WO2022106860A1

WO2022106860A1 - Recombinant peptides for use in therapy

Info

Publication number: WO2022106860A1
Application number: PCT/HU2021/050063
Authority: WO
Inventors: Antal TAPODI
Original assignee: Pécsi Tudományegyetem
Priority date: 2020-11-20
Filing date: 2021-11-20
Publication date: 2022-05-27

Abstract

The invention relates to recombinant peptides for use in therapy, in particular in the treatment, e.g.prevention of an infection or a cancer.

Description

RECOMBINANT PEPTIDES FOR USE IN THERAPY

FIELD OF THE INVENTION

The invention relates to recombinant peptides for use in therapy, in particular in the treatment, e.g. prevention of an infection or a cancer.

BACKGROUND OF THE INVENTION

New vaccine platforms are a must in an era of pathogenic agents spreading with unprecedented speed. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of the Coronaviridae family, causing Coronavirus disease 2019 (COVID-19), a life threatening disease. The Spike protein of SARS-CoV-2, which mediates cell entry and membrane fusion, is the principal target of the humoral immune response (Watanabe et al., Science 369, 330-333 (2020) 17 July). Self-organizing continuous peptidic chains are known to be capable of forming nanoparticles. Such nanoparticles may be used as a drug delivery system, e.g. in a vaccine composition (EP 1 594 469). However, the need for effective vaccine compositions against SARS-CoV-2 still exists.

SHORT DESCRIPTION OF THE INVENTION

A recombinant peptide comprising a trimeric coiled-coil peptide sequence and a second peptide sequence, for use in therapy is provided, wherein the trimeric coiled-coil peptide sequence comprises or consists of a sequence selected from the group consisting of a GCN4 sequence, sequences according to SEQ ID Nos 1-4 and functional variants and functional fragments thereof, wherein the functional variant or the functional fragment is a trimeric coiled-coil and has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with the GCN4 sequence or the sequence according to any one of SEQ ID Nos 1-4, respectively and the second peptide sequence comprises or consists of a sequence selected from the group consisting of SEQ ID No 5-8 and functional variants and functional fragments thereof, wherein the functional variant or the functional fragment has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with the sequence according to any one of SEQ ID Nos 6-9, respectively.

Preferably the second peptide sequence comprises or consists of the sequence according to

SEQ ID NO 5 or a functional or a functional fragment variant thereof, wherein the functional variant or the functional fragment is a pentameric coiled-coil and has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with SEQ ID NO 5. Preferably the second peptide sequence comprises or consists of a sequence according to SEQ ID NO 8 or a functional variant or a functional fragment thereof, wherein the functional variant or the functional fragment is capable of binding to a lipid and has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with SEQ ID NO 8. Preferably the second peptide sequence comprises or consists of a sequence according to

SEQ ID NO 6 or a functional variant or a functional fragment thereof, wherein the functional variant or the functional fragment is capable of binding to a lipid and has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with SEQ ID NO

6.

Preferably the second peptide sequence comprises or consists of a sequence according to

SEQ ID NO 7 or a functional variant or a functional fragment thereof, wherein the functional variant or the functional fragment is capable of binding to a lipid and has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with SEQ ID NO

7.

Preferably the trimeric coiled-coil peptide sequence comprises or consists of the sequence according to

SEQ ID NO 1 or a functional variant or a functional fragment thereof wherein the functional variant or the functional fragment is a trimeric coiled-coil and has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with SEQ ID NO 1.

SEQ ID NO 2 or a functional variant or a functional fragment thereof wherein the functional variant or the functional fragment is a trimeric coiled-coil and has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with SEQ ID NO 2.

SEQ ID NO 3 or a functional variant or a functional fragment thereof wherein the functional variant or the functional fragment is a trimeric coiled-coil and has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with SEQ ID NO 2.

SEQ ID NO 4 or a functional variant or a functional fragment thereof wherein the functional variant or the functional fragment is a trimeric coiled-coil and has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with SEQ ID NO 4. Preferably the trimeric coiled-coil peptide sequence comprises or consists of a GCN4 sequence.

Preferably the recombinant peptide is linked to an immunogenic moiety (IM).

Preferably the recombinant peptide comprises a linker sequence localized between the pentameric coiled-coil peptide sequence and the trimeric coiled-coil peptide sequence, preferably wherein the linker sequence is Gly- Gly.

Preferably the recombinant peptide comprises a spacer localized between the trimeric coiled-coil sequence and the IM, preferably wherein the spacer comprises or consists of the sequence according to SEQ ID NO 9 or a functional variant or a functional fragment thereof that is at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90% or preferably at least 95% identical with SEQ ID NO 9. Preferably the spacer is P (proline) or an conservative substitution of P. Preferably the spacer comprises or consists of the sequence according to SEQ ID NO 10 or a functional variant or a functional fragment thereof that is at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with SEQ ID NO 10. Preferably the spacer has a sequence that renders a configuration (3 dimensional structure) to the recombinant peptide that exposes the IM to the immune system or to the cells of the organism receiving the immunization platform.

Preferably the IM is an immunogenic amino acid sequence.

Preferably the IM is derived from SARS-CoV-2.

Preferably the IM is selected from SEQ ID Nos 11-31 and functional variants and functional fragments thereof, wherein the functional variant or functional fragment is capable of inducing an immune response, preferably against a protein or protein fragment of SARS-CoV-2.

Preferably the recombinant peptide for use is for use in a method of preventing an infection by SARS-CoV-2 or COVID-19 or a symptom of CO VID-19 or ameliorating a symptom of an infection by SARS-CoV-2.

An immunogenic construct comprising a plurality of recombinant peptide molecules as defined above is provided. Preferably the plurality of the recombinant peptide molecules form a nanoparticle and the recombinant peptide is a continous peptide chain.

A vaccine composition comprising a recombinant peptide as above or the immunogenic contract a pharmaceutically acceptable excipient is provided.

A drag delivery system comprising a recombinant peptide as defined in any one of claims 1-5, preferably further comprising a targeting moiety, is provided.

Use of the recombinant peptide in the manufacture of a medicine or an immunogenic construct, preferably a vaccine is provided.

A method for the treatment or prevention of an infection or a cancer is provided, the method comprising the administration of the recombinant peptide linked to an IM, the immunogenic construct or the vaccine composition to a subject in need thereof.

A recombinant peptide for use in therapy is provided, wherein the recombinant peptide comprises or consists of an amino acid sequence depicted in any one of SEQ ID NOs 72-87 or a functional variant or fragment thereof, wherein the functional variant or fragment is capable of inducing an immune response upon administration into a mammal, preferably a human and preferably has a sequence that is at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% identical with the corresponding sequence of SEQ ID NOs 72-87. Preferably the recombinant peptide is for use in a method of preventing an infection by SARS-CoV-2 or CO VID-19 or a symptom of CO VID-19 or ameliorating a symptom of an infection by SARS-CoV-2. A nucleic acid molecule coding for any one of the peptides according to SEQ ID NOs 72-87 is provided.

An immunogenic construct is provided, comprising a plurality of any one of the amino acid sequence depicted in any one of SEQ ID NOs 72-87 or a functional variant or fragment thereof, wherein the functional variant or fragment is capable of inducing an immune response upon administration into a mammal, preferably a human and preferably has a sequence that is at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% identical with the corresponding sequence of SEQ ID NOs 72-87. Preferably the plurality of the amino acid sequences form a nanoparticle.

In preferred embodiments the recombinant peptide, the immunogenic construct or the vaccine composition may may comprise more than one immunogenic moieties. The more than one moieties may be derived from the same pathogen or from different pathogens. For example the recombinant peptide, the immunogenic construct or the vaccine composition may comprise more than one epitope from SARS-CoV-2, for example the S2-C13 + RBM- C14 + RBD-C14 + RBD-N14 + S1S2-C14 epitopes or any combination of these epitopes or the RBM-24 + RBD-24 + S1S2-24 epitopes or any combination of these epitopes.

The invention provides an immunogenic construct comprising an immunogenic amino acid sequence linked to a nanoparticle, wherein the immunogenic amino acid sequence is derived from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

A construct comprising coiled-coil sequences is provided.

Preferably the immunogenic construct (nanoparticle) consists of a plurality of continuous peptidic chains comprising a first coiled-coil peptide sequence, a second coiled-coil peptide sequence and a peptidic linker sequence localized between the first coiled-coil peptide sequence and the second coiled-coil peptide sequence. Preferably the first coiled-coil peptide sequence is a trimeric coiled-coil.

Preferably the second coiled-coil peptide sequence is a pentameric coiled-coil.

Preferably the immunogenic amino acid sequence is linked to the first coiled-coil peptide sequence by a peptidic hinge sequence (spacer).

Preferably the trimeric coiled-coil peptide sequence is a GCN4 sequence or a functional variant thereof.

Preferably the pentameric coiled-coil peptide sequence is the pentamerization domain of the Cartilage Oligomeric Matrix Protein (COMP) or a functional variant or functional fragment thereof.

Preferably the trimeric coiled-coil peptide sequence comprises or consists of an amino acid sequence selected from a GCN4 sequence and the sequences according to SEQ ID No: 1-4 and functional variants and functional fragments thereof. Preferably the trimeric coiled-coil peptide sequence comprises or consists of the amino acid sequence according to SEQ ID No: 1 or a functional variant or fragment thereof. Preferably the trimeric coiled- coil peptide sequence comprises or consists of the amino acid sequence according to SEQ ID No: 2 or a functional variant or fragment thereof. Preferably the trimeric coiled-coil peptide sequence comprises or consists of the amino acid sequence according to SEQ ID No: 3 or a functional variant or fragment thereof. Preferably the trimeric coiled-coil peptide sequence comprises or consists of the amino acid sequence according to SEQ ID No: 4 or a functional variant or fragment thereof. Preferably the trimeric coiled-coil peptide sequence comprises or consists of a GCN4 sequence

Preferably the pentameric coiled-coil peptide sequence comprises the amino acid sequence according to SEQ ID No: 5, more preferably the trimeric coiled-coil peptide sequence consists of the amino acid sequence according to SEQ ID No: 5.

Preferably the peptidic linker segment has the amino acid sequence of GG.

Preferably the peptidic hinge sequence (linker) is GG.

Preferably the immunogenic amino acid sequence derived from SARS-CoV-2 comprises any one the sequences shown in Table 1.

The invention further provides nucleic acid sequences coding for the immunogenic construct or any part (i.e. the trimeric coiled-cil, the pentameric coiled-coil, the linker (hinge) sequence and the epitope sequence) thereof.

The invention further provides a vaccine composition comprising the immunogenic construct and a pharmaceutically acceptable excipient.

A drug delivery construct is provided, comprising the coiled-coil sequences as described herein, preferably further comprising a targeting moiety and a lipid containing agent (such as a liposome carrying a drug). Preferably the recombinant prptide or the immunogenic construct is for use in a mammal.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 Amino acid sequence of the Spike protein of SARS-CoV-2. Bold AA represent the RBM. AA in italics represent the protease binding site at S2’ cleavage. R in a circle represents the S2’ cleavage site, underlined fonts represent the HR1 domain. Italic underlined fonts represent the HR2 domain.

Figure 2 Amino acid sequence of NP-monomers used in the examples extended with SARS-CoV-2 epitopes used in the examples. Two molecule glycine (-GG-) represent the hinge between the coiled-coil structures and SARS-CoV-2 epitope.

Amino acid sequence of His tag - pentameric coiled coil - GG - trimeric coiled-coil - GG - C8-N1 epitope, SEQ ID NO: 32.

Amino acid sequence of His tag - pentameric coiled coil - GG - trimeric coiled-coil - GG - C8-N6 epitope, SEQ ID NO: 33.

Amino acid sequence of His tag - pentameric coiled coil - GG - trimeric coiled-coil - GG - C13 epitope, SEQ ID NO: 34.

Amino acid sequence of His tag - pentameric coiled coil - GG - trimeric coiled-coil - GG -N12 epitope, SEQ ID NO: 35.

Amino acid sequence of His tag - pentameric coiled coil - GG - trimeric coiled-coil - GG - HR1 epitope, SEQ ID NO: 36.

Amino acid sequence of His tag - pentameric coiled coil - GG - trimeric coiled-coil - GG - RBM epitope, SEQ ID NO: 37.

Amino acid sequence of His tag - pentameric coiled coil - GG - trimeric coiled-coil - GG - HR2 epitope; SEQ ID NO: 38

Figure 3 Amino acid sequence of NP-short (A) and long (B) platforms SEQ ID NOs 72-82 and 83-87, respectively).

Figure 4 (A): Nucleotide sequences of the NP-Short platform. Underlined nucleotides: NCol site, Bold: His-tag, Bold+underlined: linker, Italics: pentamer, Bold+underlined+italics: Glycine hinge, Bolds italics: trimer, Lowercase: Proline, XXXX: epitope (24) (B: Nucleotide sequences of the NP-Long platform (SEQ ID NO 88). Underlined nucleotides: NCol site, Bold: His-tag, Italics: pentamer, Bold+underlined+italics: Glycine hinge, Bolds italics: trimer, Underlined+italics:Smal, XXXX: epitope (14).

Figure 5 Cloning strategy of NP- SARS-CoV-2 fusion expression system.

Figure 6 SARS-CoV-2 epitope sequences conjugated with NPV370 immunization platform are immunogenic: (A): Coomassie staining of purified and assembled NPV370 and NPV370-Covid vaccine protein mono- and oligomers. (B): Identifying SARS-CoV-2 epitope sequences by Western blot. Primary antibody: anti-SARS- CoV-2-Spike-antibody. 1:200 dilution, 10 sec exposition. Secondary antibody: anti-Rabbit IgG tagged with peroxidase. (C): Identifying SARS-CoV-2 epitope sequences by Western blot. Primary antibody: serum from a CO VID-19 patient. 1:200 dilution, 10 sec exposition. Secondary antibody: anti-Human IgG tagged with peroxidase.

Figure 7: Testing of polyclonal antibodies generated by NPV370-S2-C13 vaccine with Western Blot: (A): detection of recombinant, purified SARS-CoV-2-Sl, -S2 and and TVL (Total Virus Lysate, i.e. SARS-CoV-2 infected, inactivated Vero-6 cell lysate in Laemmli buffer) antigenic proteins with the serum of rabbits immunized with NPV-370-S2-C13 vaccine. Secondary antibody: anti-Rabbit IgG tagged with peroxidase. (B) Detection of recombinant, purified SARS-CoV-2-Sl, -S2 and TVL antigenic proteins with the serum of a human individual vaccinated with the Astra Zeneca COVID-19 vaccine. Secondary antibody: anti-Human IgG tagged with peroxidase.

Figure 8 Neutralisation rate of rabbit sera 6 weeks after vaccination with NP-COVID19 combined vaccine or authorized Pfizer vaccine. Assay: SARS-CoV-2 Surrogate Virus neutralisation test Kit, Proteogenix (catalog number: KPTX02

Figure 9 Quantification of anti-Covid Spike protein SI domain antibodies in rabbit sera at 6 weeks after vaccination with NP-Covidl9 cobined vaccine or authorized Pfizer vaccine. Assay: Anti-SARS-CoV-2 QuantiVac ELISA (IgG), Euroimmun (Catalog number: El 2606-9601-10 G)

Figure 10 Nucleotide sequence of the epitopes used (SEQ ID Nos 57-71)

DETAILED DESCRIPTION OF THE INVENTION

The immunization platforms described herein are useful for eliciting an immune response to antigenic moieties (e.g. peptides), which are considered too short when using known methods. These antigenic moieties may play an essential role in an infection with a pathogen or in the immune response to a pathogen, but because of technical difficulties, are underrepresented in the immunization techniques currently used. For example, a number of CD8⁺ T cell epitope sequences (which are essential for the differentiation of memory T-cells) were identified in the viral proteins of SARS-CoV -2 (Ferretti, et al (2020) Unbiased Screens Show CD8( +) T Cells of COVID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside outside the Spike Protein. Immunity, 53: (5) 1095-1107 e3.), among which only a few were located on the S protein (Ferretti et al. Unbiased Screens Show CD8 + T Cells of CO VID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside outside the Spike Protein. Immunity. 2020 Nov 17;53(5): 1095-1107. e3. doi: 10.1016/j.immuni.2020.10.006), the main target of the vaccines developed early against the pathogen. The immunization platforms described herein provide an appropriate immunization surface for very short epitopes, such as the RBM (receptor binding motif), S1S2 cleavage site or TCE-1 (T-cell epitope 1) of SARS-CoV-2 (Mahajan, S.,et al. Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust preexisting T-cell immunity in unexposed individuals. Sci Rep 11, 13164 (2021 )., Saini et al. SARS-CoV-2 genomewide T cell epitope mapping reveals immunodominance and substantial CD8+ T cell activation in COVID- 19 patients. SCIENCE IMMUNOLOGY 14 Apr 2021Vol 6, Issue 58DOI: 10.1126/sciimmunol.abf7550 ). Thereby short epitopes can be used as effective antiviral tools and a more precise targeting of pathogens can be achieved. The term "peptide" refers to molecules comprising amino acids joined covalently by peptide bonds. The term "polypeptide" or "protein" refers to large peptides, but in general the terms "peptide", "polypeptide" and "protein" are synonyms and are used interchangeably herein. The terms “peptide”, “polypeptide” and “protein” are used in their accepted scientific meaning.

“Human cartilage oligomeric matrix protein” (“COMP”) refers to the protein identified as UniProtKB - P49747 (COMP_HUMAN). The N-terminal coiled coil region of COMP is identified in the InterPro database as IPR039081. SEQ ID NO: 5 represents the amino acid sequence of a pentameric coiled coil from COMP. A "nucleic acid" may be DNA or RNA, e.g in vitro transcribed RNA or synthetic RNA. A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. The nucleic acid can be modified e.g. by stabilizing sequences, capping, or polyadenylation.

Preferably the terms “(poly)peptide”, “protein”, “amino acid sequence”, “lipid binding domain”, “oligomerization domain”, “nucleic acid molecule”, “nucleic acid sequence”, “immunogenic moiety” includes the functional fragments and variants thereof.

The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent.

The term “functional fragment” or “functional variant”, as used herein, in particular refers to a variant sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent sequence and that is capable of fulfilling one or more of the functions of the parent or sequence, e.g., binding to a lipid molecule (i.e. the “functional fragment” or “functional variant” of a lipid binding amino acid sequence derived from a protein) or eliciting an immune response upon administration to a subject (i.e. the “functional fragment” or “functional variant” of an immunogenic sequence derived from a pathogen).

The term “fragment”, when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. A fragment is usually at least 3 amino acid long.

The term "functional fragment" or "functional variant" of an amino acid sequence or a nucleic acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence or nucleic acid sequence from which it is derived, i.e., it is functionally equivalent.

The term “functional fragment” or “functional variant”, as used herein, in particular refers to a variant sequence that comprises an amino acid sequence or nucleic acid sequence that is altered by one or more amino acids or nucleotids compared to the parent sequence and that is capable of fulfilling one or more of the functions of the parent or sequence, e.g., binding to a lipid molecule, having a coiled-coil structure, capable of oligomerization, capable of eliciting an immune response or coding an amino acid sequence that is capable of e.g. binding to a lipid molecule or eliciting an immune response.

The term “fragment”, when used in reference to a reference polypeptide or nucleic acid molecule, refers to a polypeptide or nucleic acid molecule in which amino acid residues or nucleotids are deleted as compared to the reference polypeptide or nucleic acid molecule itself, but where the remaining amino acid sequence or nucleic acid sequence is usually identical to the corresponding positions in the reference polypeptide. A fragment is usually at least 3 amino acid long or the length of the fragment of a nucleic acid molecule is at least as long as to encode 3 amino acids.

The term “functional variant” further includes conservatively substituted variants. The term “conservatively substituted variant” refers to a peptide comprising an amino acid residue sequence that differs from a reference peptide by one or more conservative amino acid substitution, and maintains some or all of the activity of the reference peptide as described herein. A “conservative amino acid substitution” is a substitution of an amino acid residue with a functionally similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another and the substitution of one hydrophilic residue for another, such as between arginine and lysine, between glutamine and asparagine, between threonine and serine. The term “conservatively substituted variant” also includes peptides wherein a residue is replaced with a chemically derivatized residue, provided that the resulting peptide maintains some or all of the activity of the reference (poly)peptide as described herein. In some embodiments, the functional variant of a (poly)peptide shares a sequence identity of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the reference peptide. In some embodiments, the functional variant of a (poly)peptide shares a sequence identity of at least 85%, 90%, 95%, or 99% with the reference (poly)peptide.

A functional variant of a reference nucleic acid refers to a nucleic acid encoding the same polypeptide as the reference nucleic acid. In some embodiments, the functional variant of a nucleic acid molecule shares a sequence identity of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the reference nucleic acid molecule.In some embodiments, the functional variant of a nucleic acid molecule shares a sequence identity of at least 85%, 90%, 95%, or 99% with the reference nucleic acid molecule. A functional variant of a reference nucleic acid refers to a nucleic acid encoding the same polypeptide as the reference nucleic acid. A functional variant of a reference nucleic acid may comprise modified nucleotides or nucleosides. A functional variant of a reference nucleic acid molecule may comprise modified nucleotides or nucleosides.

A functional variant of an amino acid sequence may comprise several amino acid substitutions relative to the original amino acid sequence, depending on the number of the amino acids in the sequence. For example a variant of an amino acid sequence that comprises less than 50 amino acids may comprise up to 25, up to 10, up to 5 or 4, 3, 2 or 1 amino acid substitutions relative to the original amino acid sequence, while a variant of an amino acid sequence that comprises more than 50 amino acids may comprise more amino acid substitutions relative to the original amino acid sequence, as long as the main function of the amino acid sequence is maintained (e.g. being immunogenic, forming an epitope, capable of oligomeruoization).

The nucleic acid may be and preferably is a modified nucleic acid molecule. The preparation and use of modified nucleotides and nucleosides are well-known in the art, e.g. from W02007024708, US 10232055, US4373071, US4458066, US5262530, US5700642, EP3294326, EP1685844, EP1392341, EP2763701, EP2600901, EP1934345, EP1934345, EP3319622, EP2918275, EP3337902. A modified nucleotide contains one or more chemical modifications (e.g., substitutions) in or on the nitrogenous base of the nucleoside. A modified nucleotide can contain chemical modifications in or on the sugar moiety of the nucleoside, or the phosphate. The nucleic acid sequences may comprise one or more modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5 -methylcytidine, 5-methyluridine), preferably pseudouridine.

The nucleic acid sequence may encode a single polypeptide or more peptides linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence. The polypeptides generated from the nucleic acid sequence may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences that may be linked by linker sequences.

The nucleic acid sequence may typically be an mRNA, having at least one open reading frame that can be translated by a cell. The translation product is a peptide or protein that may act as an antigen (immunogen). In certain embodiments the antigen is a tumor antigen. In those certain embodiments the patient to whom the pharmaceutical (vaccine) composition is to be administered, has a tumor expressing the tumor antigen or is at the risk of developing such tumor.

The term “immunogenic” refers to an agent capable of eliciting an immune response (either cellular or humoral) in a subject. Immunogenic and antigenic may be used interchangeably.

“Immunization platform” or “NP” or “NPV370” or “immunogenic construct” refers to a recombinant (poly)peptid or nucleic acid sequence, which is capable of eliciting an immune response against an antigenic agent linked to the (poly)peptid or nucleic acid sequence. For example, a polypeptide comprising the peptide sequences as described herein is an immunization platform: an antigenic agent (such as an epitope from SARS- CoV-2 or the spike protein of SARS-CoV-2 or any other known antigenic moiety) may be attached to the recombinant peptide and an immune response to the antigenic agent will be induced in a subject to whom the peptide/antigenic agent conjugate is administered. Likewise, a nucleic acid sequence, such as an mRNA sequence coding for the coiled-coil sequences as described herein is an immunization platform is an immunization platform: a nucleic acid sequence (such as an mRNA sequence) coding for an antigenic agent (such as an epitope from SARS-CoV-2 or the spike protein of SARS-CoV-2 or any other known antigenic moiety) may be attached to the nucleic acid sequence and an immune response to the antigenic agent will be induced in a subject to whom the nucleic acid sequence/ nucleic acid sequence coding for the antigenic agent conjugate is administered. The immunization platform accoding to the invention comprises peptide (preferably coiled-coil) sequences as described herein, or a functional variant or functional fragment thereof or a nucleic acid molecule coding for the peptide sequences as described herein or a functional variant or functional fragment thereof.

In an embodiment, the peptide sequences are spaced by linkers or spacers. The term "linker", as used herein, relates to a peptide added between two peptide domains such as epitopes or vaccine sequences to connect said peptide domains. It is preferred that the linker sequence reduces steric hindrance between the two peptide domains, is well translated, and supports or allows processing of the epitopes. Furthermore, the linker should have no or only little immunogenic sequence elements. Glycine and/or serine rich linkers are preferred. The linkers each may comprise 1, 2, or more, preferably up to 50 amino acids. Short linkers (e.g. up to 10 or up to 5 amino acid long) are preferred.

In an embodiment, the IM is linked to the coiled-coil sequences by a spacer. It is preferred that the spacer sequence reduces steric hindrance between the two peptide domains, is well translated, and supports or allows processing of the epitopes. Furthermore, the spacer should have no or only little immunogenic sequence elements.

The immunization platform comprises a recombinant peptide molecule comprising a trimeric coiled-coil peptide sequence and a second peptide sequence. Preferably the immunization platform comprises a continous peptidic chain comprising the trimeric coiled-coil peptide sequence and the second peptide sequence and a peptidic linker sequence localized between the trimeric coiled-coil peptide sequence and the second peptide sequence. Preferably the immunization platform comprises a plurality of such continous peptidic chains.

Preferably an epitope (immunogenic moiety (IM), such as an amino acid sequence) is linked to the immunization platform. Preferably the IM is linked to the trimeric coiled-coil peptide sequence, preferably via a spacer. Preferably the spacer comprises or consists of an amino acid sequence of SEQ ID NO 9 or a functional fragment or functional variant thereof. Preferably the spacer has a sequence that renders a configuration (3 dimensional structure) to the polypeptide (peptidic chain) that exposes the IM to the immune system or to the cells of the organism receiving the immunization platform.

The trimeric sequences may be extended, e.g. up to 46 or 45 amino acids to display a short epitope in a better orientation. An example is shown on Fig 2. Alternatively, the trimeric sequences may be shortened in case of longer epitope sequences. An example is shown on Fig 2 where the trimeric sequence is 26 AA long when a 24 AA long SARS-CoV-2 epitope is attached thereto. For amino acid sequences of NP -platform variants see Figure 2. For DNA sequence of NP -platform variants see Figure 3. The displayed epitope sequences comprise 14 or 24 amino acid (AA) epitopes (Table 1). In case of 14 AA the trimer sequence is extended up to 46 or 45 amino acid residues (Figure 2) in order to display the SARS-CoV-2 epitope in a better orientation. For the 24 amino acid epitope amino acid, the trimer sequence was reduced to 26 amino acids because the longer epitope associated with the shorter NP platform also appears in the appropriate direction (Figure 3).

It was shown that rabbits immunized with the construct comprising the immunization platform carrying the S2C13 epitope of SARS-CoV-2 are producing S2C13-specific IgGs and virus neutralizing IgGs. It was also shown that S2’ cleavage site sequence specific IgG is produced by humans immunized with an authorized CO VID-19 vaccine (from Astra Zeneca), and this IgG is immunopositive with the recombinant NP-C13 protein.

In any one of the aspects preferably the recombinant peptide is for use in a method of eliciting an immune response in a subject. The immune response is against the immunogenic agent. Preferably the (poly)peptide is for use in a method of immunizing a subject against a pathogenic agent, such as a virus or bacteria. Preferably the pathogen is an RNA virus, preferably a coronavirus, more preferably SARS-CoV-2. The subject is an animal, preferably a mammal, more preferably a human.

Preferably the length of the immunogenic amino acid sequence is up to 100 amino acids or up to 50 amino acids, preferably up to 25 amino acids, highly preferably 5-30 amino acids, highly preferably 5-25 amino acids or highly preferably 5-25 amino acids. Preferably the epitope is at least 3 amino acids long.

The term “inducing an immune response" also includes "enhancing an immune response".

Methods of treatment

A method for the prevention or treatment of an infection or a cancer is provided, wherein the method comprises administering a prophylactically or therapeutically effective amount of an antigen (such as an epitope) linked to an immunization platform described herein (e.g. in the form of a vaccine composition). The antigen may be a nucleic acid sequence coding for an antigen (such as an mRNA sequence coding for an immunogenic amino acid sequence), for example a cancer antigen. The prevention or treatment may comprise administering the antigen linked to the immunization platform more than once, e.g. in a primer and booster regime or an annual administration. In particular, a method of preventing, ameliorating or treating a disease caused by an infection with an RNA virus, preferably a coronavirus, highly preferably preventing CO VID-19 in a subject is provided, wherein an immunization platform described herein linked to an epitope from SARS-CoV-2 or a nucleic acid sequence encoding an epitope from SARS-CoV-2 is administered to the subject.

Vaccine compositions

The immunization platform is useful in vaccine compositions.

The vaccine composition may comprise an adjuvant. "Adjuvant" means any substance that increases the humoral or cellular immune response to an antigen. Adjuvants are well known in the art. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g. Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), liposomes, and immune-stimulating complexes. Examples for adjuvants are monophosphoryl-lipid-A, Saponins vitamin E, montanid, CpG oligonucleotides, and various water-in-oil emulsions which are prepared from biologically degradable oils such as squalene.

The vaccine compositions of the invention can further comprise pharmaceutically acceptable carriers, excipients and/or stabilizers (see e.g. Remington: The Science and practice of Pharmacy (2005) Lippincott Williams).

Drug delivery system

The construct comprising the coiled-coil sequences as described herein forms a polymer layer on the surface of artificial liposomes and stabilizes them. Therefore, it may be used in targeted drug delivery constructs. For example a targeting moiety (such as a specific receptor ligand) is attached to the construct comprising the coiled-coil sequences as described herein instead of an IM to “guide” the construct to the target cell. The construct to which the construct comprising the coiled-coil sequences as described herein is attached is, for example, a liposome carrying an active agent.

A construct comprising the coiled-coil sequences as described herein is provided for use in a drug delivery construct. In preferred embodiments a targeting moiety (TM) is attached to the construct comprising the coiled- coil sequences as described herein. In preferred embodiments the construct comprising the coiled-coil sequences as described herein is attached to a liposome or a lipid containing construct carrying an active agent. mRNA immunization platform

In preferred embodiments the vaccine composition comprises nucleic acid sequence(s) coding for the coiled-coil sequences, linker, spacers as defined herein, and the immunogenic epitope. The protein polymer product of the mRNA will form a stable antigen-presenting layer on the host cell membrane and on the membrane vesicles (large exosomes) exiting the host cell, leading to a strong immunity elicited by the vaccine.

SARS-CoV-2 epitopes

The list of epitope sequences of SARS-CoV-2 Spike, that were used for production of NPV370 vaccine. Eight epitopes are related to the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike. Four epitopes are designed close to the TMPRSS2 cleavage site. Three epitopes are designed to the Furin cleavage site. Six epitopes are designed to the CD8+ T-cell receptor binding sites. (Table 1)

Table 1 Examples of SARS-CoV-2 epitopes

A nucleic acid molecule comprising a nucleic acid sequence(s) encoding the immunogenic construct above is also provided. The nucleic acid molecule is preferably DNA or RNA. The nucleic acid molecule is preferably RNA. The immunogenic construct or the nucleic acid molecule are for use in a method for eliciting an immune response in a subject.

A method for the preparation for the immunogenic construct is provided, comprising the recombinant expression of the trimeric sequence, the second peptide sequence and the IM in a host organization transfected with an expression/transfection vector carrying a coding sequence for the trimeric sequence, the second peptide sequence and the IM, wherein the trimeric sequence, the second peptide sequence and the IM may be on separate expression/transfection vectors or on the same expression/transfection vector.

EXAMPLES

Experimental procedure of Nano particle (NP) based immunization of SARS-CoV-2.

I. Nanoparticle monomer:

Nano particle (NP) coding expression constructs was generous gift of Peter Burkhard (Institute of Material Science University of Connecticut, USA). NPmonomer consists of a modified pentameric coiled-coil from the cartilage oligomerization matrix protein (COMP) and a de novo designed trimeric coiledcoil peptide. Our group used NP-constructs to raise antibodies against small peptide epitope in order to monitor the proteolytic events of own target proteins. For that reason, the original epitope (HRC1) was substituted to custom peptide sequence (new epitope). The selectivity and sensitivity of new polyclonal antibodies were perfectly convincing. One of the NP -peptide was originally developed SARS-CoV-1 vaccine (Chem Biol Drug Des 2009; 73: 53-61) . The SARS-CoV-1 epitope originated from the HRC sequence (C terminal heptad repeat domain). The HRC (alternatively HR2: heptad repeat 2) has very conserved sequence and it is 100 per cent identical to the HRC sequence of SARS-CoV-2.

II. SARS-CoV-2 Spike epitope sequences fused to NP- monomers:

Best candidate epitope sequences for TFR123-SARS-CoV-2:

The most significant challenge of SARS-CoV-2 vaccine is to identify the most useful immunogen epitope sequence of the virus. Spike protein of SARS-CoV-2 seems to be the most obvious antigen. There are many strategies and challenges to use Spike protein for vaccination.

There are three remarkable problems regarding Spike protein.

Nr.l: Spike is a highly glycosylated protein (see Watanabe et al., Science 369, 330-333 (2020) 17 July). Post translational modification excludes the cost effective and high yield recombinant expression of Spike protein in bacteria. However, glycosylation might be variable changing slightly the 3D structure of the Spike. This decreases the reproducible immunogen effect of the possible vaccine. Eukaryotic expression (for instance in HEK293 cells) of Spike protein is expensive and the problem with variable glycosylation is still there.

Nr.2: The outer segment of Spike: SI -protein, which comprises the receptor binding domain (RBD) too, does not have as conserved an amino acid sequence as the S2-protein and the transmembrane domain (TM). It means that evolutional variation of SI -protein might decrease the reproducible immunogen effect of the possible vaccine.

Nr.3: Unmodified (no glycosylation) conserved amino acid sequences are located in hidden regions of Spike, which is useless for vaccination, because those amino acid structures are not well displayed on the Spike’s surface.

Investigation of the amino acid sequence of Spike protein suggest a very promising sequence: F802-F823 (PBM: protease binding motif) which comprises the S2’ proteolytic cleavage point (Figure 13). F802-F823 amino acid sequence has no Asparagine (“N”) which excludes the N-glycosylation. This is very important because N-glycosylation of spike protein is performed on Asparagine residues (Watanabe et al., Science 369, 330-333 17 July 2020). Each monomer of trimeric Spike displays 22 N-glycosylation sites (see Watanabe et al., Science 369, 330-333 17 July 2020). Glycosylation seems essential for internalization of virus into the pneumocytes, because disruption of Spike glycosylation impairs viral entry (Genetic Engineering and Biotechnology News. Retrieved 2020-05-18). Ligand binding function of ACE2 receptors of pneumocytes is very similar to lectin receptors recognising carbohydrate modification of proteins indicating an importance of glycan-protein interactions in the viral entry. However, substrate-binding of proteases prefer unmodified amino acid sequences. Proteases need clear access to the cleavage site of the substrate. In fact, glycosylation could mask this access. On the other hand, protease binding site of substrate must be very well displayed, otherwise proteolytic cleavage will not happen. The S2’ cleavage site of Spike protein might be an excellent suitable epitope to raise SARS-CoV-2 vaccine, because it is very well displayed on the outer segment of Spike protein furthermore it is not glycosylated. The optimal size of the immunogen epitope sequence contains 5-30, preferably 5-15, preferably 7-14 amino acids. Amino acid sequence of SARS-CoV-2 Spike (Figure 1). C8-N1: P807-R815: Outer loop of S-protein, which is the expected protease binding motif of S2’-site. C8-N6: P807-D820: Outer loop extended with FP sequence (fusion peptide). C13: F802-R815: Outer loop extended up to the first closest upstream glycosylation site (N801). N12: R815-V826: FP (fusion peptide) extended in direction of N-terminus. HR1: K921-I934: CoV-2 unique sequence of HRl. HR2: A1174-N1187: The epitope used by Peter Burkhard (Chem Biol Drug Des 2009; 73: 53-61). RBM: A475-C488: Receptor binding motif of S-protein, which interacts with ACE2-receptor (Shang et al. Nature | Vol581 | 14May2020.).

III. Cloning of NP-SARS-CoV-2 epitopes (Fig. 5):

Nucleotide sequence of NP -peptide monomers (core peptide) are sub-cloned into pPEP-T vector. SARS-CoV-2 epitope coding oligonucleotide sequences are designed and purchased from IDT (Integrated DNA Technologies). Single stranded oligos are hybridized to sdDNA. After hybridization, 5’-end of Sense and 3’end of Antisense displays a blunted end. Meanwhile the 5’ end of Antisense oligo displays -TTAA-5’ overhangs. Both ends of the DNA insert are compatible to the destination vector NP -pPEP-T digested with Smal-EcoRI restriction enzymes. Ligation of appropriate DNA insert provides the C-terminal extension of the recombinant core NP -monomer by SARS-CoV-2 epitope (Figure 5). IV. Expression and purification of NP-SARS-CoV-2 fusion proteins: 20 ml overnight precultures of a single BL21-NP-SARS-CoV-2 colony were started in Luria’s Broth liquid medium (LB) with 100 pg/rnl Ampicillin concentration. The tubes were incubated overnight, at 37°C and shaken at 140 rpm. The preculture then was transferred to 11 LB with lOOpg/ml Ampicillin and was incubated for 4 hours at 37°C, shaken at 140 rpm. After the cells grew until the OD600reached approximately 0.5 -0.6 the plasmid expression was induced by adding isopropol P-D-thiogalactopyranoside (IPTG) to 1 mM final concentration, then the system was incubated for 4 hours. After the expression was ready, the bacterial culture was centrifuged at 3000 x g for 20 minutes. Following the removal of the supernatant, the pellet was stored at -20°C. The pellet was thawed in room temperature, then it was resuspended in 10 ml of lysis buffer (8M Urea, 100 mM NaH2PO4, lOmM TRIS, pH=8) mixing via gentle shaking by hand. For reaching the desired solubility, the cells were sonicated for 6 minutes, at 40% power, with 50% pulsation on ice. The mixture was then centrifuged down with Sorwall RC 5B Plus, at 2000 x g, for 40 minutes at 4°C. Meanwhile 1.5 ml of HISSelect Nickel Affinity Gel was rinsed by distilled water 3 times and equilibrated once with lysis buffer. The rinsed beads were incubated with the bacterial supernatant for 1 hour while shaking. The supernatant/nickel- NTA slurry was then used to fill a column for purification. The process contained 4 washing steps, 10 ml each, initially with lysis buffer, followed by washing buffer 1 (8M Urea, 100 mM NaH₂PO₄, pH=6.8); washing buffer 2 (8M Urea, 20mM citrate, pH=5.9); washing buffer 3 (8M Urea, 20mM citrate, pH=4.5). Finally, the purified polypeptide monomers were eluted from the column withlO ml lysis buffer containing IM imidazole (pH=8). The samples were then kept in 15 ml tubes at - 80°C until further application.

IV. Expression and purification of NP-SARS-CoV-2 fusion proteins:

20 ml overnight precultures of a single BL21-NP-SARS-CoV-2 colony were started in Luria’s Broth liquid medium (LB) with 100 pg/rnl Ampicillin concentration. The tubes were incubated overnight, at 37°C and shaken at 140 rpm. The preculture then was transferred to 11 LB with lOOpg/ml Ampicillin and was incubated for 4 hours at 37°C, shaken at 140 rpm. After the cells grew until the GD600reached approximately 0.5 -0.6 the plasmid expression was induced by adding isopropol P-D-thiogalactopyranoside (IPTG) to 1 mM final concentration, then the system was incubated for 4 hours. After the expression was ready, the bacterial culture was centrifuged at 3000 x g for 20 minutes. Following the removal of the supernatant, the pellet was stored at - 20°C. The pellet was thawed in room temperature, then it was resuspended in 10 ml of lysis buffer (8M Urea, 100 mM NaH2PO4, lOmM TRIS, pH=8) mixing via gentle shaking by hand. For reaching the desired solubility, the cells were sonicated for 6 minutes, at 40% power, with 50% pulsation on ice. The mixture was then centrifuged down with Sorwall RC 5B Plus, at 2000 x g, for 40 minutes at 4°C. Meanwhile 1.5 ml of HISSelect Nickel Affinity Gel was rinsed by distilled water 3 times and equilibrated once with lysis buffer. The rinsed beads were incubated with the bacterial supernatant for 1 hour while shaking. The supernatant/nickel-NTA slurry was then used to fill a column for purification. The process contained 4 washing steps, 10 ml each, initially with lysis buffer, followed by washing buffer 1 (8M Urea, 100 mM NaH2PO4, pH=6.8); washing buffer 2 (8M Urea, 20mM citrate, pH=5.9); washing buffer 3 (8M Urea, 20mM citrate, pH=4.5). Finally, the purified polypeptide monomers were eluted from the column with 10 ml lysis buffer containing IM imidazole (pH=8). The samples were then kept in 15 ml tubes at - 80°C until further application.

V. Assembly of NP-SARS-CoV-2 fusion proteins

The frozen nanoparticle sample was thawed in a water bath (60°C) until it melted. Then, 2 ml of suspension was diluted to 0.1 pg/ml avoiding the aggregation of the monomers.40 ml of the diluted sample was placed to a dialysis tube in order to remove 8 M urea forcing the nanoparticle monomers to assemble themselves into spheroids. The dialysis membranes were pre-treated in a 10 mM EDTA, 10 mM NaHCO3boiling solution for 5 minutes. 40 ml of diluted nanoparticle monomer suspension was taken into the dialysis tube. The tube was placed in 2 1 lysis buffer containing 6 M urea while puddling with a magnetic shaker for 2 hours. Afterwards 1 1 of lysis buffer was added containing 0 M urea, hence creating 3 1 of 4 M urea solution. After 2 hours of incubation 1.5 1 of solution was taken out and replaced by 0 M solution, resulting in a 2 M dialysis buffer. This dilution step was repeated 2 times leading to the assembly of the nanoparticles. When the dialysis was done, we re-concentrated the nanoparticles back to 2 mg/ml via a size exclusion centrifugal tube, with a limit of 10 kDa.

VI. Immunization of rabbits

For the immunization, we use 3-month-old rabbits. The rabbits receive an immunizing shot of 0.5 mg antigen, extended to 1.2 ml. The combined shots are extended with 400 pl CFL. Each rabbit receives 4x300 ml of subcutaneous injection in the neck area. Following the initial step, the animals receive a 0.25 mg boosting shot after two weeks, and another 0.25 mg shot one more week later. Rabbits will be bled two weeks later. Blood will be centrifuge and rabbit sera will be stored on ice and analysed by serological investigations (see Chapter VIII).

VII. Quantification of immune response of rabbits a. Detection of Immunoglobulin titters of sera after each boost of immunization:

IgG titter of diluted sera will be qualified by ELISA.

VIII. Serological investigation of the sensitivity of rabbit sera b. ELISA or Western blot (Fig. 6) with immobilized recombinant peptides used for immunization:

The appropriate antigens (NP-SARS-CoV-2) used for immunization, will be immobilized on ELISA plate. The same experiment will be performed as in previous chapter (VIII. a) c. Immunocytochemistry of Vero6 cells infected with SARS-CoV-2 using sera of immunized rabbits. Infected Vero6 cells will be fixed with 4% PFA, permeabilised with digitonin, and blocked in 10% donkey sera. Primary antibody will be the serum of immunized rabbits. Secondary antibody will be anti-rabbit-AlexaFluor- 488 fluorescens antibody. Fluorescent photographs will be taken by confocal microscope. See Fig. 7.

IX. Serological investigation of the sensitivity of human sera taken from individuals infected with SARS- CoV-2 and having no symptoms or who had been already healed.

To prove our concept, that outer loop of protease binding motif (PBM=NP-P807-R815) of SARS-CoV-2 Spike is a potential best epitope sequence for vaccination, we intend to test human sera taken from individuals infected with SARS-CoV-2 virus. The appropriate antigens (NP-SARS-CoV-2) used for immunization, will be immobilized on ELISA plate and incubated with serial diluted human sera (1: 1, 1:2, 1:4, 1:8, 1:16 and 1:32).

X. Investigation of the effectivity of NP-CoV-2 vaccine in hamster a. Investigation of immune response of control NP and NP-SARS-CoV-2 vaccines in hamster. To quantify immune response of hamster injected with appropriate antigens, we intend to measure the changes of pro- inflammatory cytokines (TNF-alpha, IL2, IL6 etc.) by validated inflammatory ELISA kit. Detection of Immunoglobulin titters of sera after each boost of immunization: IgM and IgG titter of diluted sera will be qualified by ELISA. b. Quantification of virus copy number in hamster infected with SARS-CoV2 c. Quantification of virus copy number in hamster injected with NP-CoV2 thereafter infected with SARS-CoV2 Summary

We have cloned a recombinant NPV370 protein family comprising short (14 AA) and long (24 AA) epitope sequences from SARS-CoV-2. Each member of the protein family exposes an epitope sequences from SARS- CoV-2 on a huge surface.

Recombinant, N-terminally poly-His-tagged NP monomers were purified on a His-Select affinity column in the presence of 8M urea. With stepwise removal of the urea (dialysis) the NP monomers self-assemble, thereby forming the desired immunization surface with the appropriate antigen sequences.

NPV370-Covid vaccines were concentrated (2 mg/ml), filtered with a 0.22 pm to sterilize, and stored at -80 °C until the start of the immunization procedure.

4-5 week old female rabbits weighing 1-1.5 kg were used for immunization (n=4 in each group). A preimmunization blood sample (PIS: pre-immune serum) was drawn from each rabbit before immunization. Members of the control group were not immunized. Members of the negative control group received the NPV370 platform without the SARS-CoV-2 epitope, while members of the positive control group received Comirnaty (Pfizer-BioNTech Covid-19 vaccine).

NPV370 and NPV370-Covid vaccines were administerd subcutaneously into the skin flap of the neck, while the Pfizer-BioNTech vaccine was administered intramuscularly. No adjuvant treatment was used. The first vaccination (primer) was repeated twice: booster 1 on day 14 and booster 2 on day 28 after the primer. Rabbits were terminated 6 weeks after the primer. Serum and tissue samples were stored at -80°C.

Anti-SARS-CoV-2 Spike antibodies immunopositively reacted with SARS-CoV-2 epitope sequences presented on the surface of the NPV370-Covid vaccines.

Antigenic sequences (S2-C8N1, S2-C8N6, S2-C13, RBM-C14, RBD-N14, RBD-C14) were identified by Western blot. The SARS-CoV-2 epitopes all reacted immunopositively with primary anti-SARS-CoV-2-Spike antibodies. The NPV370 protein without a SARS-CoV-2 epitope is not immunogenic, therefore the detected chemiluminescent signal indicates the SARS-CoV-2 sequences. (Fig. 6).

Antibodies recognizing the short SARS-CoV-2 epitope sequences used for the NPV370-Covid vaccines were detected in the serum of a human subject having been infected with SARS-CoV-2. The serum of a patient recovered from CO VID-19 was used as primary antibody in a dilution of 1:200. It is clear from the results, that the short sequences used for the immunization, especially S2-C13 play an important role in the immune response against the pathogen. (Fig. 6).

Anti-SARS-CoV-2 antibodies from rabbits vaccinated with NPV370-S2-C13 showed an immunopositive reaction with the recombinant SARS-CoV-2 spike protein (Fig.6).

To show the specifity of the immune response induced by NPV370-S2-C13, we used blood samples from immunized rabbits drawn on the second week following the third vaccination with NPV370-S2-C13 (i.e. six weeks after the first shot). As a positive control, a serum sample from a subject vaccinated with Astra-Zeneca- COVID-19 vaccine (four weeks after the second shot) was used. The specifity of the antibodies was proven with purified, recombinant SARS-CoV-2-Sl, SARS-CoV-2-S2 and TVL (Total Viral lysate, i.e. inactivated lysate from Vero-6 cells infected with SARS-CoV-2) antigenic proteins.

The serum sample from the subject vaccinated with the Astra-Zeneca-COVID-19 vaccine gave an immunopositive signal with both SARS-CoV-2-Sl and SARS-CoV-2-S2 (Fig. 7). Anti-SARS-CoV-2 antibodies from rabbits showed an immunopositive reaction with the recombinant SARS-CoV-2-S2 antigen and the endogenous SARS-CoV-2-S2 protein in the TVL (Fig. 7).

Immunization of rabbits: Rabbits were immunized with below mentioned NPV370-Covid vaccine respectively as well as in combination.

The following groups were formed:

1. Rabbits receving NPV370

2. Rabbits receving NPV370-S2-C13

3. Rabbits receving NPV370-RBM-C14

4. Rabbits receving NPV370-RBD-C14

5. Rabbits receving NPV370-RBD-N14

6. Rabbits receving NPV370-S1S2-C14

7. Rabbits receving a combined vaccine of:

Ctrl group: NPV370;

Short-NP treatment groups: NPV370-S2-C13 + NPV370-RBM-C14 + NPV370-RBD-C14 +

NPV370-RBD-N14 + NPV370-S1S2-C14.

Long-NP treatment groups: NP-RBM-24 + NP-RBD-24 + NP-S1S2-24

For combined vaccination, NP-monomers carrying the different epitopes were isolated separately and then assembled in equivalent amounts.

Both the short (Short: NP: RBD-N14, RBD-C14, RBM-C14, S1S2-C14 es S2-C14) and the long (LongNP: RBM-24, RBD-24 and S1S2-24) epitopes were tested in this protocol.

Serum samples, i.e. samples were drawn on the 2^nd week after the 3^rd immunization. NP vaccines were administered subcutaneous into the neck skin flap, while the Pfizer vaccine (Comirnaty) was administered i.m. Surrogate neutralization ELISA assay:

Recombinant Spike-RBD (Receptor binding domain) is immobilized on the bottom of the ELISA plate. We investigated whether Igs (IgG and IgM) induced by our new vaccines are capable to displace HRP -labelled recombinant ACE2. Negative control: pre-immune sera (PIS), i.e. serum from blood drawn before immunization. Control: ELISA + puffer without IgG. Positive control: serum from rabbits immunized with Comirnaty. N=3 in every group. Both the short (MAA) and the long (24 AA) SARS-CoV-2 epitope carrying NP platform were able to displace ACE2 (Fig. 8).

Spike-specific IgG titer measured by ELISA:

In this case, full length recombinant Spike protein is immobilized on the bottom of the ELISA plate. The amount of spike-specific IgGs present in the blood of the immunized rabbits were quantified with validated, commercially available anti-spike antibody dilution. Antigen specific IgG production was similar by the NP- vaccines and by Comirnaty.

Claims

1. A recombinant peptide comprising a trimeric coiled-coil peptide sequence and a second peptide sequence, for use in therapy, wherein the trimeric coiled-coil peptide sequence comprises or consists of a sequence selected from the group consisting of a GCN4 sequence, sequences according to SEQ ID Nos 1-4 and functional variants and functional fragments thereof, wherein the functional variant or the functional fragment is a trimeric coiled-coil and has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with the GCN4 sequence or the sequence according to any one of SEQ ID Nos 1-4, respectively and the second peptide sequence comprises or consists of a sequence selected from the group consisting of SEQ ID No 5-8 and functional variants and functional fragments thereof, wherein the functional variant or the functional fragment has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with the sequence according to any one of SEQ ID Nos 5-8, respectively.

2. The recombinant peptide for use according to claim 1, wherein the second peptide sequence comprises or consists of the sequence according to

SEQ ID NO 5 or a functional or a functional fragment variant thereof, wherein the functional variant or the functional fragment is a pentameric coiled-coil and has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with SEQ ID NO 5.

3. The recombinant peptide for use according to claim 1, wherein the second peptide sequence comprises or consists of a sequence selected from the group consisting of SEQ ID No 6-8 and functional variants and functional fragments thereof, wherein the functional variant or the functional fragment has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with the sequence according to any one of SEQ ID Nos 6-8, respectively.

4. The recombinant peptide for use according to claim 1, wherein the second peptide sequence comprises or consists of a sequence according to

SEQ ID NO 8 or a functional variant or a functional fragment thereof, wherein the functional variant or the functional fragment is capable of binding to a lipid and has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with SEQ ID NO 8.

5. The recombinant peptide for use according to any one of claim 1-4, wherein the trimeric coiled-coil peptide sequence comprises or consists of the sequence according to

6. The recombinant peptide for use according to any one of claim 1-4, wherein the trimeric coiled-coil peptide sequence comprises or consists of the sequence according to SEQ ID NO 2 or a functional variant or a functional fragment thereof wherein the functional variant or the functional fragment is a trimeric coiled-coil and has a sequence that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with SEQ ID NO 2.

7. The recombinant peptide for use according to any one of the preceding claims, wherein an immunogenic moiety (IM) is linked to the recombinant peptide.

8. The recombinant peptide for use according to any one of the preceding claims, wherein the recombinant peptide comprises a linker sequence localized between the pentameric coiled-coil peptide sequence and the trimeric coiled-coil peptide sequence, preferably wherein the linker sequence is Gly-Gly.

9. The recombinant peptide for use according to any one of the preceding claims, wherein the recombinant peptide comprises a spacer localized between the trimeric coiled-coil sequence and the IM, preferably wherein the spacer comprises or consists of the sequence according to SEQ ID NO 9 or a functional variant or a functional fragment thereof that is at least 80%, preferably at least 85%, preferably at least 90% or preferably at 95% identical with SEQ ID NO 9.

10. The recombinant peptide for use according to any one of claims 7-9, wherein the IM is an immunogenic amino acid sequence.

11. The recombinant peptide for use according to claim 10, wherein the IM is derived from SARS-CoV-2.

12. The recombinant peptide for use according to claim 11, wherein the IM is selected from SEQ ID Nos

13. Immunogenic construct comprising a plurality of the recombinant peptides as defined in any one of the proceeding claims.

14. Vaccine composition comprising a recombinant peptide as defined in any one of the proceeding claims or the immunogenic contract of claim 13 and a pharmaceutically acceptable excipient.

15. A drug delivery system comprising a recombinant peptide as defined in any one of claims 1-6, preferably further comprising a targeting moiety.

16. A recombinant peptide for use in therapy, wherein the recombinant peptide comprises or consists of an amino acid sequence according to any one of SEQ ID NOs 72-87, or a functional variant or fragment thereof, wherein the functional variant or fragment is capable of inducing an immune response upon administration into a mammal, preferably a human and has a sequence that is at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% identical with the corresponding recombinant peptide according to SEQ ID NOs 72-87.