more preferably 99% with a sequence selected from the group consisting of SEQ ID NO: 2-7 and 51-54.

In a preferred embodiment, said polypeptide of the invention consists of an amino acid sequence of SEQ ID NO: 1.

In a preferred embodiment, SEQ ID NO: 1 is a sequence selected from the group consisting of SEQ ID NO: 2-7 and 51-54, preferably SEQ ID NO: 5-7, 53 and 54. In a preferred embodiment, SEQ ID NO: 1 is SEQ ID NO: 5 or 54. In a preferred embodiment, said polypeptide comprises an amino acid sequence of SEQ ID NO: 1, wherein SEQ ID NO: 1 is selected from the group consisting of SEQ ID NO: 2-7 and 51-54, preferably SEQ ID NO: 5-7, 53 and 54. In a preferred embodiment, said polypeptide comprises an amino acid sequence of SEQ ID NO: 1, wherein SEQ ID NO: 1 is SEQ ID NO: 5 or 54. In a preferred embodiment, said polypeptide consists of an amino acid sequence of SEQ ID NO: 1, wherein SEQ ID NO: 1 is selected from the group consisting of SEQ ID NO: 2-7 and 51-54 preferably SEQ ID NO: 5-7, 53 and 54. In a preferred embodiment, said polypeptide consists of an amino acid sequence of SEQ ID NO: 1, wherein SEQ ID NO: 1 is SEQ ID NO: 5 or 54. Table 1: Amino acid sequences included in the polypeptide

In a preferred embodiment, SEQ ID NO: 1 or the fragment of SEQ ID NO: l is a sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6 and 7 and said variant of SEQ ID NO: 1 or of the fragment of SEQ ID NO: l is a variant of a sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6 and 7, wherein in said variant at most 33 amino acids are exchanged as compared to SEQ ID NO: 2, 3, 4, 5, 6 and 7, respectively. In another preferred embodiment, at most 25 amino acids are exchanged in said variant of each of SEQ ID NO: 2, 3, 4, 5, 6 or 7 or in a fragment thereof, as compared to SEQ ID NO: 2, 3, 4, 5, 6 and 7, respectively. In another preferred embodiment, at most 20 amino acids are exchanged in said variant of each of SEQ ID NO: 2, 3, 4, 5, 6 or 7 or in a fragment thereof (i.e. a fragment of SEQ ID NO: 2, 3, 4, 5, 6 or 7), as compared to SEQ ID NO: 2, 3, 4, 5, 6 and 7, respectively. In another preferred embodiment, at most 15 amino acids are exchanged in said variant of each of SEQ ID NO: 2, 3, 4, 5, 6 or 7 or in a fragment thereof as compared to SEQ ID NO: 2, 3, 4, 5, 6 and 7, respectively. In another preferred embodiment, at most 10 amino acids are exchanged in said variant of each of SEQ ID NO: 2, 3, 4, 5, 6 or 7 or in a fragment thereof as compared to SEQ ID NO: 2, 3, 4, 5, 6 and 7, respectively. In another preferred embodiment, at most 5 amino acids are exchanged in said variant of each of SEQ ID NO: 2, 3, 4, 5, 6 or 7 or in a fragment thereof, as compared to SEQ ID NO: 2, 3, 4, 5, 6 and 7. In another preferred embodiment, at most 4 amino acids are exchanged in said variant as compared to SEQ ID NO: 2, 3, 4, 5, 6 and 7, respectively. In another preferred embodiment, at most three amino acids are exchanged in said variant of each of SEQ ID NO: 2, 3, 4, 5, 6 or 7 or in a fragment thereof, as compared to SEQ ID NO: 2, 3, 4, 5, 6 and 7, respectively. In a further preferred embodiment, at most two amino acids are exchanged in said variant of each of SEQ ID NO: 2, 3, 4, 5, 6 or 7 or in a fragment thereof, as compared to SEQ ID NO: 2, 3, 4, 5, 6 and 7, respectively. In a further preferred embodiment, one amino acid is exchanged in said variant of each of SEQ ID NO: 2, 3, 4, 5, 6 or 7 or in a fragment thereof, as compared to SEQ ID NO: 2, 3, 4, 5, 6 and 7, respectively.

More preferably, in said variant of SEQ ID NO: 2 amino acids at positions 15, 17, 25, 43, 44, 56, 60 and 66 are conserved, in said variant of SEQ ID NO: 3 amino acids at positions 15, 17, 20 and 26 are conserved, in said variant of SEQ ID NO: 4 amino acids at positions 2, 10, 28, 29, and 41 are conserved, in said variant of SEQ ID NO: 5 amino acids at positions 17, 19, 27, 45, 46, 58, 62 and 68 are conserved, in said variant of SEQ ID NO: 6 amino acids at positions 17, 19, 22 and 28 are conserved, in said variant of SEQ ID NO: 7 amino acids at positions 3, 11, 29, 30, and 42 are conserved.

In a preferred embodiment, SEQ ID NO: 1, the fragment of SEQ ID NO: 1 is a sequence selected from the group consisting of SEQ ID NO: 2-7 and 51-54, and said variant of SEQ ID NO: 1 or of the fragment of SEQ ID NO: 1 is a variant of SEQ ID NO: 2-7 and 51-54, wherein in said variant at most 33 amino acids are exchanged as compared to SEQ ID NO: 2-7 and 51- 54, respectively. In another preferred embodiment, at most 25 amino acids are exchanged in said variant as compared to SEQ ID NO: 2-7 and 51-54, respectively. In another preferred embodiment, at most 20 amino acids are exchanged in said variant as compared to SEQ ID NO: 2-7 and 51-54, respectively. In another preferred embodiment, at most 15 amino acids are exchanged in said variant as compared to SEQ ID NO: 2-7 and 51-54, respectively. In another preferred embodiment, at most 10 amino acids are exchanged in said variant as compared to SEQ ID NO: 2-7 and 51-54, respectively. In another preferred embodiment, at most 5 amino acids are exchanged in said variant as compared to SEQ ID NO: 2-7 and 51-54. In another preferred embodiment, at most 4 amino acids are exchanged in said variant as compared to SEQ ID NO: 2-7 and 51-54, respectively. In another preferred embodiment, at most three amino acids are exchanged in said variant as compared to SEQ ID NO: 2-7 and 51-54, respectively. In a further preferred embodiment, in said variant at most two amino acids are exchanged as compared to SEQ ID NO: 2-7 and 51-54, respectively. In a further preferred embodiment, one amino acid is exchanged in said variant as compared to SEQ ID NO: 2-7 and 51-54, respectively.

More preferably, in said variant of SEQ ID NO: 2 amino acids at positions 15, 17, 25, 43, 44, 56, 60 and 66 are conserved; in said variant of SEQ ID NO: 3 amino acids at positions 15, 17, 20 and 26 are conserved; in said variant of SEQ ID NO: 4 amino acids at positions 2, 10, 28, 29, and 41 are conserved; in said variant of SEQ ID NO: 5 amino acids at positions 17, 19, 27, 45, 46, 58, 62 and 68 are conserved; in said variant of SEQ ID NO: 6 amino acids at positions 17, 19, 22 and 28 are conserved; in said variant of SEQ ID NO: 7 amino acids at positions 3, 11, 29, 30, and 42 are conserved; in said variant of SEQ ID NO: 51 amino acids at positions 17, 19, 22, 28 are conserved; in said variant of SEQ ID NO: 52 amino acids at positions 2, 4, 12, 30, 31, 43, 46 are conserved; in said variant of SEQ ID NO: 53 (mimetic 4) amino acids at positions 15, 17, 20, 26 are conserved; and in said variant of SEQ ID NO: 54 (mimetic 5) amino acids at positions 1, 3, 11, 29, 30, 42, 45 are conserved.

In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1 consist of a sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 7. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1, the variants of SEQ ID NO: 1 and the fragments of SEQ ID NO: 1 and of said variants of SEQ ID NO: 1 each consists of a sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 7. In a preferred embodiment, said SEQ ID NO: 1 consist of a sequence selected from the group consisting of SEQ ID NO: 2 to 7 and 51 to 54, preferably SEQ ID NO: 5-7, 53 and 54. In another preferred embodiment, said amino acid sequence of SEQ ID NO: 1, the variants of SEQ ID NO: 1 and the fragments of SEQ ID NO: 1 and of said variants of SEQ ID NO: 1, each consists of a sequence selected from the group consisting of SEQ ID NO: 2 to 7 and 51 to 54, preferably SEQ ID NO: 5-7, 53 and 54. In another preferred embodiment, said amino acid sequence of SEQ ID NO: 1, said variants and fragments, each consists of a sequence of SEQ ID NO: 5 or 54. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1 and the fragments of SEQ ID NO: 1, each consists of a sequence selected from the group consisting of SEQ ID NO: 2 to 7 and 51-54, preferably SEQ ID NO: 5-7, 53 and 54, and in the variants of SEQ ID NO: 1 or of the fragments of SEQ ID NO: 1 at most 33 amino acids are exchanged as compared to the corresponding non-modified sequences. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1 and the fragments of SEQ ID NO: 1, each consists of a sequence selected from the group consisting of SEQ ID NO: 2 to 7 and 51-54, preferably SEQ ID NO: 5 or 54, and in the variants at most 33 amino acids are exchanged as compared to the corresponding non-modified sequences.

In a preferred embodiment, (i) SEQ ID NO: 1 and (ii) the variant of SEQ ID NO: 1 each consists of SEQ ID NO: 2 or 5; and (iii) the fragment of SEQ ID NO: 1 and the fragment of said variant of SEQ ID NO: 1 each consists of SEQ ID NO: 3, 4, 6 or 7. In a preferred embodiment, (i) SEQ ID NO: 1 and (ii) the variant of SEQ ID NO: 1 each comprises SEQ ID NO: 2 or 5; and (iii) the fragment of SEQ ID NO: 1 and the fragment of said variant of SEQ ID NO: 1 each comprises SEQ ID NO: 3, 4, 6 or 7.

In a preferred embodiment, SEQ ID NO: 1 consists of SEQ ID NO: 2 or 5, and the fragment of SEQ ID NO: 1 consists of a sequence selected from the group consisting of SEQ ID NO: 3, 4, 6 and 7, wherein in the variant of SEQ ID NO: 1 and in the fragment of the variant of SEQ ID NO: 1 at most 33 amino acids are exchanged as compared to the corresponding non- modified SEQ ID NO: 2, 3, 4, 5, 6 or 7. In a preferred embodiment, SEQ ID NO: 1 consists of SEQ ID NO: 2 or 5, or the fragment of SEQ ID NO: 1 consists of a sequence selected from the group consisting of SEQ ID NO: 3, 4, 6 and 7, wherein in the variant of SEQ ID NO: 1 and in the fragment of the variant of SEQ ID NO: 1 at most 33 amino acids are exchanged as compared to the corresponding non-modified SEQ ID NO: 2, 3, 4, 5, 6 or 7. In a preferred embodiment, SEQ ID NO: 1 consists of SEQ ID NO: 2 or 5, wherein in the variant of SEQ ID NO: 1 at most 33 amino acids are exchanged as compared to the corresponding non-modified SEQ ID NO: 2 or 5. In a preferred embodiment, the fragment of SEQ ID NO: 1 consists of a sequence selected from the group consisting of SEQ ID NO: 3, 4, 6 and 7, wherein in the fragment of the variant of SEQ ID NO: 1 at most 33 amino acids are exchanged as compared to the corresponding non- modified SEQ ID NO: 3, 4, 6 or 7.

In a preferred embodiment, SEQ ID NO: 1 and the variant of SEQ ID NO: 1 each consists of a sequence SEQ ID NO: 2 or 5, and the fragment of SEQ ID NO: 1 and the fragment of the variant of SEQ ID NO: 1 each consists of a sequence selected from the group consisting of SEQ ID NO: 3, 4, 6 and 7. In a preferred embodiment, SEQ ID NO: 1 and the variant of SEQ ID NO: 1 each consists of a sequence SEQ ID NO: 2 or 5, and the fragment of SEQ ID NO: 1 and the fragment of the variant of SEQ ID NO: 1 each consists of a sequence selected from the group consisting of SEQ ID NO: 3, 4, 6 and 7, wherein none amino acid is exchanged in the variant of SEQ ID NO: 1 as compared to SEQ ID NO: 1.

In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1, the variants of SEQ ID NO: 1 and the fragments of SEQ ID NO: 1 and of said variants of SEQ ID NO: 1 each consists of a sequence selected from the group consisting of SEQ ID NO: 2, 3 and 4. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1, the variants of SEQ ID NO: 1 and the fragments of SEQ ID NO: 1 and of said variants of SEQ ID NO: 1 each consists of a sequence selected from the group consisting of SEQ ID NO: 2, 3 and 4. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1, said fragments or variants each consists of SEQ ID NO: 2. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1, the variants of SEQ ID NO: 1 and the fragments of SEQ ID NO: 1 and of said variants of SEQ ID NO: 1 each consists of SEQ ID NO: 3. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1, the variants of SEQ ID NO: 1 and the fragments of SEQ ID NO: 1 and of said variants of SEQ ID NO: 1 each consists of SEQ ID NO: 4 In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1, the variants of SEQ ID NO: 1 and the fragments of SEQ ID NO: 1 and of said variants of SEQ ID NO: 1 each consists of SEQ ID NO: 5. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1, the variants of SEQ ID NO: 1 and the fragments of SEQ ID NO: 1 and of said variants of SEQ ID NO: 1 each consists of SEQ ID NO: 6. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1, said fragments or variants each consists of SEQ ID NO: 7. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1, said fragments or variants each consists of SEQ ID NO: 51. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1, said fragments or variants each consists of SEQ ID NO: 52. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1, said fragments or variants each consists of SEQ ID NO: 53. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1, said fragments or variants each consists of SEQ ID NO: 54.

In a preferred embodiment, the polypeptide of the invention consists of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 7. In another preferred embodiment, the polypeptide of the invention consists of a sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 7. In another preferred embodiment, the polypeptide of the invention consists of a sequence selected from the group consisting of SEQ ID NO: 5, 6 or 7. In a preferred embodiment, the polypeptide of the invention consists of SEQ ID NO: 1. In a preferred embodiment, the polypeptide of the invention consists of a sequence selected from the group consisting of SEQ ID NO: 1 to 7 and 51-54. In another preferred embodiment, the polypeptide of the invention consists of a sequence selected from the group consisting of SEQ ID NO: 2 to 7 and 51-54. In another preferred embodiment, the polypeptide of the invention consists of a sequence selected from the group consisting of SEQ ID NO: 5, 6, 7, 53 and 54. In another preferred embodiment, the polypeptide of the invention consists of a sequence of SEQ ID NO: 5 or 54.

In a preferred embodiment, the polypeptide of the invention consists of SEQ ID NO: 1.

In a preferred embodiment, the polypeptide of the invention comprises a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 7. In another preferred embodiment, the polypeptide of the invention comprises a sequence each selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 7. In another preferred embodiment, the polypeptide of the invention comprises a sequence each selected from the group consisting of SEQ ID NO: 5, 6 or 7. In a preferred embodiment, the polypeptide of the invention comprises a sequence selected from the group consisting of SEQ ID NO: 2-7 and 51-54. In another preferred embodiment, the polypeptide of the invention comprises a sequence each selected from the group consisting of SEQ ID NO: 2-7 and 51-54. In another preferred embodiment, the polypeptide of the invention comprises a sequence each selected from the group consisting of SEQ ID NO: 5, 6, 7, 53 and 54. In another preferred embodiment, the polypeptide of the invention comprises a sequence of SEQ ID NO: 5 or 54.

In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1 consists of a sequence selected from the group consisting of SEQ ID NO: 2-7, and the C-terminus of the polypeptide of the invention is either an amide (CONEh) or a free carboxyl terminus, more preferably CONEh. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1 consists of a sequence selected from the group consisting of SEQ ID NO: 2-7, wherein the C- terminus of the sequence of SEQ ID NO: 1 is an amide (CONEh). In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1 consists of a sequence selected from the group consisting of SEQ ID NO: 2-7 and 51-54, and the C-terminus of the polypeptide of the invention is either an amide (CONEh) or a free carboxyl terminus, more preferably CONEh. In a preferred embodiment, said amino acid sequence of SEQ ID NO: 1 consists of a sequence selected from the group consisting of SEQ ID NO: 2-7 and 51-54, wherein the C-terminus of the sequence of SEQ ID NO: 1 is an amide (CONH₂).

It will be apparent to the person skilled in the art that, for attaching the polypeptide of the invention to the peptide moiety, a large variety of suitable moieties and strategies exist, which include but are not limited to attachment based on dicarboxylic acid residues, residues containing one or multiple ethylene glycol units, amino acid residues (including alpha-, beta-, gamma-, omega-amino acids), or sugar (carbohydrate) units, or residues containing heterocyclic rings.

Attachment residue

In a preferred embodiment, said polypeptide comprising amino acid sequence of SEQ ID NO: 1, or a variant of SEQ ID NO: 1, or a fragment of SEQ ID NO: 1 or of the variant of SEQ ID NO: 1 further comprises an attachment residue, which is attached to the N-terminus of the polypeptide of the invention. Said attachment residues can be used for attaching the polypeptide of the invention to a lipopeptide building block (LBB).

In a preferred embodiment, said attachment residue could be a fatty acid of formula N₃- CH₂(CH₂)_nCOOH or N₃-CH₂(CH₂)_nCHNH₂COOH wherein n is from 0 to 10, or said attachment residue could be a PEG moiety of formula N3- CH₂CH₂0(CH₂CH₂0)_m(CH₂)_pC00H, wherein m is from 1 to 30 and p is from 1 to 20, preferably m is from 1 to 24 and p is from 1 to 16, more preferably m is from 5 to 20 and p is from 5 to 10. In a very preferred embodiment, said attachment residue could be a fatty acid of formula N3-CH₂(CH₂)_nCOOH or N3-CH₂(CH₂)_nCHNH₂COOH wherein n is from 0 to 10, or said attachment residue could be a PEG moiety of formula N3- CH₂CH₂0(CH₂CH₂0)_m(CH₂)_pC00H, wherein m is from 1 to 24 and p is from 1 to 16.

Preferably, said attachment residue comprises or preferably consist of a synthetic amino acid comprising an azido (-N3) group. In a preferred embodiment, said azido group is incorporated into a side chain of an amino acid of the polypeptide of the invention. In a preferred embodiment, the -NEE group of an amino acid side chain is substituted by an azido group. A lipopeptide building block including a coupling moiety can be conjugated to the polypeptide of the invention through the side chain of the azidolysine residue Z (Lys(Ns)) at the N-terminus of the polypeptide.

In a preferred embodiment, the -NEE group of lysine is substituted by an azido group. In a more and very preferred embodiment, said attachment residue is L-azidolysine (L-Lys(N₃)) (fV)-2-Amino-6-azidohexanoic acid), having the following formula, mentioned herein as Z residue. Preferably, said Z is attached to the N-terminus of the polypeptide of the invention, preferably via a peptide bond.

L- Lys(N₃) o

Z

In a preferred embodiment, said polypeptide of the invention comprises an attachment residue attached to the N-terminus of a sequence selected from the group consisting of SEQ ID NO: 2-7 and 51-54. In a preferred embodiment, said polypeptide of the invention comprises, preferably consists of an attachment residue and a sequence selected from the group consisting of SEQ ID NO: 5, 6, 7, 53 and 54, wherein said attachment residue is attached to the N-terminus of one of the sequences selected from the group consisting of SEQ ID NO: 5, 6, 7, 53 and 54. More preferably, said attachment residue is said Z residue.

Thus, in a very preferred embodiment, said polypeptide of the invention comprises an attachment residue Z which is attached to the N-terminus of a sequence selected from the group consisting of SEQ ID NO: 2-7 and 51-54. In a more preferred embodiment, said polypeptide of the invention comprises, preferably consists of SEQ ID NO: 5, 6, 7, 53 and 54, wherein the attachment residue Z is attached to the N-terminus of each of said sequences SEQ ID NO: 5, 6, 7, 53 or 54. In a preferred embodiment, said polypeptide of the invention comprises or preferably consists of a formula selected from the group consisting of Z-mimetic-1, Z-mimetic- 2, Z-mimetic-3, Z-mimetic-4, Z-mimetic-5, wherein Z is L-azidolysine (L-Lys(N3)):

Z-mimetic-1

Z-GWCSNNLDSKVGGNYNYLYrPQSYGFGPTNGVGYQPCR-NH₂

Z-mimetic-2

Z-GWGSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPCR-NHa

Z-mimetic-3 I I

Z-GWCSNNLDSKVGGNYNYCY-^DPro-^LPro-QCYGFQPTNGVGYQPCR-NH₂

I _ J

Z-mimetic-4

I I

Z-KCERLFRKSNLK-^LPro-^DPro-ERDISTEIYQAGSTPCNGVEGFNCYFPLQCR-NH₂

! _ I

Z-mimetic-5

In a preferred embodiment, said polypeptide of the invention comprises or preferably consists of Z-mimetic-3 or Z-mimetic-5.

In further aspect, the polypeptide of the invention comprises a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 7 or SEQ ID NO: 1-7 and 51-54. In another preferred embodiment, the polypeptide of the invention comprises a sequence, each selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 7 or SEQ ID NO: 2-7 and 51-54. In another preferred embodiment, the polypeptide of the invention comprises a sequence each selected from the group consisting of SEQ ID NO: 5, 6, 7, 53, and 54. In another preferred embodiment, the polypeptide of the invention comprises a sequence of SEQ ID NO: 5 or 54. In a preferred embodiment, the polypeptide of the invention comprises SEQ ID NO: 1.

In a further aspect, the invention relates to a nucleic acid molecule encoding the polypeptide of the invention. In a further aspect, the invention relates to a vector comprising at least one nucleic acid molecule according to the invention.

In a further aspect, the invention relates to a host comprising at least one vector according to the invention. Preferably, said host is a cell, more preferably a bacterial or mammalian cell.

In a further aspect, the invention relates to a conjugate comprising

(i) the polypeptide of the invention,

The covalently linked peptide moiety and lipid moiety of the invention form a lipopeptide of the invention referred to herein also as lipopeptide building block (LBB). Said conjugate is able to aggregate to a synthetic virus-like particle (sVLP), presenting the polypeptide of the invention or part thereof on the surface of the sVLP.

Peptide moiety (PM)

Said peptide moiety of the conjugate of the invention (herein mentioned also as PM) comprises at least one coiled coil peptide chain segment. In a preferred embodiment, said coiled coil peptide chain segment of said peptide moiety consists of 3 to 8 repeat units including 3, 4, 5, 6, 7, 8 repeat units. The upper number of repeat units in the peptide moiety influences the stability of the coiled coil. In a preferred embodiment, said coiled coil peptide chain segment of said peptide moiety comprises or preferably consists of 4 repeat units. In a most preferred embodiment, said coiled coil peptide chain segment of said peptide moiety consists of 4 repeat units. In a preferred embodiment, said 4 repeat units are consecutively linked to each other.

Coiled coil peptide chain segments of the invention are based on canonical repeat units, typically and preferably canonical tandem heptad repeats that form right-handed amphipathic alpha-helices, which then assemble to form helical bundles with left-handed coiled coils. Design rules are discussed in more detail, for example, in Woolfson, D.N., Adv. Prot. Chem. 2005, 70, 79-112.

In the invention, said repeat unit of the coiled coil peptide chain segments consists of seven amino acids (a, b, c, d, e, f and g). Preferably, in the coiled-coil peptide chain segment, positions a and d in each heptad motif (abcdefg) comprise alpha-amino acids with small to medium-sized hydrophobic side chains and/or aromatic or heteroaromatic side chains; wherein none, one or two of all the a and d positions comprise an amino acid with a polar non-charged residue; and wherein none or one of all the a and d positions comprise an amino acid with a polar cationic residue or an acylated derivative thereof, or with a polar anionic residue, or glycine. Preferably, alpha-amino acids with small to medium-sized hydrophobic side chain are alanine, isoleucine, leucine, methionine and valine; alpha-amino acids with aromatic or heteroaromatic side chain are phenylalanine, tyrosine, tryptophan and histidine; alpha-amino acids with polar non-charged residue are asparagine, cysteine, glutamine, serine and threonine; alpha-amino acids with polar cationic residue are arginine, lysine and histidine; and alpha- amino acids with polar anionic residue are aspartic acid and glutamic acid.

In a preferred embodiment, said heptad motif consist of the sequence IEKKIE-X0 (SEQ ID NO: 50), wherein X0 represents an amino acid. In a preferred embodiment, said repeat unit consists of the sequence IEKKIE-X0, wherein X0 represents an amino acid provided that said X0 is not proline. In another preferred embodiment, said repeat unit consists of the sequence IEKKIE-X0, wherein X0 represents an amino acid, wherein said amino acid is a naturally occurring amino acid, wherein said naturally occurring amino acid is in its L-configuration, in its D-configuration, or in a mixture of any ratio thereof, provided that said amino acid is not proline. In another preferred embodiment, said repeat unit consists of the sequence IEKKIE- X0, wherein X0 represents an amino acid, wherein said amino acid is a naturally occurring amino acid in its L-configuration.

In a preferred embodiment, said repeat unit consists of the sequence selected from IEKKIEG (SEQ ID NO: 34), IEKKIEA (SEQ ID NO: 35) or IEKKIES (SEQ ID NO: 36). In a more preferred embodiment, said repeat unit consists of the sequence selected from IEKKIEA (SEQ ID NO: 35) or IEKKIES (SEQ ID NO: 36). In another preferred embodiment, said repeat unit consists of the sequence IEKKIEG (SEQ ID NO: 34). In a very preferred embodiment, said repeat unit consists of the sequence IEKKIEA (SEQ ID NO: 35). In an even more preferred embodiment, said repeat unit consists of the sequence IEKKIES (SEQ ID NO: 36).

In a preferred embodiment, said coiled coil peptide chain segment comprises the sequence selected from (IEKKIEG)₄ (SEQ ID NO: 37), (IEKKIEA)4 (SEQ ID NO: 38) or (IEKKIES)₄ (SEQ ID NO: 39). In another preferred embodiment, said coiled coil peptide chain segment consists of the sequence selected from (IEKKIEG)₄ (SEQ ID NO: 37), (IEKKIEA)4 (SEQ ID NO: 38) or (IEKKIES)₄ (SEQ ID NO: 39). In a very preferred embodiment, said coiled coil peptide chain segment comprises, or preferably consists of, the sequence SEQ ID NO: 38 or 39. In a very preferred embodiment, said coiled coil peptide chain segment of the peptide moiety consists of the sequence SEQ ID NO: 38 or 39. In a very preferred embodiment, said coiled coil peptide chain segment comprises the sequence (IEKKIES)₄ (SEQ ID NO: 39). In a very preferred embodiment, said coiled coil peptide chain segment of the peptide moiety consists of the sequence (IEKKIES)₄ (SEQ ID NO: 39).

In a preferred embodiment, said peptide moiety further comprises a T-helper cell epitope. In a preferred embodiment, said peptide moiety further comprises a T-helper cell epitope, wherein said T-helper cell epitope comprises or preferably consists of a sequence selected from the group consisting of (i) SEQ ID NO: 8 to SEQ ID NO: 33 and (ii) SEQ ID NO: 8 to SEQ ID NO: 33, wherein one, two, or three amino acids are exchanged by other amino acids or are deleted. In a preferred embodiment, said peptide moiety further comprises a T-helper cell epitope, wherein said T-helper cell epitope consists of a sequence selected from the group consisting of (i) SEQ ID NO: 8 to SEQ ID NO: 33 and (ii) SEQ ID NO: 8 to SEQ ID NO: 33, wherein one, two, or three amino acids are exchanged by other amino acids or are deleted. In a preferred embodiment, said peptide moiety further comprises a T-helper cell epitope, wherein said T-helper cell epitope comprises a sequence selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 33. In a preferred embodiment, said T-helper cell epitope consists of a sequence selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 33.

In one embodiment, said peptide moiety further comprises an amino acid sequence which includes one or more T-helper cell epitopes, and/or strings of polar residues that promote the solubility of the lipopeptide building block in water. Suitable T-helper cell epitopes are known to the skilled person in the art and are described, e.g., in Weber et al, Advanced Drug Delivery Reviews, 2009, 61:11, 965-976; Caro-Aguilar et al, Infect. Immun., 2002, 70:7, 3479-3492; Mishra et al, Immunology, 1993, 79:3, 362-367; Kobayashi et al, Cancer Research, 2000, 60:18, 5228-523; Fraser et al, Vaccine, 2014, 32:24, 2896-2903; Grabowska et al, Int. J. Cancer, 2015, 136:1, 212-224 and WO1998/023635A1. More preferred T-helper cell epitopes included in the peptide moiety are those listed in WO 2015/082501 such TT830-843, TT1064- 1079, TT1084-1099, TT947-968, TT1174-1189, DTD271-290, DTD321-340, DTD331-350, DTD351-370, DTD411-430, DTD431-450, TT632-651, CTMOMP36-60, TraTl, TraT2, TraT3, HbcAg50-69, HbSAgl9-33, HA307-319, MA17-31, MVF258-277, MVF288-302, CS.T3, SM Th, PADREl and PADRE2 as well as variants thereof in which one, two, or three amino acids are inserted, replaced by other amino acids or deleted.

Preferred T-helper epitopes that can be incorporated into said peptide moiety are any one selected from the group listed in Table 2 below, and variants thereof in which one, two, or three amino acids are replaced by other amino acids or are deleted. Table 2. T helper epitopes

In a very preferred embodiment, said peptide moiety further comprises a T-helper cell epitope, wherein said T-helper cell epitope comprises or preferably consists of the amino acid sequence of SEQ ID NO: 8. In a preferred embodiment, said peptide moiety further comprises a T-helper cell epitope, wherein said T-helper cell epitope consists of the amino acid sequence of SEQ ID NO: 8.

In a preferred embodiment, said peptide moiety has a length of 12 to 200 amino acids, more preferably of 21 to 120 amino acids, again more preferably of 21 to 80 amino acids, again more preferably of 21 to 70 amino acids again more preferably of 21 to 60 amino acids again more preferably of 21 to 50 amino acids, again more preferably said peptide moiety has a length of 28 to 48 amino acids. Preferred, said peptide moieties are non-human sequences to avoid the risk of autoimmune disorders when applied in the vaccination of humans.

In a further preferred embodiment, the last C terminal amino acid of said peptide moiety is a D-amino acid, preferably D-Ala. Preferably, the C-terminus is either an amide (CONEh) or a free carboxyl terminus, more preferably CONEh.

In a further preferred embodiment, said peptide moiety comprises (i) an N-terminal amino acid sequence, wherein said N-terminal amino acid sequence comprises or preferably consists of fibroblast-stimulating lipopeptide FSL-1 (S-(2,3-bispalmitoyloxypropyl)- or PAM2-Cys- Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe; SEQ ID NO: 40), FSL-2 (S-(2,3- bispalmitoyloxypropyl)- or FSL-3 (S-(2,3-bisstearyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His- Pro-Lys-Ser-Phe; SEQ ID NO: 41), Mycoplasma fermentans-derived peptide MALP-2 (S-(2,3- bispalmitoyloxypropyl)- or PAM2-Cys-Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu- Lys; SEQ ID NO: 42), or GG; and/or GX where X is Asx or Ser and/or (ii) a C-terminal amino acid sequence, wherein said C-terminal amino acid sequence comprises or preferably consists of a sequence recognized by an enzyme as cleavage site; wherein preferably said C-terminal amino acid sequence comprises or preferably consists of sequence KKKCa (SEQ ID NO: 43) or wherein preferably said C-terminal amino acid sequence is an amino acid sequence of 5 consecutive amino acids. In a very preferred embodiment, said peptide moiety comprises (i) an N-terminal amino acid sequence, wherein said N-terminal amino acid sequence comprises or preferably consists of GG and/or GX where X is Asx or Ser and/or (ii) a C-terminal amino acid sequence, wherein said C-terminal amino acid sequence comprises or preferably consists of sequence KKKCa (SEQ ID NO: 43). In a more preferred embodiment, said peptide moiety comprises (i) an N-terminal amino acid sequence, wherein said N-terminal amino acid sequence comprises or preferably consists of GG and (ii) a C-terminal amino acid sequence, wherein said C-terminal amino acid sequence comprises or preferably consists of sequence KKKCa (SEQ ID NO: 43). In an even more preferred embodiment, said peptide moiety comprises (i) an N- terminal amino acid sequence, wherein said N-terminal amino acid sequence consists of GG and (ii) a C-terminal amino acid sequence, wherein said C-terminal amino acid sequence consists of sequence KKKCa (SEQ ID NO: 43).

In another preferred embodiment, said peptide moiety comprises (i) an N-terminal amino acid sequence consisting of GG, (ii) a C-terminal amino acid sequence consisting of sequence KKKCa (SEQ ID NO: 43), a T-helper epitope of SEQ ID NO: 8 and a coiled coil peptide chain segment consisting of the sequence (IEKKIES)₄ (SEQ ID NO: 39). In a very preferred embodiment, said peptide moiety comprises or preferably consists of a sequence selected from the group consisting of SEQ ID NO: 44:

GG(IEKKIEA)4lEKKIAKMEKAS S VFNVVN SKKKCa, SEQ ID NO: 45:

GG(IEKKIEA)4lEKKIAKMEKAS S VFNVVN SKKKCa-NFh_, SEQ ID NO: 46: GG(IEKKIES)4lEKKIAKMEKAS S VFNVVN SKKKCa, SEQ ID NO: 47:

GG(IEKKIES)4lEKKIAKMEK AS S VFNVVN SKKKCa-NFh . In another very preferred embodiment, said peptide moiety comprises or preferably consists of SEQ ID NO: 47 or 46. In an even more preferred embodiment, said peptide moiety comprises or preferably consists of (SEQ ID NO: 47): GG(ffiKKffiS)₄ffiKKIAKMEKAS S VFNVVN SKKKCa-NEh

Lipid moiety (LM)

The term "hydrocarbyl" as used herein preferably means a straight or branched, preferably straight alkyl or alkenyl group of at least 7 carbon atoms. More preferably, straight alkyl or alkenyl consists of between 8 and 50 C atoms, preferably between 8 and 25 C atoms. Alkenyl has preferably one, two or three double bonds in the chain, each with E or Z geometry, as is customarily found in natural fatty acids and fatty alcohols. Branched alkyl or alkenyl are preferably alkyl bearing a methyl or ethyl substituent at the second or third carbon atom counted from the end of the chain, as e.g. as in 2-ethyl -hexyl. In a preferred embodiment, said lipid moiety of the conjugate of the invention (herein mentioned also as LM) preferably consisting of, the formula LM-I

wherein R¹ and R² are independently Cn-isalkyl, preferably R¹ and R² are independently - C11H23, -C13H27 or -C15H31, and further preferably R¹ and R² are -C15H31; and R³ is hydrogen or -C(0)Cn-i₅alkyl, preferably R³ is H or -C(0)Ci₅H_3i.

In another preferred embodiment, said lipid moiety of the conjugate of the invention (herein mentioned also as LM) preferably consisting of, the formula LM-II

The inventors found that lipopeptide building blocks comprising PaimCys or PaimCys moieties with the (Reconfiguration at the 2-propyl carbon atom and further comprising as coiled coil peptide chain segment several units of the sequence IEKKIE-X0 with preferably X0 being Gly, Ala or Ser, most preferably Ser, showed increased avidity of the antibodies generated against antigens linked to the inventive lipopeptide building blocks and comprised by the inventive conjugates or SVLPs, respectively.

Said lipid moiety is linked to said peptide moiety, wherein the wavy line in formula LM- I and LM-II and other LM formulas mentioned herein indicates the linkage site to said peptide moiety.

In a preferred embodiment, said R¹ and R² are independently -C₁₁H₂₃, -C₁₃H₂₇ or - C₁₅H₃₁. In a very preferred embodiment, said R¹ and R² are -C₁₅H₃₁. In a preferred embodiment, said R³ is H or -C(0)Ci₅H_3i. In a preferred embodiment, said R¹ and R² are independently - C₁₁H₂₃, -C₁₃H₂₇ or -C₁₅H₃₁, and R³ is hydrogen or -C(0)Cn-i₅alkyl. In a very preferred embodiment, said R¹ and R² are -C₁₅H₃₁, and R³ is hydrogen or -C(0)Cn-i₅alkyl. OIn a preferred embodiment, said R¹ and R² are independently -C₁₁H₂₃, -C₁₃H₂₇ or -C₁₅H₃₁, and R³ is H or - C(0)Ci₅H_3i. In a very preferred embodiment, said R¹ and R² are -C₁₅H₃₁, and R³ is H or - C(0)Ci₅H3i.

In a preferred embodiment, said lipid moiety is linked to the N-terminus of said peptide moiety. This conveniently allows that said linking can be performed on-resin after assembly of the peptide chain of said peptide moiety by solid phase peptide synthesis. Linking of said lipid moiety to the C-terminus of said peptide moiety is also encompassed within the present invention and is possible using linkage chemistry known by the skilled person in the art.

A preferred lipid moiety is di-palmitoyl-S-glycerylcysteinyl (PaimCys) or tripalmitoyl- S-glyceryl cysteine (PaimCys), more preferably, PaimCys. More preferably, PaimCys or PaimCys are both with the //-configuration at the chiral 2-propyl carbon atom and the R- configuration of the chiral carbon of the cysteinyl moiety. The lipid moiety PaimCys or PaimCys is preferably conjugated to the N-terminus of the lipid moiety.

In a preferred embodiment, said lipid moiety comprises, preferably consists of, the formula LM-I*

wherein R³ is hydrogen or -C(0)Cn-i₅alkyl, preferably H or -C(0)Ci₅H_3i; wherein preferably said lipid moiety is linked to the N-terminus of said peptide moiety.

In a preferred embodiment, said lipid moiety comprises, preferably consists of, the formula LM-II*

In a preferred embodiment, said lipid moiety consists of the formula LM-I* or LM-II*, wherein R³ is hydrogen or -C(0)Cn-i₅alkyl. In a preferred embodiment, said lipid moiety comprises, preferably consists of, the formula LM-I* or LM-II*, wherein R³ is H or - C(0)C_I5H_3I. In a preferred embodiment, said lipid moiety comprises, preferably consists of, the formula LM-I* or LM-II*, wherein R³ is H or -C(0)Ci₅H_3i and wherein said lipid moiety is linked to the N-terminus of said peptide moiety. In a preferred embodiment, said lipid moiety consists of, the formula LM-I* or LM-II*, wherein R³ is H or -C(0)Ci₅H_3i and wherein preferably said lipid moiety is linked to the N-terminus of said peptide moiety.

In a very preferred embodiment, said lipid moiety comprises, preferably consists of, the formula LM-P1 or LM-I*2. In a very preferred embodiment, said lipid moiety consists of the formula LM-I*1 or LM-I*2. In a very preferred embodiment, said lipid moiety consists of the formula LM-I*1.

In a very preferred embodiment, said lipid moiety consists of the formula LM-IP1.

In a very preferred embodiment, said lipid moiety consists of the formula LM-P2.

Very preferred lipid moieties of the present invention are, thus, PaimCys LM-IP2, i.e. tripalmitoyl-S-glyceryl cysteine (N-palmitoyl-S-[(2,3-bis-(0-palmitoyloxy)-(2-propyl)]- cysteinyl-) or Par^Cys LM-IP1, i.e. dipalmitoyl-S-glyceryl cysteine (S-[2,3-bis-(0- palmitoyloxy)-(2-propyl)]-cysteinyl-). In a further very preferred embodiment, said lipid moiety is N-a-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2-propyl)]-cysteine or S-[2,3- bis(palmitoyloxy)-(2-propyl)]-cysteine, thus LM-IP1.

In a very preferred embodiment, said lipid moiety consists of the formula LM-IP2.

Very preferred lipid moieties of the present invention are, thus, (/^/^-ParmCys LM-IP2, i.e. tripalmitoyl-S-glyceryl cysteine (N-palmitoyl-S-[2,3-bis-(0-palmitoyloxy)-(2f?)-propyl]- (i?)-cysteinyl-) and (R,R)~ Par^Cys LM-IP1, i.e. dipalmitoyl-S-glyceryl cysteine (S-[2,3-bis- (0-palmitoyloxy)-(2f?)-propyl]-(f?)-cysteinyl-). Thus, in a further very preferred embodiment, said lipid moiety is N-a-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2f?)-propyl]-(f?)-cysteine or S- [2,3-bis(palmitoyloxy)-(2f?)-propyl]-(f?)-cysteine, thus LM-IP1.

In a preferred embodiment, the lipid moiety is linked to the peptide moiety, either directly or via a connecting moiety. Preferably, the lipid moiety is linked to the peptide moiety at or near one terminus, i.e. the N-terminus or the C-terminus, preferably the N-terminus. In a preferred embodiment, the lipid moiety is linked to the first, second, third, fourth or fifth amino acid of the peptide moiety, calculated from the N-terminus or C-terminus of the peptide moiety. The lipid moiety may be linked, directly or through a connecting moiety, to the backbone or to the side chain of one of the amino acids of the peptide moiety, preferably said amino acid is near to the terminus, more preferably it is the first, second, third, fourth or fifth amino acid of the peptide moiety.

The lipopeptide building block of the invention comprises the lipid moiety and the peptide moiety of the invention, wherein the lipid moiety is attached to the peptide moiety either directly or via a connecting moiety. If the peptide moiety and the lipid moiety are directly linked, this is preferably accomplished through an amide bond between a lipid moiety carbonyl function and an amino function, e.g. the N-terminal amino function, of the peptide moiety. It will be apparent to the skilled person in the art that a large variety of suitable connecting moieties and strategies exist, which include but are not limited to connecting moieties based on dicarboxylic acid derivatives, connecting moieties containing one or multiple ethylene glycol units, amino acid residues (including alpha-, beta-, gamma-, omega-amino acids), or sugar (carbohydrate) units, or containing heterocyclic rings.

In a preferred embodiment, said lipid moiety and said peptide moiety are linked via a connecting moiety. In a preferred embodiment, said lipid moiety and said peptide moiety are linked via a connecting moiety, wherein said connecting moiety is an amino acid linker consisting of 2-15 amino acids. Examples hereto include the amino acid linker sequences comprised by FSL-1, FSL-2, FSL-3, PAM2 or MALP-2 moieties. In a preferred embodiment, said lipid moiety and said peptide moiety are linked via a connecting moiety, wherein said connecting moiety is an amino acid linker consisting of 2-10 amino acids. In a preferred embodiment, said lipid moiety and said peptide moiety are linked via a connecting moiety, wherein said connecting moiety is an amino acid linker consisting of 2-5 amino acids. In a preferred embodiment, said lipid moiety and said peptide moiety are linked via a connecting moiety, wherein said connecting moiety is an amino acid. In a preferred embodiment, said lipid moiety and said peptide moiety are linked via a connecting moiety, wherein said connecting moiety is a Gly-Gly moiety.

In a preferred embodiment, said direct linking of said lipid moiety and said peptide moiety is by way of an amide bond between a carbonyl function of said lipid moiety and an amino function of said peptide moiety. In a preferred embodiment, said linking of said lipid moiety and said peptide moiety via said connecting moiety is by way of an amide bond between a carbonyl function of said lipid moiety and an amino function of said connecting moiety.

In a preferred embodiment, said linking of said lipid moiety and said peptide moiety via said connecting moiety is by way of an amide bond between a carbonyl function of said lipid moiety and an amino function of said connecting moiety, wherein said connecting moiety is an amino acid linker, preferably consisting of 2-15 amino acids, preferably 2-10 amino acids, and wherein said amino function is the N-terminal amino function of said connecting moiety. In a preferred embodiment, said linking of said lipid moiety and said peptide moiety via said connecting moiety is by way of an amide bond between a carbonyl function of said lipid moiety and an amino function of said connecting moiety, wherein said connecting moiety is an amino acid linker, preferably consisting of 2-5 amino acids, and wherein said amino function is the N- terminal amino function of said connecting moiety.

In a preferred embodiment, said direct linking of said lipid moiety and said peptide moiety is by way of an amide bond between a carbonyl function of said lipid moiety and an amino function of said peptide moiety, wherein said amino function is the N-terminal amino function of said peptide moiety.

In a preferred embodiment, two Gly residues are included as connecting moiety between the lipid moiety, preferably said Pam?Cys moiety LM-P1 or LM-II* of the present invention and the start of the coiled-coil heptad repeats, typically and preferably the coiled coil peptide chain segment comprising, preferably consisting of, the sequence IEKKIES or IEKKIEA. In a more preferred embodiment, two Gly residues are included as link connecting moiety between the lipid moiety, preferably said (R,R)- Par^Cys moiety LM-II* 1 of the present invention and the start of the coiled-coil heptad repeats, typically and preferably the coiled coil peptide chain segment comprising, preferably consisting of, the sequence IEKKIES or IEKKIEA.

The introduction of an amino acid linker, and preferably a short amino acid linker consisting of two amino acids, preferably glycine, allows during peptide synthesis that after each amino acid connecting, a capping step can be performed with acetic anhydride. This has the practical advantage that after completion of peptide assembly, and connecting of the lipid moieties, preferably the lipid moieties consisting of the formula LM- 1 [ParroCys moiety] or formula LM-P2 [PaimCys moiety], more preferably the lipid moieties consisting of the formula LM-I 1 [(/(ri'l-PaimCys moiety] or formula LM-IP2 [(/^/^-PaimCys moiety] to the free N-terminus, the HPLC retention time of the peptide is dramatically altered by lipidation, thus greatly facilitating HPLC purification of the desired lipopeptide building block of the present invention.

Lipopeptide building block (LBB)

The covalently linked peptide moiety and lipid moiety of the invention form a lipopeptide of the invention referred to herein also as lipopeptide building block (LBB).

In a very preferred embodiment, said lipopeptide building block is of the formula LBB-1 to LBB-6, preferably of LBB-1 to LBB-3, more preferably LBB-2 and 3, again more preferably

LBB-2. In an again more preferred embodiment, said lipopeptide building block is of formula LBB-4 to LBB-6, more preferably of LBB-5 or LBB-6, most preferably LBB-6.

LBB-6 In a further very preferred embodiment, said lipopeptide building block is of the formula LBB-4. In a more preferred embodiment, said lipopeptide building block is of the formula

LBB-5.

In further very preferred embodiment, the present invention provides a lipopeptide building block consisting of

(i) a peptide moiety comprising a coiled coil peptide chain segment, wherein said coiled coil peptide chain segment comprises 3 to 8 repeat units, and wherein said repeat unit consists of the sequence IEKKIE-XO (SEQ ID NO:58), wherein X0 represents an amino acid, and wherein preferably said repeat unit consists of the sequence selected from IEKKIEG (SEQ ID NO:59), IEKKIEA (SEQ ID NO: 12) or IEKKIES (SEQ ID NO: 13), and wherein further preferably said repeat unit consists of the sequence IEKKIES (SEQ ID NO: 13);

(ii) a lipid moiety comprising, preferably consisting of, the formula LM-I or LM-II, wherein R¹ and R² are independently Cn-isalkyl, wherein preferably R¹ and R² are independently -C11H23, -C13H27 or -C15H31, and wherein further preferably R¹ and R² are - C15H31; and wherein R³ is hydrogen or -C(0)Cn-i₅alkyl, and wherein preferably R³ is H or - C(0)Ci5H3i; and wherein said lipid moiety is linked to said peptide moiety, wherein the wavy line in formula LM-I indicates the linkage site to said peptide moiety, and wherein preferably said lipid moiety is linked to the N-terminus of said peptide moiety.

In a further aspect, the present invention provides a conjugate comprising (a) a lipopeptide building block of the present invention and (b) an antigen, wherein said antigen is connected, directly or via a linker, to said lipopeptide building block.

(i) a peptide moiety comprising a coiled coil peptide chain segment, and wherein said coiled coil peptide chain segment comprises, preferably consists of, the sequence of SEQ ID NO: 38 or 39;

(ii) a lipid moiety comprising, preferably consisting of, the formula LM-I or LM-II, wherein R¹ and R² are independently Cn-isalkyl, wherein preferably R¹ and R² are independently -C₁₁H₂₃, -C₁₃H₂₇ or -C₁₅H₃₁, and wherein further preferably R¹ and R² are -C₁₅H₃₁; and wherein R³ is hydrogen or -C(0)Cn-i₅alkyl, and wherein preferably R³ is H or -C(0)Ci₅H_3i; and wherein said lipid moiety is linked to said peptide moiety, wherein the wavy line in formula LM-I and LM-II indicates the linkage site to said peptide moiety, and wherein preferably said lipid moiety is linked to the N-terminus of said peptide moiety. In an even more preferred embodiment, the present invention provides a lipopeptide building block consisting of

(ii) a lipid moiety comprising, preferably consisting of, the formula LM-II, wherein R¹ and R² are independently Cn-isalkyl, wherein preferably R¹ and R² are independently -C11H23, -C13H27 or -C15H31, and wherein further preferably R¹ and R² are -C15H31; and wherein R³ is hydrogen or -C(0)Cn-i₅alkyl, and wherein preferably R³ is H or -C(0)Ci₅H_3i; and wherein said lipid moiety is linked to said peptide moiety, wherein the wavy line in formula LM-II indicates the linkage site to said peptide moiety, and wherein preferably said lipid moiety is linked to the N-terminus of said peptide moiety.

Coupling moiety

In order to form the conjugate of the invention, one or more polypeptides of the invention may be conjugated to the peptide moiety of the lipopeptide building block, either directly or through a coupling moiety, either via the N- or C-terminus of the polypeptide of the invention, and is connected either to the N- or to the C-terminal of the peptide moiety or optionally to one or more amino acid side chains of the peptide moiety. Alternatively, the polypeptide of the invention is conjugated to the peptide moiety of the lipopeptide building block through a side chain residue of the polypeptides of the invention, such as a terminal or internal aspartic acid, glutamic acid, lysine, ornithine or cysteine side chain.

Coupling or conjugation procedures were used to attach the polypeptide of the invention functioning as antigens or epitope mimetics to the peptide moiety of the lipopeptide building block of the invention. Free amino groups in the side chains of amino acids in the peptide moiety may be coupled to reactive esters in the polypeptide of the invention (e.g. N- hydroxysuccinimide esters prepared from carboxylic acids); thiols in the peptide moiety may be coupled to maleimide groups in the polypeptide of the invention; azides may be incorporated into the side chains of amino acid residues in the polypeptide of the invention and coupled to acetylene groups of the peptide moiety using copper catalyzed cycloaddition reactions; and other nucleophiles (e.g. hydrazino, hydroxylamino, vic-aminothiol groups) in the peptide moiety may be coupled to electrophiles (e.g. aldehydes, ketones, active esters) in the polypeptide of the invention. It will be obvious that it is possible, in principle, to reverse the positions of the two reactive groups in the peptide moiety and polypeptide in order to achieve selective coupling. Other linking and conjugation procedures that may be used to attach the polypeptide of the invention to the lipopeptide building block can be found in the art (see for example Hermanson, G.T, Bioconjugate Techniques, 2nd edition, Academic Press, 2008).

In a preferred embodiment, the polypeptide of the invention is conjugated via its N- terminus to an amino acid side chain or the C-terminus of the peptide moiety of the lipopeptide building block, either directly or through a coupling moiety. Preferably, the polypeptide of the invention is attached to the peptide moiety of the lipopeptide building block via the attachment residue of the polypeptide, either directly or through a coupling moiety. In a preferred embodiment, the polypeptide of the invention is conjugated to the peptide moiety of the lipopeptide building block via the N terminal amino acid or the N terminal attachment residue of the polypeptide of the invention, preferably the N terminal Z residue. Said N-terminal attachment residue is preferably conjugated to one of the amino acid side chains of the peptide moiety or to the C-terminus of the peptide moiety, preferably to one of the amino acid side chains, either directly or through a coupling moiety. More preferably, said amino acid side chain of the peptide moiety belongs to an amino acid between the very last C terminal amino acid and an amino acid which is minus 10, preferably minus 5 positions to the N direction from said very last C terminal amino acid (i.e. 5 amino acids from the very last C terminal amino acid towards the N-terminus). The functional N3 group of said Z residue is preferably conjugated to a functional group in one of the side chains or the terminus of the peptide moiety, or to a functional group of said coupling moiety, preferably to a functional group of said coupling moiety.

Coupling moieties considered for coupling the polypeptide of the invention to the peptide moiety of the lipopeptide building block are alkylene chains, short peptides of 1 to 20 amino acids, hydroxyalkyl- or aminoalkyl-carboxylic acids, substituted or unsubstituted polyalkylenoxy glycols, preferably containing one to twelve C2 and/or C3 alkylenoxy units, polyalkylenoxy glycol block co-polymers (e.g. pluronics), mono-, di-, tri- and oligo saccharides, which may comprise acetyl, glycerol-phosphate or other substituents at one or more positions, proteinogenic or non-proteinogenic amino acids, and C1-C8 saturated or unsaturated hydrocarbons; and may comprise one or more of the following functional groups: a disulfide bond, azide, amine, amide, acetal, ester, ether, thioether, hydrazone, hydrazide, imine, oxime, urea, thiourea, carbonate, iminocarbonate, amidine, amide, imide, an alkyl succinimide, which may also be hydrolyzed to an amide, sulphonamide, sulfone, or a heterocyclic ring comprising one or more atoms selected from nitrogen and oxygen, preferably a triazole. In a preferred embodiment, said coupling moiety is capable of cross-linking the polypeptide of the invention with a thiol group of a second peptide.

In a preferred embodiment, the polypeptide of the invention is conjugated to the peptide moiety of the lipopeptide building block via the N terminal attachment residue of the polypeptide of the invention, which is the N terminal Z residue, wherein the functional N3 group of said Z residue is conjugated to one or more of the amino acid side chains of the peptide moiety or the N-terminus of the peptide moiety, each through a coupling moiety. More preferably, said Z residue is conjugated to one or more of the amino acid side chains, wherein said side chain is cysteine. In a further preferred embodiment, said coupling moiety comprises a maleimide moiety and/or alkyne group. In a more preferred embodiment, said coupling moiety comprises or preferably consists of a maleimide moiety linked to an alkyne group via a polyethylenglycol (PEG) linker.

In a further preferred embodiment, said coupling moiety comprises or preferably consists of maleimido-(CH₂CH₂0)q(CH₂)_wCCH of formula BO, wherein q is from 1 to 24 and w is from 1 to 12:

BO maleimido-(CH₂CH₂0)_q(CH₂)_wCCH

In a very preferred embodiment, said coupling moiety has formula Bl.

chain of Cys

In a preferred embodiment, said coupling moiety comprises or preferably consists of one of the following formulas:

wherein q is from 1 to 24 and w is from 1 to 12 in B2. Sad coupling moieties are preferably attached to the polypeptide of the invention via copper catalyzed click-reaction.

In an even more preferred embodiment, said coupling moiety is of formula B2, wherein q is from 1 to 24 and w is from 1 to 12, preferably q is from 1 to 12 and w is from 1 to 6, more preferably q is from 1 to 8 and w is from 1 to 3, again more preferably q is from 1 to 6 and w is from 1 to 3.

Again more preferably, said coupling moiety is of formula B3. Cys

The wavy line in B1 and B3 after coupling (shown above) indicates the attachment site of the peptide moiety. Preferably, the maleimido group of the coupling moiety BO, Bl, B2 or B3 is covalently bound to a sulphur atom in a side chain thiol group of a Cys of the peptide moiety. B0-B3 are preferably attached to the polypeptide of the invention via copper catalyzed click-reaction.

In a very preferred embodiment, azides are included in the side chains of amino acid residues or the attachment residue of the polypeptide of the invention and coupled to acetylene groups of the coupling moiety in order to link the polypeptide of the invention to the peptide moiety using copper catalyzed cycloaddition reactions. In a preferred embodiment, the polypeptide of the invention comprises said Z residue attached to the N-terminus of said polypeptide, and said polypeptide is conjugated to the peptide moiety via a coupling moiety having formula BO, such asBl or B2, such as B3, wherein BO, Bl, B2 or B3 is linked via the thiol group to the cysteine side chain of the peptide moiety.

In a further very preferred embodiment, said lipopeptide building block of the invention comprises a coupling moiety. Preferably, said coupling moiety is BO or B 1. In another preferred embodiment, said coupling moiety is BO, such as Bl, or B2, such as B3. More preferably, said coupling moiety is B2, again more preferably, B3.

In a very preferred embodiment, said lipopeptide building block is selected from the group consisting of LBB-1, LBB-2, LBB-3, LBB-4, LBB-5 and LBB-6 of the invention and comprises said coupling moiety BO or Bl. In a very preferred embodiment, said lipopeptide building block is selected from the group consisting of LBB-1 to LBB-6 and comprises said coupling moiety BO, such as Bl, or B2, such as B3; preferably said coupling moiety is Bl or B3.

In another embodiment, a lipopeptide building block selected from the group consisting of LBB-1 to LBB-6 comprises said coupling moiety Bl resulting in LBB-1B1 to LBB-6-B1, exemplarily depicted below for LBB-1 -B. In a more preferred embodiment, the lipopeptide building block is selected from the group consisting of LBB-1 to LBB-6 and comprises said coupling moiety Bl or B3, preferably B3 resulting in LBB-1 -B3 to LBB-6-B3. B0-B3 are indicated as B in the formula of LBB-1 -B below.

LBB-1 -B In a preferred embodiment, said coupling moiety comprises or preferably consists of one of formulas CM 1 to CM 19:

shown with the connecting functional group C=0 or X, wherein X is O or NH, m is between 1 and 45 and n is between 1 and 45, preferably n is between 1 and 20, and the terminal wavy lines indicate the attachment site to the polypeptide of the invention or its attachment residue and the peptide moiety of the LBB.

In further embodiments, said coupling moiety is selected from the following formulas:

wherein n is an integer of 1 to 45, preferably 6 to 8, and the terminal wavy line indicates the attachment site to said polypeptide of the invention or SEQ ID NO: 1. Further preferred, said n is 6.

In case the compounds of the present invention, hereby including said coupling moiety, comprise one or more double bonds, said double bonds can be of either the (£)- or (Z)- configuration, or mixtures thereof in any ratio. The same applies for the preferred coupling moiety comprising an oxime moiety. Thus, the preferred coupling moiety comprising an oxime moiety thus may include either said linker with said oxime moiety in its syn-configuration (and thus as syn-isomer), said linker with said oxime moiety in its anti-configuration (and thus as anti-isomer) and mixtures thereof in any ratio. Within the chemical formulas presented herein for said double bond or said oxime moiety, this is typically and preferably represented by an internal wavy line, unless otherwise stated.

Not only are the disclosure of PCT/EP2018/065714 incorporated herein in its entirety by way of reference, but all the disclosures of PCT/EP2018/065714, in particular, the disclosure related to the specific linking, coupling, attaching and connecting moieties/ residues, spacers, lipid and peptide moieties conjugates and other component moieties, and the generated biological data hereto are specifically incorporated herein in its entirety by way of reference.

In a very preferred embodiment, the lipopeptide building block (LBB) is of formula LBB- 4 to LBB-6, more preferably of LBB-5 or LBB-6, most preferably LBB-6, wherein a coupling moiety B1-B3, preferably B3, is attached to the LBB, wherein the maleimido group of the coupling moiety is covalently bound to a sulphur atom in a side chain thiol group of a Cys of the peptide moiety of the LBB (exemplarily indicated below for LBB6 and B3).

Conjugate

In a further preferred embodiment, said conjugate comprises (i) the polypeptide comprising an amino acid sequence of SEQ ID NO: 1 :

Xi SNNLD SK V GGNYN YX₂ YRLFRK SNLKPFERDIS TEI Y Q AGS TPCN GVEGFN C YFPLQX3YGFQPTNGVGYQPX4, wherein each of XI to X4 is independently at least one amino acid; or a variant of SEQ ID NO: 1, wherein in said variant at most 33 amino acids are exchanged as compared to SEQ ID NO: 1; or a fragment of SEQ ID NO: 1 or a fragment of the variant of SEQ ID NO: 1; and (ii) a lipopeptide building block selected from the group consisting of LBB-1, LBB-2, LBB-3, LBB-4, LBB-5 and LBB-6, more preferably selected from the group consisting of LBB-1, LBB-2, LBB-4, and LBB-5, again more preferably LBB-4 or LBB-5.

In a further preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises a sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6 and 7, and (ii) a lipopeptide building block selected from the group consisting of LBB- 1, LBB-2, LBB-3, LBB-4, LBB-5 and LBB-6, more preferably selected from the group consisting of LBB-1, LBB-2, LBB-4, and LBB-5, again more preferably LBB-4 or LBB-5. In a further preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises, preferably consists of a sequence selected from the group consisting of SEQ ID NO: 5, 6 and 7, and (ii) a lipopeptide building block selected from the group consisting of LBB-1, LBB-2, LBB-3, LBB-4, LBB-5 and LBB-6, more preferably selected from the group consisting of LBB-1, LBB-2, LBB-4, and LBB-5, again more preferably LBB-4 or LBB-5. In a further preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises, preferably consists of a sequence selected from the group consisting of SEQ ID NO: 5, 6 and 7, and (ii) a lipopeptide building block selected from the group consisting of LBB-4, LBB-5, and LBB-6, more preferably LBB-4 or LBB-5.

In a further preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises, preferably consists of a sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6 and 7, and the attachment residue Z which is independently attached to the N-terminus of each of these sequences SEQ ID NO: 2 to 7, and (ii) a lipopeptide building block selected from the group consisting of LBB-1, LBB-2, LBB-3, LBB-4, LBB-5 and LBB- 6. In a further preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises, preferably consists of a sequence selected from the group consisting of SEQ ID NO: 5, 6 and 7, and the attachment residue Z which is independently attached to the N-terminus of each of these a sequences of SEQ ID NO: 5, 6 and 7, and (ii) a lipopeptide building block selected from the group consisting of LBB-1, LBB-2, LBB-3, LBB-4, LBB-5 and LBB-6. In a further preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises, preferably consists of a sequence selected from the group consisting of SEQ ID NO: 5, 6 and 7 and the attachment residue Z which is independently attached to the N-terminus of each of these a sequences of SEQ ID NO: 5, 6 and 7, and (ii) a lipopeptide building block selected from the group consisting of LBB-4, LBB-5, and LBB-6, more preferably LBB-4 or LBB-5. In a further very preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises, preferably consists of a sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6 and 7, and the attachment residue Z which is independently attached to the N-terminus of each of these sequences of SEQ ID NO: 2 to 7, and (ii) a lipopeptide building block which is selected from the group consisting of LBB-1, LBB-2, LBB-3, LBB-4, LBB-5 and LBB-6 and which further comprises said coupling moiety BO or Bl. In a further very preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises, preferably consists of a sequence selected from the group consisting of SEQ ID NO: 5, 6 and 7, and the attachment residue Z which is independently attached to the N-terminus of each of these sequences of SEQ ID NO: 5, 6 and 7, and (ii) a lipopeptide building block which is selected from the group consisting of LBB-1, LBB-2, LBB- 3, LBB-4, LBB-5 and LBB-6 and which further comprises said coupling moiety BO or Bl. In a further very preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises, preferably consists of a sequence selected from the group consisting of SEQ ID NO: 5, 6 and 7, and the attachment residue Z which is independently attached to the N-terminus of each of these sequences of SEQ ID NO: 5, 6 and 7, and (ii) a lipopeptide building block which is selected from the group consisting of LBB-4, LBB-5, and LBB-6, more preferably LBB-4 or LBB-5, and which further comprises said coupling moiety BO or Bl.

In a further preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises a sequence selected from the group consisting of SEQ ID NO: 2-7 and 51-54, and (ii) a lipopeptide building block selected from the group consisting of LBB-1, LBB-2, LBB-3, LBB-4, LBB-5 and LBB-6, more preferably selected from the group consisting of LBB-1, LBB-2, LBB-4, and LBB-5, again more preferably LBB-4 or LBB-5. In a further preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises, preferably consists of a sequence selected from the group consisting of SEQ ID NO: 5, 6, 7, 53 and 54, and (ii) a lipopeptide building block selected from the group consisting of LBB-1, LBB-2, LBB-3, LBB-4, LBB-5 and LBB-6, more preferably selected from the group consisting of LBB-1, LBB-2, LBB-4, and LBB-5, again more preferably LBB-4 or LBB-5.

In a further preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises, preferably consists of a sequence selected from the group consisting of SEQ ID NO: 2-7 and 51-54, and the attachment residue Z which is attached to the N-terminus of each of these sequences SEQ ID NO: 2-7 and 51-54, and (ii) a lipopeptide building block selected from the group consisting of LBB-1, LBB-2, LBB-3, LBB-4, LBB-5 and LBB-6. In a further preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises, preferably consists of a sequence selected from the group consisting of SEQ ID NO: 5, 6, 7, 53 and 54; and the attachment residue Z which is independently attached to the N-terminus of each of these a sequences of SEQ ID NO: 5, 6, 7, 53 and 54, and (ii) a lipopeptide building block selected from the group consisting of LBB-1, LBB-2, LBB-3, LBB-4, LBB-5 and LBB-6, preferably of LBB-4, LBB-5, and LBB-6, more preferably of LBB-4 or LBB-5.

In a further very preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises, preferably consists of a sequence selected from the group consisting of SEQ ID NO: 2-7 and 51-54, and the attachment residue Z which is independently attached to the N-terminus of each of these sequences of SEQ ID NO: 2-7 and 51-54, and (ii) a lipopeptide building block which is selected from the group consisting of LBB-1, LBB-2, LBB-

3, LBB-4, LBB-5 and LBB-6 and which further comprises said coupling moiety selected from the group consisting of BO, Bl, B2 or B3, preferably B1 or B3. In a further more preferred embodiment, said conjugate comprises (i) the polypeptide of the invention which comprises, preferably consists of a sequence selected from the group consisting of SEQ ID NO: 5, 6, 7, 53 and 54, and the attachment residue Z which is independently attached to the N-terminus of each of these sequences of SEQ ID NO: 5, 6, 7, 53 and 54, and (ii) a lipopeptide building block which is selected from the group consisting of LBB-4, LBB-5, and LBB-6, more preferably LBB-4 or LBB-5, and which further comprises said coupling moiety Bl or B3.

In a further preferred embodiment, said conjugate is selected from any one of the following formula of conjugate 1 to conjugate 9:

Conjugate 1

5

Conjugate 3

Conjugate 4

Conjugate 5

Conjugate 6

10

20 Conjugate 7

Conjugate 9

In a more preferred embodiment, said conjugate is selected from any one of conjugate 4 to conjugate 9. In an again more preferred embodiment, said conjugate is selected from conjugate 7 to conjugate 9. Conjugates 1 to 23 are composed as indicated in the formulas herein and in the following table below:

Conjugates 10 and 11 have the same formula as conjugates 7-9, except that Z-mimetics 4 and 5 are attached instead of Z-mimetics 1-3. Conjugates 12 and 13 has the same formula as conjugates 4-6, except that Z-mimetics 4 and 5 are attached instead of Z-mimetics 1-3. Conjugates 14-17 have the same formula as conjugate 18 (scheme-7), except that Z-mimetics 1-4 are attached instead of Z-mimetic 5. Conjugates 19-21 and 23 have the same formula as conjugate 22 (scheme-6), except that Z-mimetics 1-3 and 5 are attached instead of Z-mimetic 4.

O 18)

Formulas of conjugates 14-18

Formulas of conjugates 19-23

In another preferred embodiment, said conjugate is selected from the group consisting of conjugates 1 to 23, preferably conjugates 4 to 23. In another preferred embodiment, said conjugate is selected from the group consisting of conjugates 14 to 23, preferably conjugates 19 to 23. In another preferred embodiment, said conjugate is selected from the group consisting of conjugates 7 to 11 and 19 to 23. In another preferred embodiment, said conjugate is selected from the group consisting of conjugates 4-6 and 14 to 18. In another preferred embodiment, said conjugate is selected from the group consisting of conjugates 3, 6, 9, 11, 13, 16, 18, 21 and 23. In another preferred embodiment, said conjugate is selected from the group consisting of conjugates 9, 11, 21 and 23. In another preferred embodiment, said conjugate is conjugate 21 and 23.

All embodiments and preferred and very preferred embodiments of the polypeptide, the lipid building block, and the conjugate of the invention, and all of its components including linker, fusion moiety, attachment moiety, coupling moiety etc. described herein are applicable to all aspects of the present invention, even though not all embodiments and preferred and very preferred embodiments are necessarily again repeated and reiterated.

In another aspect, the present invention provides for a bundle of conjugates comprising 2, 3, 4, 5, 6 or 7 of the inventive conjugate. In another aspect, the present invention provides for a bundle of conjugates comprising 2, 3, 4 or 5 of the inventive conjugate. In another very preferred aspect, the present invention provides for a bundle of conjugates comprising 3 of the inventive conjugate.

In another aspect, the present invention provides for a method of preparing the conjugate of the invention. Preferably, said method for preparing the conjugate of the invention comprises the steps of (i) preparing a lipopeptide building block of the invention (LBB) by linking a lipid moiety of the invention to a peptide moiety of the invention, wherein said peptide moiety comprises at least one cysteine, (ii) covalently linking the coupling moiety BO, Bl, B2 or B3 to said LBB, (iii) subsequently conjugating the polypeptide of the invention comprising residue Z to the LBB via covalently linking Z residue and BO, Bl, B2 or B3, preferably via a copper- catalyzed click reaction .

In a more preferred embodiment of the method of preparing the conjugate of the invention, said lipopeptide building block of the invention is selected from the group consisting of LBB-1, -2, -3, -4, -5, and -6. In a more preferred embodiment, said LBB is LBB-4 or 5. In another preferred embodiment, said polypeptide of the invention comprising attachment residue Z is Z-mimetic-1 to Z-mimetic-5. Again more preferably, in the conjugate of the invention, said LBB is LBB-4 or -5 and the polypeptide of the invention comprising attachment residue Z is Z-mimetic-1 to Z-mimetic-5.

This order has the advantage that the intermediates LBB+BO, Bl, B2 or B3 and polypeptide comprising Z are more stable and thus the reaction is more efficient leading to a higher yield.

In another aspect, the present invention provides for a bundle of conjugates comprising 2, 3, 4, 5, 6 or 7 of the inventive conjugates. Preferably, said conjugate is selected from conjugate 1, 2 or 3. In another very preferred embodiment, the present invention provides for a bundle of conjugates comprising 3, preferably exactly 3, of the inventive conjugates. Preferably, said conjugate is selected from conjugate 1, 2 or 3.

According to a preferred embodiment, in said bundle, the coiled coil peptide chain segments of said peptide moieties comprised by said conjugates are coiled together, preferably said coiled coil peptide chain segments are helically coiled together, more preferably said coiled coil peptide chain segments are alpha-helically coiled together. In a preferred embodiment, said coiled coil peptide chain segments of said peptide moieties are coiled together left-handed or right-handed. According a preferred embodiment, in said bundle, said coiled coil peptide chain segments of said peptide moieties form an alpha-helical left-handed coil.

In a preferred embodiment, said coiled coil peptide chain segments have a parallel orientation, i.e. they run in the same direction; or they have an anti-parallel orientation, i.e. they run in directions opposite to each other; wherein the first option is preferred. The term “direction” is based on the direction of a peptide chain having on one side an N-terminus and on the other side a C-terminus. In a preferred embodiment of said inventive bundle, said coiled coil peptide chain segments of said peptide moieties form a left-handed alpha-helical coiled coil, wherein the coiled coil peptide chain segments have a parallel orientation in said coiled coil. Preferably, said bundle comprises 2 to 7 (e.g. dimer, trimer, tetramer, pentamer, hexamer or heptamer), more preferably 2, 3, 4 or 5, again more preferably 3 helically twisted coiled coil peptide chain segments, having a parallel orientation in said coiled coil.

In another aspect, the present invention provides for a synthetic virus-like particle (sVLP) comprising at least one bundle of conjugates of the present invention. Said bundle comprises, preferably consists two, three, four, five, six or seven conjugates of the invention. In a preferred embodiment, said sVLP comprises 20-30 bundles of conjugates of the present invention. In a preferred embodiment of the present invention said synthetic virus-like particle comprises at least one bundle, preferably 20-30 bundles of conjugates of the present invention, wherein said conjugate is selected from conjugate 1 to 9. In a preferred embodiment of the present invention said synthetic virus-like particle comprises 20-30 bundles of conjugates of the present invention, wherein said conjugate is selected from conjugate 1 to 9, preferably conjugate 4-9, more preferably conjugate 7-9. In a preferred embodiment of the present invention said synthetic virus-like particle consists of helical lipopeptide bundles comprising two, three, four, five, six or seven conjugates of the invention.

The invention also relates to a method of preparing the synthetic virus-like particles of the invention. Synthetic virus-like particles (sVLP) may be produced by a self-assembly process, e.g. in aqueous solution. Preferably, said conjugates of the invention self-associate in aqueous buffer to form sVLPs in the range 10-100 nm, and preferably 20-40 nm.

This method may involve dissolving the lipopeptide building block in a suitable carrier, preferably an aqueous buffer system (e.g. buffered saline or unbuffered saline). The solvent may be removed after preparation of the synthetic virus-like particles, for example by lyophilization or spray drying. Conjugates including the specific combination of the polypeptide of the invention and the lipopeptide building block of the invention self-assemble to bundles and further to synthetic virus-like particles (sVLPs).

As used herein, the term “effective amount” refers to an amount necessary or sufficient to realize a desired biologic effect. Preferably, the term “effective amount” refers to an amount of the polypeptide of the present invention, the conjugate of the present invention or the synthetic virus like particle of the present invention that (i) treats or prevents the particular disease, medical condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, medical condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, medical condition, or disorder described herein. An immunogenically effective amount, as herein understood, is an amount that is capable of modulating, preferably enhancing the response of the immune system of a subject to an antigen or pathogen.

The invention further relates to the conjugate or the synthetic virus like particle of the invention for use as a vaccine. A vaccine, as used herein, is a pharmaceutical composition that is used to modulate, preferably to stimulate the response of the body’s immune system to a particular antigen or pathogen. In a preferred embodiment, the pharmaceutical composition or preferably the vaccine is used for preventing or reducing the risk of a SARS-CoV or SARS- CoV-2 infection in a subject, preferably a human, more preferably a child or elderly people.

In again another aspect, the present invention provides for the conjugate of the present invention or the synthetic virus like particle of the present invention for use as a medicament, preferably for use in a method for preventing a disease or for reducing the risk of a disease, wherein further preferably said disease is an infectious disease, a cancer or an allergy, and again more preferably wherein said disease is a SARS-CoV or SARS-CoV-2 infection.

In again another aspect, the present invention provides for the conjugate of the present invention or the synthetic virus like particle of the present invention for use in a method for preventing a disease or for reducing the risk of a disease or for treating a disease, wherein further preferably said disease is an infectious disease, and again more preferably wherein said disease is a SARS-CoV or SARS-CoV-2 infection. In again another aspect, the present invention provides for the conjugate of the present invention or the synthetic virus like particle of the present invention for use in a method for preventing of an infectious disease or reducing the risk of an infectious disease, preferably for use in a method for preventing or reducing the risk of an infectious disease associated with or caused by a SARS-CoV or SARS-CoV-2 virus. As used herein, the term treating refers to therapy and to a therapeutic treatment.

The present invention provides for the conjugate of the invention, the synthetic virus like particle of the invention, or the pharmaceutical composition of the invention for use as a medicament, preferably for use in a method for preventing an infectious disease or for reducing the risk of an infectious disease, more preferably for use in a method for preventing or reducing the risk of an infectious disease associated with or caused by a SARS-CoV or SARS-CoV-2 virus. The present invention provides for the conjugate of the invention, the synthetic virus like particle of the invention, or the pharmaceutical composition of the invention for use as a medicament, preferably for use in a method for preventing an infection or for reducing the risk of an infection, more preferably for use in a method for preventing or reducing the risk of an infection caused by a SARS-CoV or SARS-CoV-2 virus. The present invention provides for the conjugate of the invention, the synthetic virus like particle of the invention, or the pharmaceutical composition of the invention for use as a medicament, preferably for use in a method for preventing an infection or for reducing the risk of an infection, more preferably for use in a method for preventing or reducing the risk of a SARS-CoV or SARS-CoV-2 virus infection.

The invention further relates to a method of vaccination against or treatment of a SARS- CoV or SARS-CoV-2 virus infection wherein an immunogenically effective amount of the polypeptide of the invention, the lipopeptide building block of the invention, the synthetic virus like particle of the invention, or the pharmaceutical composition of the invention is administered to a patient in need thereof. The invention further relates to a method of eliciting or modulating an immune response or to a method of limiting the risk of developing a disease, preferably an infection, more preferably an infection associated with or caused by a SARS-CoV or SARS-CoV-2 virus, wherein an immunogenically effective amount of the conjugate or the synthetic virus like particle of the invention is administered to a subject, preferably a human, more preferably a child or elderly people. The invention further relates to a method for treating a disease, preferably an infection, more preferably an infection associated with or caused by a SARS-CoV or SARS-CoV-2 virus, comprising administering an immunogenically effective amount of the conjugate or the synthetic virus like particle of the invention to a subject, preferably a human, more preferably a child or elderly people. Moreover, the invention relates to the conjugate or the synthetic virus like particle of the invention for use in treating a disease, preferably an infection associated with or caused by a SARS-CoV or SARS-CoV-2 virus. Moreover, the invention relates to the conjugate or the synthetic virus like particle of the invention for use in treating a disease, preferably an infection caused by a SARS-CoV or SARS- CoV-2 virus. Moreover, the invention relates to the polypeptide of the invention, the conjugate or the synthetic virus like particle of the invention for use in treating a disease, preferably a SARS-CoV or SARS-CoV-2 virus infection.

The invention further relates to use of the polypeptide of any one of the invention, the lipopeptide building block of any one of the invention, the synthetic virus like particle of invention, or the pharmaceutical composition of the invention in preventing and treating SARS- CoV or SARS-CoV-2 diseases and in preparing antibodies.

The invention further relates to a diagnostic test method for determining the presence of antibodies against SARS-CoV or SARS-CoV-2 virus in a sample, comprising the steps of (i) putting said sample into contact with a polypeptide according to the invention or a conjugate according to the invention and (ii) determining whether antibodies bind to said peptide. Preferably, said sample is from a human subject potentially infected with a SARS-CoV or SARS-CoV-2 virus.

The invention further relates to a diagnostic test method for determining the presence of SARS-CoV or SARS-CoV-2 virus in a sample, comprising the steps of (i) putting a sample with a SARS-CoV or SARS-CoV-2 antibody to be tested into contact with a polypeptide according to the invention or a conjugate according to the invention and (ii) determining whether the antibody binds to the polypeptide or a conjugate according to the invention. Preferably, said sample is from a human subject potentially infected with a SARS-CoV or SARS-CoV-2 virus.

EXAMPLES

EXAMPLE 1: Design and synthesis of mimetics of SARS-CoV-2 polypeptides

A Z-Mimetic-1

L-Lys(N₃)

In Z-mimetic-1, the residue Z at the N-terminus is L-azidolysine (L-Lys(N3)), with an azido group in its side chain, as shown. The 4 cysteine residues in the peptide sequence are linked through two disulfide bonds, as depicted in the formula above. The C-terminus of the peptide is an amide (CONFb).

The synthesis of Z-mimetic 1 was carried out using Fmoc solid phase peptide synthesis (SPPS). Solvents were purchased in HPLC or peptide synthesis grade quality. N- Methylpyrrolidone (NMP) and piperidine were used without further purification. Dimethylformamide (DMF) was redistilled under vacuum from ninhydrin in order to remove amine impurities. Di-isopropylethylamine (DIPEA) was first redistilled from ninhydrin and subsequently from KOH. Rink amide resin (100-200 mesh) was used. Fmoc protected amino acid (all with the L-absolute configuration except where noted) used were Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc- Cys(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(N3)-OH, Fmoc-Pro-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH and Fmoc-Val-OH if not stated otherwise. Coupling reagents were:

HBTU 2-(lH-benzotriazol- 1 -yl)- 1 , 1 ,3 ,3 -tetramethyluronium hexafluorophosphate HOBt hydroxybenzotri azole

The methods of solid-phase peptide synthesis are well-known to those knowledgeable in the field. A typical method follows here. The peptide was synthesized on an automated peptide synthesizer with a UV/Vis detector on a 0.25 mmol scale. For each amino acid coupling, a protected amino acid (4 eq, 1 mmol) was activated with 0.45 M HBTU/HOBt (3.6 eq) in DMF and 2 M DIPEA (6.0 eq) in NMP. The coupling was performed in NMP for 20 min. After washing with NMP, the resin was capped using a solution of acetic anhydride and 2,6-lutidine in DMF (5:6:89) for 5 min. The N-terminal Fmoc group was removed with a solution of 20% piperidine in NMP for 10 min and the deprotection was monitored by detection of the released fulvene derivative by UV at 301 nm. The deprotection cycle was repeated until the absorption was less than 4% compared to the previous cycle.

The synthetic route to Z-mimetic-1 is shown in the scheme- 1 below:

Fmoc peptide synthesis

Z — YCYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQCY-NH₂

Scheme-1. Route for the synthesis of Z-mimetic-1.

The synthesis of Z-mimetic-1 was started (step-1) by coupling Fmoc-Tyr(tBu)-OH to the resin, and the assembly was continued by the repeated sequential coupling of each amino acid, until the final coupling of Fmoc-Lys(N3)-OH to the resin-bound peptide. The N-terminal Fmoc group was then removed by treatment with 20% piperidine in NMP for 10 min. The resin was washed with DMF (2 x 3 mL) and dichloromethane (DCM) (2 x 3 mL). The peptide was deprotected and cleaved from the resin (step-2) with a solution of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and water (90/9/1, 10 ml). The resin was shaken under argon atmosphere for 3.5 h. After washing the resin with DCM (5 ml, 3 x 2 min), the peptide was precipitated with cold diethylether (Et₂0) (40 ml) and collected by centrifugation. The pellet was washed with Et₂0 (3 x 50 ml) and air-dried overnight. The resulting peptide (step-3) was redissolved in a mixture of water and dimethylsulfoxide (DMSO) (20mL) and stirred in air for 3 h, during which time the first disulfide bond was formed between the Cys residues indicated in the scheme above. When complete, the solution was then freeze dried. Subsequently, the peptide (step-4) was redissolved in a mixture of acetic acid and water (1:1, 50 mL) and iodine (25x excess) was added with stirring. When the reaction is complete, the excess iodine was neutralized with 1M ascorbic acid in water. The solution was then freeze dried and the Z- mimetic-1 was purified by preparative HPLC.

The product was dissolved in ThO/MeCN (2:1) and purified by RP-HPLC on a C18 preparative column. The fractions containing the product were combined and lyophilized to give the product. The peptide was then analysed by reverse-phase HPLC (RP-HPLC) on a Cl 8 analytical column using a gradient of 0-100% of eluent A (H2O+0.1% TFA) and eluent B (60% MeCN+40% H2O and 0.1% TFA) over 40 min. (t_R = 28.0 min), purity: > 94%. Electrospray ionization MS (ESI-MS) m/z 5473.06 [M+H]⁺.

B Z-Mimetic-2

Z-GWCSNNLDSKVGGNYNYLYrPQSYGFQPTNGVGYQPCR ^~NH 2

L-Lys(N₃) O

In Z-mimetic-2, the residue Z at the N-terminus is L-azidolysine (L-Lys(N3)), with an azido group in its side chain, as shown. The two cysteine residues in the peptide sequence are linked through a disulfide bond, as depicted in the formula above. The residue in the sequence denoted as "r" is a D-amino acid (indicated by the lower-case letter), in this case D-Arg. The C -terminus of the peptide is an amide (CONH2).

The synthetic route to mimetic-2 and Z-mimetic-2 is shown in the scheme-2 below. The methods required for the synthesis of Z-mimetic-2 are the same as those described above for the synthesis of Z-mimetic-1, except that step-4 in scheme- 1 is not required. The synthesis of Z-mimetic-2 is started (step-1) by coupling Fmoc-Arg(Pbf)-OH to the resin, and the assembly is continued by the repeated sequential coupling of each amino acid, until the final coupling of Fmoc-Lys(N3)-OH to the resin-bound peptide. A single D-Arg residue (denoted with small case "r") is incorporated during the assembly process, as shown in Scheme-2. Fmoc peptide synthesis

L-Lys(N₃) v

Iris Biotech

Z-GWCSNNLDSKVGGNYNYLYrPQSYGFQPTNGVGYQPCR-NHj

Scheme-2. Route for the synthesis of Z-mimetic-2.

After removal of the N-terminal Fmoc protecting group, the peptide is cleaved from the resin and side chain protecting groups are removed (step-2) with a solution of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and water (90/9/1). The resulting peptide is redissolved in a mixture of water and dimethylsulfoxide (DMSO) (20mL) and stirred in air for 3 h (step-3), during which time the first disulfide bond is formed between the Cys residues indicated in Scheme-2. The product is dissolved in FhO/MeCN (2:1) and purified by RP-HPLC on a C18 preparative column. The fractions containing the product are combined and lyophilized to give the product. The peptide is then analysed by RP-HPLC on a C18 analytical column using a gradient (0%B to 100%B) comprising eluant-A (water + 0.1% TFA) and eluant-B (60%/40% MeCN/water+0.1% TFA) over 40 min at 1 mL/min (t_R = 24.5 min), purity: > 92%. ESI-MS m/z 4456.82 [M+H]⁺.

C. Z-Mimetic-3

In Z-mimetic-3, the residue Z at the N-terminus is L-azidolysine (L-Lys(N3)), with an azido group in its side chain, as shown. The 4 cysteine residues in the peptide sequence are linked through two disulfide bonds, as depicted in the formula above. The C-terminus of the peptide is an amide (CONH2).

The synthetic route to Z-mimetic-3 is shown in the scheme-3.1 below. The methods required for the synthesis of Z-mimetic-3 are the same as those described above for the synthesis of Z-mimetic-1. The synthesis of Z-mimetic-3 is started (step-1) by coupling Fmoc- Arg(Pbf)-OH to the resin, and the assembly is continued by the repeated sequential coupling of each amino acid, until the final coupling of Fmoc-Lys(N3)-OH to the resin-bound peptide.

Fmoc peptide synthesis

Z-GWCSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPCR-NH, I - 1

Scheme-3.1 Route for the synthesis of Z-mimetic-3.

After removal of the N-terminal Fmoc protecting group, the peptide is cleaved from the resin and side chain protecting groups are removed (step-2) with a solution of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and water (90/9/1). After removing the resin, the solution is freeze dried. The resulting peptide is redissolved in a mixture of water and dimethylsulfoxide (DMSO) (20mL) and stirred in air for 3 h (step-3), during which time the first disulfide bond is formed between the Cys residues indicated in Scheme-3.1. When complete, the solution is then freeze dried. Subsequently, the peptide (step-4) is redissolved in a mixture of acetic acid and water (1:1) and iodine (25x excess) is added with stirring. When the reaction is complete, the excess iodine is neutralized with 1M ascorbic acid in water. The solution is then freeze dried and the Z-mimetic-3 is purified by preparative HPLC, using RP-HPLC on a Cl 8 preparative column using a gradient (0%B to 100%B) comprising eluant-A (water + 0.1% TFA) and eluant- B (60%/40% MeCN/water+0.1% TFA) over 40 min at 1 mL/min (t_R = 28.9 min), purity: > 90%. MALDI-MS m/z 8682.98 [M+H]⁺. D. Z-Mimetic-4 GFQPTNGVGYQPCR-NH₂

In Z-mimetic-4, the residue Z at the N-terminus is L-azidolysine (L-Lys(N₃)), with an azido group in its side chain, as shown. The 4 cysteine residues in the peptide sequence are linked through two disulfide bonds, as depicted in the formula above. The mimetic contains D- proline and L-proline in the sequence as shown. The C-terminus of the peptide is an amide (CONFh).

The synthetic route to Z-mimetic-4 is shown in the scheme 3.2 below. The methods required for the synthesis of Z-mimetic-4 are the same as those described for the synthesis of Z-mimetic-1. The synthesis of Z-mimetic-4 is started (step-1) by coupling Fmoc-Arg(Pbf)-OH to the resin, and the assembly is continued by the repeated sequential coupling of each amino acid, until the final coupling of Fmoc-Lys(N3)-OH to the resin-bound peptide.

Fmoc peptide synthesis

₁ I Fmoc peptide synthesis 1 normal side chain protection

Acm Trt

1 I H

Z — GWCSNNLDSKVGGNYNYCY-^DPro-^LPro-QCYGFQPTNGVGYQPCR — N — .0

Z-GWCSNNLDSKVGGNYNYCY-DPro-LPro-QCYGFQPTNGVGYQPCR-NH₂ Scheme-3.2 Route for the synthesis of Z-mimetic-4.

After removal of the N-terminal Fmoc protecting group, the peptide is cleaved from the resin and side chain protecting groups are removed (step-2) with a solution of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and water (90/9/1). After removing the resin, the solution is freeze dried. The resulting peptide is redissolved in a mixture of water and dimethylsulfoxide (DMSO) (20mL) and stirred in air for 3 h (step-3), during which time the first disulfide bond is formed between the two internal Cys residues indicated in Scheme-3.2. When complete, the solution is then freeze dried. Subsequently, the peptide is redissolved in a mixture of acetic acid and water (1:1) and iodine (25x excess) is added with stirring (step-4). When the second disulfide bond formation is complete, the excess iodine is neutralized with 1M ascorbic acid in water. The solution is then freeze dried and the Z-mimetic-4 is purified by preparative HPLC, using RP-HPLC on a Cl 8 preparative column using a gradient (0%B to 100%B) comprising eluant-A (water + 0.1% TFA) and eluant-B (60%/40% MeCN/water+0.1% TFA) over 40 min at 1 mL/min (t_R = 23.6 min), purity: > 90%. MALDI-MS m/z 4399.9 [M+H]⁺.

E Z-Mimetic-5

Z-KCERLFRKSNLK-^LPro-^DPro-ERDISTEI

In Z-mimetic-5, the residue Z at the N-terminus is L-azidolysine (L-Lys(N3)), with an azido group in its side chain, as shown for Z-mimetic-4. The 4 cysteine residues in the peptide sequence are linked through two disulfide bonds, as depicted in the formula above. The mimetic contains L-proline and D-proline in the sequence as shown. The C-terminus of the peptide is an amide (CONFb). The synthetic route to Z-mimetic-5 is shown in the scheme 3.3 below. The methods required and applied for the synthesis of Z-mimetic-5 are the same as those described for the synthesis of Z-mimetic-1 and -4.

Z

Z-KCERLFRKSNLK-^LPro-^DPro-ERDISTEIYQAGSTPCNGVEGFNCYFPLQCR— NH₂

Scheme-3.3 Route for the synthesis of mimetic-5.

EXAMPLE 2: Lipopeptide building block with coupling moiety

A. Lipopeptide+coupling moiety B1 (EBB-1-B1)

This lipopeptide building block contains a coiled-coil domain ((IEKKIEA)₄) with four heptad repeats (IEKKIEA). The C-terminus of the peptide moiety is a D-Ala (written "a" in the formula above), with an amide (CONEb) instead of a free carboxyl terminus. The lipid group Pani2Cys is conjugated to the N-terminus of the lipid moiety. Attached to the side chain of the Cys residue is the coupling moiety (B), in this case coupling moiety B1 as shown, with the sulphur of Cys conjugated to a maleimido group and a chain terminating in an alkyne group, as shown in the formula.

For the synthesis of LBB-1-B1, the lipopeptide 1 shown below is used (see Scheme-4A). The lipopeptide 1 was synthesized and purified by RP-HPLC, essentially as described in WO 2008/068017. The lipopeptide 1 was analyzed by analytical RP-HPLC and matrix-assisted laser-desorption ionization time-of-flight MS (MALDI-TOF). Analytical RP-HPLC (Agilent VariTide reverse phase column, 0 to 95% MeCN in ¾0 (+ 0.1% TFA) over 63 min.): Purity= 95%, t_R = 22.71 min. MALDITOF: m/z 6795 [M+l]⁺. The lipopeptide of step (1) (7 mg) was dissolved in 0.5 ml ¾0 and added to a solution of the coupling moiety (2) (0.4 mg) of step, as shown in Scheme-4A (1.1 eq.) in 2 ml 50% MeCN. The pH was adjusted to pH = 6.5 with 0.1 N NaOH and 0.1 N HC1 and the mixture was stirred at r.t. for2.5 h. The product lipopeptide building block LBB-1-B1 was purified by HPLC on a C8 column. The TFA was removed using AG-X2 anion exchange resin (acetate form). The conjugate was analyzed by analytical UPLC and MS. UPLC (ACQUITY UPLC C8 column, 1.7pm, 2.1x150 mm 40 to 80% MeCN in H₂0 (+ 0.1 % TFA) over 50 min., 40°C): Purity 92%, t_R = 20.8 min. ESI-MS: m/z 7106.1 [M+H]⁺.

Scheme-4A. Synthesis of the lipopeptide+coupling moiety (LBB-1-B1)

B. Synthesis of the further lipopeptides

In LBB-2 the lipid Pam2-Cys of LBB-2 has the R-configuration at the chiral 2-propyl carbon atom and the R-configuration of the chiral carbon of the cysteinyl moiety. LBB-2 was synthesized and purified by RP-HPLC as described above for LBB-1 as described in WO 2008/068017 (Agilent VariTide reverse phase column, 0 to 95% MeCN in FLO (+ 0.1% TFA) over 63 min.): Purity= 95%, t_R = 22.71 min. MALDI-TOF: m/z 6795 [M+l]⁺.

LBB-4

LBB-4 contains a coiled-coil domain, which has serine in the “c” positions of the heptad repeat “defgabc” IEKKIES. LBB-4 was synthesized and purified by RP-HPLC as described above for LBB-1 and 2 but with serine in the “c” positions of the heptad repeat “defgabc” and analyzed by analytical RP-HPLC and MALDI-MS. HPLC (Zorbax C8 column, 30 to 100% MeCN in H₂0 (+ 0.1% CHOOH) over 10 min): Purity: 90%, t_R = 4.81 min.; MALDI-MS: m/z 6860.61 [M+H]⁺.

This lipopeptide building block LBB-5 corresponds to LBB-4 except that the lipid Pam₂- Cys has the //-configuration at the chiral 2-propyl carbon atom and the //-configuration of the chiral carbon of the cysteinyl moiety. LBB-5 was synthesized and purified by RP-HPLC as described above for LBB-1, -2 or -3 (Agilent VariTide RPC, 0 to 95% MeCN in H₂0 (+ 0.1% TFA) over 63 min.): Purity 97.0%, t_R = 45.58 min. MALDI-MS: 6860.46 Da [M+H]⁺.

C. Lipopeptide LBB-5+coupling moiety B3 (LBB-5-B3)

For the synthesis of LBB-5-B3, the lipopeptide LBB-5 and coupling moiety 3 are used (see Scheme-4B). The lipopeptide was synthesized and purified by RP-HPLC, essentially as described in WO 2008/068017 and Ghasparian, Riedel et al., (Chembiochem, 2011, 12, 100- 109). The lipopeptide was analyzed by analytical RP-HPLC and matrix-assisted laser- desorption ionization time-of-flight MS (MALDI-TOF). Analytical RP-HPLC (Agilent VariTide reverse phase column, 0 to 95% MeCN in H2O (+ 0.1% TFA) over 63 min.): Purity= 95%, t_R = 23.0 min. MALDI-TOF: m/z 6859 [M+l]⁺.

The lipopeptide of step (1) (7 mg) was dissolved in 0.5 ml FhO and added to a solution of the coupling moiety (B3) (0.4 mg), as shown in Scheme-4B (1.1 eq.) in 2 ml 50% MeCN. The pH was adjusted to pH = 6.5 with 0.1 N NaOH and 0.1 N HC1 and the mixture was stirred at r.t. for 2.5 h. The product lipopeptide building block LBB-5 was purified by HPLC on a C8 column. The TFA was removed using AG-X2 anion exchange resin (acetate form). The conjugate LBB-2 was analyzed by analytical UPLC and MS. UPLC (ACQUITY UPLC C8 column, 1.7pm, 2.1x150 mm 40 to 80% MeCN in H2O (+ 0.1 % TFA) over 50 min., 40°C): Purity 92%, t_R = 23.0 min. ESI-MS : m/z 7241 [M+H]⁺.

Scheme-4B. Synthesis of the lipopeptide LBB-5+coupling moiety B3 (LBB-5-B3) EXAMPLE 3: Conjugates

A. Coniugates- -4 and -7 comprising B1 and mimetic 1

The lipopeptide building block+coupling moiety LBB-1-B1, LBB-4-B1 or LBB-5-B1 is each independently conjugated to Z-mimetic-1 through the side chain of the azidolysine residue Z (Lys(N₃)) at the N-terminus of the mimetic, to give conjugate-1, -4 or -7. Conjugate 1 is exemplarily shown below. Conjugate-4 and -7 are synthesized as conjugate- 1:

Exemplarily conjugate-1 LBB-1-B1 coupled to Z-mimetic-1

The coupling reaction is exemplarily depicted in Scheme-5 for conjugate 1.

Scheme-5. Exemplarily preparation of conjugate-1 (LBB-1-B1 coupled to Z-mimetic-1)

For the coupling, the Z-mimetic-1 (6 mg) was dissolved in phosphate buffered saline (PBS) (1 mL). This was added to a solution of the lipopeptide LBB-1, -4 or -5 + coupling moiety (LBB-1, -4, -5 + Bl) (7 mg) dissolved in phosphate buffered saline (PBS) (1 mL) containing sodium ascorbate (0.1M, 100 pL) and CuSCL (0.1M, 100 pL). The solution was stirred at room temp. After lh another aliquot of sodium ascorbate (70 pL) and CuSCL (70 pL) was added and stirring continued for 1 h. To the reaction was then added water with 0.1% TFA (1 mL). The product, conjugate-1 using LBB-1+B1 and Z-mimetic-1, conjugate-4 using LBB-

4+B1 and Z-mimetic-1 and conjugate-7 using LBB-5+B1 and Z-mimetic-7, was then purified by preparative RP-HPLC on a C2 semi-preparative HPLC column using a gradient of 40-70% MeCN in water containing 0.1% v/v TFA over 40 min. The conjugate was then analysed by RP-HPLC on a C2 analytical column using a gradient of 20 to 100% MeCN in H2O (+0.1% TFA) over 17 min at 1 mL/min (t_R = 12 min), purity > 90%. MALDI-TOF: m/z 12577 [M+H]⁺.

B Coniugates-2. -5 and -8 comprising B1 and Z-mimetic-2

The lipopeptide building block+coupling moiety, LBB-1, -4, -5 + B1 is conjugated to Z- mimetic-2 through the side chain of the azidolysine residue Z (Lys(N₃)) at the N-terminus of the mimetic. For synthesis of conjugate-2, -5 and -8, the same method is used for the coupling as used above for the synthesis of conjugate-1, -4 and -7. The product is purified by preparative RP-HPLC on a C2 semi-preparative HPLC column using a gradient of 40-70% MeCN in water containing 0.1% v/v TFA over 40 min. The conjugate is analysed by RP-HPLC on a C2 analytical column using a gradient of 20 to 100% MeCN in H2O (+0.1% TFA) over 17 min at 1 mL/min (t_R = 14 min), purity > 90%. MALDI-TOF: m/z 11561 [M+H]⁺.

Exemplarily conjugate-2 (LBB-1-B1 coupled to Z-mimetic-2) C. Coniugates-3. -6 and 9 comprising B1 and Z-mimetic-3

The lipopeptide building block+coupling moiety, LBB-1, -4 or -5 + B1 is conjugated to Z-mimetic-3 through the side chain of the azidolysine residue Z (Lys(N₃)) at the N-terminus of the mimetic. For synthesis of conjugate-3, -6 and -9, the same method is used for the coupling as used above for the synthesis of conjugate-1, -4 and -7. The product is purified by preparative RP-HPLC on a C2 semi-preparative HPLC column using a gradient of 40-70% MeCN in water containing 0.1% v/v TFA over 40 min. The conjugate is analysed by RP-HPLC on a C2 analytical column using a gradient of 20 to 100% MeCN in FhO (+0.1% TFA) over 17 min at 1 mL/min (t_R = 10 min), purity > 90%. MALDI-TOF: m/z 15784 [M+H]⁺.

Exemplarily conjugate-3 (LBB-1-B1 coupled to Z-mimetic-3)

D. Conjugates 14 to 23 comprising B3 and Z-mimetic-1 to 5

The lipopeptide building block LBB-4 or LBB-5+coupling moiety B3 is conjugated to Z- mimetics-1, 2, 3, 4, and 5 through the side chain of the azidolysine residue Z (Lys(N3)) at the N-terminus of the mimetic, to give conjugates 14-23. Conjugate 22 is exemplarily shown below in scheme 6. The coupling reaction is depicted in Schemes-6 and -7 exemplarily for mimetic 4 and 5 resulting in conjugates 22 and 18.

Mimetic-4 + phosphate buffer + MeCN CuS0₄, sodium ascorbate

o

H₂N

Scheme-6. Exemplarily preparation of conjugate-22 (LBB-5-B3 coupled to Z-mimetic-4)

For the coupling, mimetic-1, 2, 3, 4 or 5 (6 mg) was dissolved in phosphate buffered saline (PBS) (1 mL). This was added to a solution of the lipopeptide+coupling moiety B3 (7 mg) dissolved in phosphate buffered saline (PBS) (1 mL) containing sodium ascorbate (0.1M, 100 pL) and CuS04 (0.1M, 100 pL). The solution was stirred at room temp. After lh another aliquot of sodium ascorbate (70 pL) and CuS04 (70 pL) was added and stirring continued for 1 h. To the reaction was then added water with 0.1% TFA (1 mL). The product was then purified by preparative RP-HPLC on a C2 semi-preparative HPLC column using a gradient of 40-70% MeCN in water containing 0.1% v/v TFA over 40 min. The conjugate was then analysed by RP-HPLC on a C2 analytical column using a gradient of 20 to 100% MeCN in H20 (+0.1% TFA) over 17 min at 1 mL/min (tR = 21 min), purity > 90%. MALDI-TOF : m/z 11641 [M+H]+.

Z-KCERLFRKSNLK-^LPro-^DPro-ERDISTEIYQAGSTPCNGVEGFNCYFPLQCR— NH₂

Z-mimetic-5

Scheme-7. Exemplarily preparation of conjugate-18 (LBB-4-B3 coupled to Z-mimetic-5) EXAMPLE 4: Immunological studies

For the analysis of antibody responses, 6-8 week-old female BALB/c mice (5 - 6 animals per group) are subcutaneously immunized three times in three-week intervals subcutaneously with 0.1 ml of a formulations of conjugate-1, or conjugate-2, or conjugate-3, each dissolved in phosphate-buffered saline (PBS), with the amounts of administered immunogen in the range 2- 100 pg. Control animals are immunized with PBS.

Blood is collected before the first and 10 days after the third immunization, and the sera are analyzed by enzyme linked immunosorbent assay (ELISA) for antibodies binding to the corresponding conjugate- 1, or -2 or -3. The ELISA is performed essentially as described using MaxiSorp 96-well microtitre plates (Nunc, Fischer Scientific) which are coated at 4°C overnight with 5 mΐ/ml solutions of each conjugate in PBS, pH 7.2 in 50 mM sodium carbonate buffer. The wells are washed with PBS containing 0.05% Tween 20 (PBST) and blocked with PBS containing 5% skimmed milk powder for 1 h at r.t. After blocking, the wells are washed three times with PBST and incubated with serial four fold-dilutions of mouse sera in PBS containing 0.05% Tween 20 and 0.5% skimmed milk powder (MPBST) for 2 h at r.t., followed by three washes with PBST. The plates are then incubated with anti -Mouse IgG (Fc specific)- peroxidase antibody produced in goat (Sigma, St. Louis, MO), diluted E15Ό00 in MPBST for

1 h at r.t., washed again three times with PBST and incubated in the dark with 3,3 ',5,5'- Tetramethylbenzidine (TMB) solution (T0440, Sigma) for 15 min. The color reaction is stopped by addition of 0.16 M H2S04 and the absorbance in the wells is read at 450 nm on a plate reader. IgG titers are calculated as reciprocal serum dilutions corresponding to half-maximal binding concentrations (EC50) are. Mean titers (loglO) ± one standard errors for each of the conjugates are in the range 3.5±0.1 to 5.2 ± 0.2.

An ELISA is also performed to measure antibody binding to commercially available recombinant SARS-CoV2 spike glycoprotein S manufactured in HEK293 mammalian cells. The same method is used as above, except that the microtitre plates are coated with the S spike glycoprotein. The assay revealed strong binding of antibodies in the mouse sera, with mean titers (loglO) ± one standard errors, for conjugate-1 in the range 3.0±0.3 to 4.1 ± 0.2, for conjugate-2 in the range 3.1±0.3 to 4.0 ± 0.3, and for conjugate-3 in the range 3.5±0.2 to 4.5 ± 0.3.

To measure neutralizing activity of the mouse anti-sera for each conjugate, a SARS-CoV-

2 pseudovirus neutralization assay is used. The assay uses 293T cells expressing the ACE2 receptor. Such cell lines are commercially available. The protocol used is based on published procedures (J. Vis. Exp. 2019, (145):doi: 10.3791/59010). Briefly, SARS-CoV2 spike fusion protein pseudotyped particles are generated with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure with the widely available HEK-293T cell line. Using the luciferase assay, transduced cells can be easily quantified. The luciferase luminescence value reaches up to 106 RLU after pseudovirus infection. For neutralization assays, the IgG fraction is purified from mouse sera immunized with the three conjugates and used in serial dilutions in the range 0.05 - 1.0 mg/mL IgG. Neutralizing activity is observed with IgG isolated from all the mice immunized with conjugate- 1, -2 or -3. An IC50 value for inhibition of pseudovirus infection varied typically in the range 0.2-10 pg/mL of IgG.

EXAMPLE 5: Assessing immunogenicity in mice

Mouse immunogenicity studies were used to evaluate the mimetics-1 to -5.

Immunogenicity study

Female Balb/c mice (4 per group) were subcutaneously immunized with 50 pg or 100 pg SVLPs carrying epitope mimetics-1 to -5 (Ml to M5; SVLP-M1 to -M5), as well as ‘naked’-SVLPs. The immunization was performed twice, on days 1 and 21 (Prime-boost immunizations). Blood samples were collected on days 0, 14, 20, 28 and 35 for IgG antibody analyses. Readouts were IgG titers and Pseudovirus neutralization assay.

ELISA measurements of IgG titers

Plates were coated with 5 pg/mL of free peptide mimetics 1 to 5 (Ml to M5) in PBS, overnight at 4°C. The following day plates were washed three times with wash buffer (lxPBS, 0.05 % Tween-20) and further blocked with blocking buffer (lxPBS, 3% BSA). Sera from blood samples were serially diluted (1 : 100 to 1 :6400) in lxPBS buffer and incubated on the plates for lh at 37°C. Following three times washing with wash buffer, plates were incubated with secondary anti-mouse antibody 1:20000 for lh at 37°C. Plates were washed three times and treated with TMB substrate reagent set. Reactions were stopped with 0.16M sulfuric acid. Absorbance was measured at 450 nm with a Perkin Elmer, Multimode Plate Reader EnSpire. Pseudovirus neutralization assay

Spike-pseudotyped lenti viruses from the D614G strain carrying a firefly luciferase (FLuc) reporter gene were generated by plasmid co-transfection in 293T cells. Pseudovirus supernatants were collected 48 h post-transfection, filtered through a 0.45pm filter, titrated with ELISA (titers were expressed as relative luminescence unit per mL of pseudovirus supernatants (RLU/ml)) and either used immediately or stored at -80°C. The pseudovirus neutralization assays were performed with 293T cells stably expressing the ACE2 receptor. Cells were seeded at 104 cells per well into 96-well plates in DMEM medium with 10% FCS. An original 1:100 dilution of serum samples, and subsequently three-fold dilutions were prepared and pre incubated for lh at 37oC with an equal volume of pseudovirus in DMEM plus 10% FCS, before being added to the cells. After 48h of incubation at 37 °C, a luciferase assay was performed to monitor pseudoviral infection. The serum dilution causing a 50% and 80% reduction of RLE1 compared to control (ID50 and ID80, respectively) were reported as the neutralizing antibody titers.

Results and conclusions: Immunization of mice with all five different SVLP formulations, namely SVLP-M1, SVLP- M2, SVLP -M3, SVLP-M4 and SVLP-M5, results in measurable levels of IgG responses, as determined by ELISA against the respective epitope mimetics (cf. Figures 4 and 5). The antibody titers increase over time, and till the study endpoint, also suggesting the positive effect of the boost (second) immunization. In particular, SVLP -M3 and SVLP-M5 formulations show the highest antibody responses.

Claims

1. A polypeptide comprising (i) an amino acid sequence of SEQ ID NO: 1 :

Xi SNNLD SK V GGNYN YX₂ YRLFRK SNLKPFERDIS TEI Y Q AGS TPCN GV EGFNCYFPLQX3YGFQPTNGVGYQPX4, wherein each of XI to X4 is independently at least one amino acid; or

(ii) a variant of SEQ ID NO: 1, wherein in said variant at most 33 amino acids are exchanged as compared to SEQ ID NO: 1; or

(iii) a fragment of SEQ ID NO: 1 or a fragment of the variant of SEQ ID NO: 1.

2. The polypeptide of claim 1, wherein SEQ ID NO: 1, said variant or said fragment comprises 2 to 8 cysteines, preferably 2 to 4 cysteines.

3. The polypeptide of any one of the preceding claims, wherein XI, X2 X3 and X4 is independently of each other 1 to 5 amino acids, more preferably 1 to 3 amino acids, most preferably 1 or 2 amino acids.

4. The polypeptide of claim 1 or 2, wherein (i) each of XI and X4, (ii) each of X2 and X3, or (iii) each of XI, X2, X3 and X4 is a cysteine.

5. The polypeptide of any of the preceding claims, wherein said fragment is at least 20 amino acids long, preferably said fragment is between 25 and 80 amino acids long.

6. The polypeptide of any of the preceding claims, wherein said fragment of SEQ ID NO: 1 or of a variant of SEQ ID NO: 1 consists of a single continuous sequence stretch or two discontinuous sequence stretches of SEQ ID NO: 1 or the variant of SEQ ID NO: 1, wherein

(i) the first of said two discontinuous sequence stretches or the continuous sequence stretch starts between amino acid positions 13 and 18 and the second discontinuous sequence stretch or the continuous sequence stretch ends between amino acid positions 56 and 60 of SEQ ID NO: 1 or the variant thereof, and each of said first and second discontinuous sequence stretches has a length between 3 and 40 amino acids; or (ii) the first of said two discontinuous sequence stretches starts between amino acid positions 1 and 3 and ends between amino acid positions 16 and 20 of SEQ ID NO: 1 or said variant, and the second of said two discontinuous sequence stretches starts between amino acid positions 55 and 59 and ends between amino acid positions 70 and 72 of SEQ ID NO: 1 or said variant.

7. The polypeptide of any of the preceding claims, wherein amino acids at positions 15, 25, 43, 44, 56, 60 and 66 of SEQ ID NO: 1 are conserved in said variant of SEQ ID NO: 1 or in the fragment of said variant of SEQ ID NO: 1, if included.

8. The polypeptide of any of the preceding claims, wherein amino acids at positions 4, 14, 15, 25, 29, 31, 32, 43, 44, 56, 60, 61, 64 and 66 of SEQ ID NO: 1 are conserved in said variant of SEQ ID NO: 1 or in the fragment of said variant of SEQ ID NO: 1, if included, preferably amino acids at positions 4, 6, 11-15, 18, 21, 25, 27-33, 43, 44, 51-53, 55, 56, 59, 60, 61, 64, 66 and 68-71 of SEQ ID NO: 1 are conserved in said variant of SEQ ID NO: 1 or in the fragment of said variant of SEQ ID NO: 1, if included.

9. The polypeptide of any of the preceding claims, wherein SEQ ID NO: 1 consists of SEQ ID NO: 2 or 5, and the fragment of SEQ ID NO: 1 consists of a sequence selected from the group consisting of SEQ ID NO: 3, 4, 6, 7, and 51-54, wherein in the variant of SEQ ID NO: 1 and in the fragment of the variant of SEQ ID NO: 1 at most 33 amino acids are exchanged as compared to the corresponding non-modified SEQ ID NO: 2, 3, 4, 5, 6, 7, or 51-54.

10. The polypeptide of any of the preceding claims, wherein SEQ ID NO: 1 and the variant of SEQ ID NO: 1 each consists of a sequence SEQ ID NO: 2 or 5, and the fragment of SEQ ID NO: 1 and the fragment of the variant of SEQ ID NO: 1 each consists of a sequence selected from the group consisting of SEQ ID NO: 3, 4, 6, 7 and 51-54.

11. The polypeptide of any one of the preceding claims, wherein the amino acid sequence of SEQ ID NO: 1, the variant of SEQ ID NO: 1, the fragment of SEQ ID NO: 1 and the fragment of said variant of SEQ ID NO: 1 is capable of eliciting an antibody against residues 437-508 of a receptor binding domain of SARS-CoV-2 or against residues 424- 494 of a receptor binding domain of SARS-CoV.

12. A nucleic acid molecule encoding the polypeptide of any one of the preceding claims.

13. A conjugate comprising (i) the polypeptide of claims 1 to 11,

(iii) a lipid moiety comprising two or three, preferably two hydrocarbyl chains, wherein the peptide moiety is covalently linked at one end to the polypeptide of claims 1 to 11 and at the other end to the lipid moiety, either directly or through a coupling moiety.

14. A synthetic virus-like particle (sVLP) consisting of helical lipopeptide bundles comprising two, three, four, five, six or seven conjugates of claim 13.

15. The polypeptide, the conjugate, or the synthetic virus like particle of any one of the preceding claims for use as a vaccine against SARS-CoV diseases and SARS-CoV-2 diseases and for use in preventing or treating SARS-CoV diseases and SARS-CoV-2 diseases.