WO2024061759A1

WO2024061759A1 - Stabilized coronavirus s proteins

Info

Publication number: WO2024061759A1
Application number: PCT/EP2023/075414
Authority: WO
Inventors: Johannes Petrus Maria Langedijk; Mark Johannes Gerardus BAKKERS; Jaroslaw JURASZEK
Original assignee: Janssen Vaccines and Prevention BV
Current assignee: Janssen Vaccines and Prevention BV
Priority date: 2022-09-23
Filing date: 2023-09-15
Publication date: 2024-03-28
Anticipated expiration: 2025-03-23

Abstract

The present invention relates to stabilized HCoV-NL63 S proteins, or fragments thereof, nucleic acid sequences encoding such proteins, as well as to uses thereof.

Description

STABILIZED CORONAVIRUS S PROTEINS

The present invention relates to the field of medicine. The invention, in particular, relates to stabilized recombinant prefusion Coronavirus spike (S) proteins, in particular to NL63 S proteins, to nucleic acid molecules encoding said NL63 S proteins, and uses thereof, e.g. in vaccines.

BACKGROUND OF THE INVENTION

Coronaviruses (CoVs) are a family of enveloped, single-stranded positive-sense RNA viruses belonging to the order Nidovirales, which can infect a broad range of mammalian and avian species, causing respiratory or enteric diseases. Coronaviruses possess large, trimeric spike glycoproteins that mediate binding to host cell receptors as well as fusion of viral and host cell membranes. The Coronavirus family contains the genera Alphacoronavirus (a-CoV), Betacoronavirus (0-CoV), Gammacoronavirus (y-CoV), and Deltacoronavirus (5-CoV). The host range of these viruses is primarily determined by the viral spike protein (S) protein. Coronaviruses that can infect humans (hCoV) are found both in the genus a-CoV, namely NL63 and 229E, and in the genus p-CoV, namely SARS-CoV, MERS-CoV, HCoV- OC43,HCoV-HKUl, and SARS-CoV-2. The currently identified human coronaviruses have been shown to originate from previous zoonotic transmissions, with rodents and bats likely serving as the source of most a-CoVs and 0-CoVs, while birds are the main reservoir of y- CoVs and 5-CoVs (Su et al. (2016), Trends Microbiol, 24:490-502).

In recent years, several of such zoonotic events have caused outbreaks of severe respiratory disease and high mortality, like SARS, MERS, and COVID-19 (Zhu et al. (2020), Respir Res, 21 :224). In contrast, the human endemic seasonal coronaviruses such as NL63, 229E, OC43, and HKU1 cause 15-29% of all common colds and mostly result in mild respiratory or gastrointestinal infections, although in immunocompromised individuals and the elderly more severe disease can occur (Kistler and Bedford (2021), Elife, 10:e64509. doi: 10.7554/eLife.64509; Su et al. (2016), Trends Microbiol, 24:490-502; Tortorici and Veesler (2019), Adv Virus Res, 105:93-116). Of these cases, NL63 and OC43 are reported to be the most common infections that lead to hospitalization (Chiu et al. (2005), Clin Infect Dis, 40: 1721-1729; Van Der Hoek et al. (2006), FEMS Microbiol Rev, 30:760-773).

Characteristic of coronaviruses are the membrane-bound spike (S) fusion proteins, visible on electron-microscope images as crown-like structures on the virus. S proteins are comprised of a S 1 and S2 subunit and form homotrimers on the viral membrane. The aminoterminal SI subunit contains the N-terminal domain (NTD) and receptor binding domain (RBD) which is responsible for host-receptor attachment. SARS-CoV-2, SARS-CoV, and HCoV-NL63 S bind to human angiotensin-converting enzyme 2 (ACE2) via their receptorbinding domain (RBD) (Hoffmann et al. (2020), Cell, 181 :271-280; Wrapp et al. (2020), Science, 367: 1260-1263, Hoffmann et al. (2005), Proc Natl Acad Sci USA, 102:7988-7993). The carboxy-terminal S2 subunit contains the fusion machinery and is required for fusion of the viral and host membranes (Heald- Sargent and Gallagher (2012), Viruses, 4:557-580).

Coronavirus S proteins are class I fusion proteins that are inherently metastable. Their metastable nature is a prerequisite for their ability to undergo extensive conformational changes from a labile prefusion to a stable post-fusion conformation, a pivotal event that drives membrane fusion. The prefusion conformation of S, as present on the infectious viral particle, contains the epitopes for neutralizing antibodies and thus holds most promise as a vaccine immunogen (Chen et al. (2020), Hum Vaccin Immunother, 16: 1239-1242; Liu et al. (2020), Nature, 584:450-456; Yuan et al. (2020), Science, 368;630-633; Brouwer et al. (2020), Science, 369;643-650; Hsieh et al. (2020), Science, 369: 150-1505; Robbiani et al. (2020), Nature, 584:437-442.). Therefore, for development of robust efficacious vaccine components it is desirable that the metastable fusion protein is maintained in its prefusion conformation. In recent years several attempts have been made to stabilize various class I fusion proteins, including coronavirus S proteins.

A particularly successful approach to enhance prefusion stability was shown to be the stabilization of the so-called hinge loop preceding the central helix (CH) in the HR1 region, which has been applied to a range of viral fusion glycoproteins (WO2017/037196; Krarup et al. (2015) Nat Comm 6, 8143; Rutten el al. (2020) Cell Rep 30, 4540-4550; Hastie el al. (2017) Science 356, 923-928). This has also proved successful for coronavirus S proteins, by mutation of two consecutive residues to prolines (2P), as shown for SARS-CoV, MERS-CoV and SARS- CoV-2 (Pallesen et al. (2017) Proc Natl Acad Sci USA 114, E7348-E7357; Wrapp et al. (2020) Science 367, 1260-1263). The HR1 region in the S2 subunit consists of several discontinuous a-helices in the prefusion structure, which rearrange to a single extended a-helix in the postfusion conformation (Walls et al. (2017), Proc Natl Acad Sci USA, 17: 11157-11162). The 2P substitution prevents this reorganization and consequently stabilizes the S protein in the prefusion conformation (Hsieh et al. (2020), Science, 369:150-1505; Juraszek et al. (2021) Nat Commun, 12: 244; Pallesen et al. (2017) Proc Natl Acad Sci USA, 114:E7348-E7357). In general, stabilizing spike proteins in the prefusion conformation increases recombinant expression levels which may be a result of a more efficient folding process and reduced misfolding of the protein (Graham el al. (2019), Annu Rev Med 70: 91-104).

Although vaccine development against P-CoVs has taken flight since the COVID-19 pandemic (Burton and Walker (2020), Cell Host Microbe, 27:695-698), no vaccine against a-

CoV NL63 currently exists. The present invention aims at providing means for obtaining a stable prefusion NL63 S protein for use in vaccinating against HCoV-NL63. BRIEF DESCRIPTION OF THE FIGURES

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended figures. It should be understood that the invention is not limited to the precise embodiments shown in the examples.

FIGURE 1: Domain organization of NL63 spike protein

(A) Schematic representation of the conserved elements of the NL63 spike (S) protein in both the full-length membrane bound protein (‘full-length’, top panel) and in the mature, soluble ectodomain (‘ectodomain’, bottom panel). The N-terminal domain is preceded by a signal peptide sequence (SP) that is cleaved off during protein maturation (indicated by arrow). S protein domains include N-terminal domain (NTD), receptor-binding domain (RBD), S1/S2 protease cleavage site (S1/S2), S2’ protease cleavage site (S2’) (indicated by arrows), heptadrepeat region 1 (HR1), heptad-repeat region 2 (HR2), transmembrane-domain (TMD), cytosolic tail (CT). For the soluble ectodomain S, the TMD and CT are replaced by a GCN4 trimerization domain (GCN4).

FIGURE 2: Expression profile of NL63 S proteins with proline substitutions in the «13al4 loop of the HR1 region in crude cell supernatant.

(A) Schematic overview of NL63 S designs. (B) Biolayer interferometry expression analysis of NL63 S protein in supernatant of cells transfected with constructs listed in (A). Samples were subjected to qOctet with biotinylated anti-C-tag nanobody immobilized to streptavidin sensors. Initial binding rate is plotted. Transfections are performed with n=4. The average per design is reported with error bar indicating the standard deviation. FIGURE 3: Expression profile of NL63 S proteins with proline substitutions in the hinge loop region at the C-terminus of HR1 in crude cell supernatant.

(A) Schematic overview of NL63 S designs. (B) Biolayer interferometry expression analysis of NL63 S protein in supernatant of cells transfected constructs listed in (A). Samples were subjected to qOctet with biotinylated anti-C-tag nanobody immobilized to streptavidin sensors. Initial binding rate is plotted. Transfections are performed with n=l or n=4. The average (for n>l) per design is reported with error bar indicating the standard deviation.

FIGURE 4: Expression profile in crude supernatant and characterization of purified NL63 S proteins with proline substitutions A996P and S1052P.

(A) Schematic overview of NL63 S designs. (B) Biolayer interferometry expression analysis of NL63 S protein in supernatant of cells transfected with constructs listed in (A). Samples were subjected to qOctet with biotinylated anti-C-tag nanobody immobilized to streptavidin sensors. Initial binding rate is plotted. Transfections are performed with n=2. The average per design is reported with error bar indicating the standard deviation.

(C) Analytical SEC profile of purified NL63 S trimers. (D) Characterization of purified NL63 trimers by SEC-MALS. (E) Melting temperature of purified NL63 S trimers by DSF. n=3 replicate measurements, and individual and average values are reported as grey and black solid lines, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Human coronavirus NL63 (HCoV-NL63) is a species of coronavirus from the Alphacoronavirus genus. It was identified in late 2004 in patients in the Netherlands. The virus is an enveloped, positive-sense, single-stranded RNA virus which enters its host cell by binding to ACE2. Infection with the virus has been confirmed worldwide and is associated with symptoms that include mild to moderate upper respiratory tract disease, severe lower respiratory tract disease, croup and bronchiolitis.

The virus is found primarily in young children, the elderly, and immunocompromised patients with acute respiratory illness. It also has a seasonal association in temperate climates. A study performed in Amsterdam estimated the presence of HCoV-NL63 in approximately 4.7% of common respiratory illnesses. Viruses closely related to NL63 are found in palm civets and bats, which thus form natural reservoirs.

Like for SARS-CoV-2, the spike protein (S) of HCoV-NL63 is involved in fusion of the viral membrane with a host cell membrane, which is required for infection. HCoV-NL63 S RNA is translated into a 1356 amino acid precursor protein, which contains a signal peptide sequence at the N-terminus (e.g. amino acid residues 1-15 of SEQ ID NO: 1, predicted by SignalP 6.0) which is removed by a signal peptidase in the endoplasmic reticulum. Priming of the S protein typically involves cleavage by host proteases at the boundary between the SI and S2 subunits (S1/S2) in a subset of coronaviruses (including HCov-NL63), and at a conserved site upstream of the fusion peptide (S2’) in all known coronaviruses.

The prefusion conformation of S, as present on the infectious viral particle, contains the epitopes for neutralizing antibodies and thus holds most promise as a vaccine immunogen (Chen et al. (2020), Hum Vaccin Immunother, 16: 1239-1242; Liu et al. (2020), Nature, 584:450-456; Yuan et al. (2020), Science, 368;630-633; Brouwer et al. (2020), Science, 369;643-650; Hsieh et al. (2020), Science, 369: 150-1505; Robbiani et al. (2020), Nature, 584:437-442.). Since class I proteins are metastable proteins, increasing the stability of the prefusion conformation is desirable for use of fusion proteins as vaccine components. In addition, stabilization of prefusion S is desired for a soluble, subunit-based vaccine, which requires truncation of the viral fusion protein by deletion of the transmembrane (TM) and the cytoplasmic region. The remaining ectodomain of the fusion protein is considerably more labile due to removal of the membrane anchor and will either not form as a trimeric protein or even more readily refold into the post-fusion end-state. Finally, stabilization of fusion proteins generally increases the expression level of the protein, because less protein will be misfolded and more protein will successfully transport through the secretory pathway. For manufacturability purposes, high S protein expression and stable S trimeric conformation is critical for protein-based vaccines.

The present invention provides stabilized HCoV-NL63 S proteins, or fragments thereof, comprising a proline substitution in the al3al4 (amino acid residues 992-1001) loop and/or in the hinge loop region (amino acid residues 1049-1054) of HR1, wherein the numbering of the amino acid positions in said NL63 S protein is according to the numbering of the amino acid positions in SEQ ID NO: 1.

In a particular embodiment, the proteins comprise a proline substitution in the al3al4 (amino acid residues 992-1001) loop and in the hinge loop region (amino acid residues 1049- 1054) ofHRl.

In certain embodiments, the proline is introduced in the al3al4 loop at position 993, 996, 997, 998, and/or 1000, wherein the numbering of the amino acid positions in said NL63 S protein is according to the numbering of the amino acid positions in SEQ ID NO: 1.

In a preferred embodiment, the proline in the al3al4 loop is introduced at position 996.

In certain embodiment, the proline is introduced in the hinge loop region at position 1051, 1052, 1053, and/or 1054, wherein the numbering of the amino acid positions in said

NL63 S protein is according to the numbering of the amino acid positions in SEQ ID NO: 1.

In a preferred embodiment, the proline in the hinge loop is introduced at position

1052. According to the present invention, it has been demonstrated that one or both proline substitutions in the HR1 region of HCoV-NL63 increase expression of soluble trimeric HCoV-NL63 ectodomain.

In certain embodiments, the protein according to the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3-8 and 12-16, or a fragment thereof. In a preferred embodiment, the protein according to the invention comprises an amino acid sequence of SEQ ID NO: 16, or a fragment thereof.

The numbering of the positions of the amino acid residues is according to the numbering of the amino acid residues in the amino acid sequence of SEQ ID NO: 1. According to the invention it has been demonstrated that the presence of the specific amino acids at the indicated positions increases the stability of the S proteins in the prefusion conformation and/or increases trimer yields. According to the invention, the specific amino acids may be already present in the amino acid sequence of the S protein or may be introduced by substitution (mutation) of a naturally occurring amino acid residue at that position into the specific amino acid residue according to the invention. According to the invention, the proteins comprise one or more mutations in their amino acid sequence as compared to the amino acid sequence of a wild type S protein. The wording ‘the amino acid at position 996” thus refers to the amino acid residue that is at position 996 in SEQ ID NO: 1.

In certain embodiments, the multimeric HCoV-NL63 S proteins are trimeric, i.e. comprise three monomers comprising identical amino acid sequences.

An amino acid residue according to the invention can be any of the twenty naturally occurring (or ‘standard’ amino acids) or variants thereof, such as e.g. D-amino acids (the D- enantiomers of amino acids with a chiral center), or any variants that are not naturally found in proteins, such as e.g. norleucine. The standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties such as cysteine, that can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, proline that induces turns of the polypeptide backbone, and glycine that is more flexible than other amino acids. Table 1 shows the abbreviations and properties of the standard amino acids.

It will be appreciated by a skilled person that the mutations can be made to the protein by routine molecular biology procedures.

The term "fragment" as used herein refers to a peptide that has an amino-terminal and/or carboxy-terminal and/or internal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence of a HCoV-NL63 S protein, for example, the full-length sequence of a HCoV-NL63 S protein. It will be appreciated that for inducing an immune response and in general for vaccination purposes, a protein does not have to be full length nor have all its wild type functions, and fragments of the protein are equally useful. A fragment according to the invention is an immunologically active fragment, and typically comprises at least 15 amino acids, or at least 30 amino acids of the HCoV-NL63 S protein. In certain embodiments, it comprises at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 amino acids, of the HCoV-NL63 S protein. In certain embodiment, the fragment is the HCoV-NL63 S ectodomain.

In certain embodiments, the proteins according to the invention are soluble proteins, i.e. S protein ectodomains. Thus, in certain embodiments, the S proteins comprise a truncated S2 domain. As used herein a “truncated” S2 domain refers to a S2 domain that is not a full length S2 domain, i.e. wherein either N-terminally or C-terminally one or more amino acid residues have been deleted. According to the invention, at least the transmembrane domain and cytoplasmic domain have been deleted to permit expression as a soluble ectodomain, corresponding to the amino acids 1-1283 (or 16-1283 without signal peptide) of SEQ ID NO:

1.

For the stabilization of soluble HCoV-NL63 S proteins in the pre-fusion conformation, a heterologous trimerization domain, such as a GCN4 trimerization domain, may be fused to the C-terminus of the HCoV-NL63 S protein ectodomain (Walls et al. (2016) Nat Struct Mol Biol, 23:899-905). Thus, in certain embodiments, the transmembrane region has been replaced by a heterologous trimerization domain. In certain embodiments, the heterologous trimerization domain is GCN4 domain comprising the amino acid sequence of SEQ ID NO: 2. However, it is to be understood that according to the invention other trimerization domains are also possible. It is also possible that the stabilized S proteins do not comprise a heterologous trimerization domain. Thus, in certain preferred embodiments, the soluble HCoV-NL63 S proteins do not comprise a heterologous trimerization domain.

The pre-fusion HCoV-NL63 S proteins, or fragments thereof, according to the invention are trimeric and stable, i.e. do not readily change into the post-fusion conformation upon processing of the proteins, such as e.g. upon purification, freeze-thaw cycles, and/or storage etc.

The proteins according to the invention may comprise a signal peptide, also referred to as signal sequence or leader peptide, corresponding to amino acids 1-15 of SEQ ID NO: 1 (as predicted by SignalP v6.0). Signal peptides are short (typically 5-30 amino acids long) peptides present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. In certain embodiments, the proteins according to the invention do not comprise a signal peptide. The proteins according to the invention may comprise a linker and C-terminal tag (C- tag), such as GSGEPEA (SEQ ID NO: 17). In certain embodiments, the proteins do not comprise a C-tag.

The present invention further provides nucleic acid molecules encoding the HCoV- NL63 S proteins according to the invention. The term “nucleic acid molecule” as used in the present invention refers to a polymeric form of nucleotides (i.e. polynucleotides) and includes both DNA (e.g. cDNA, genomic DNA) and RNA (e.g. mRNA, modified RNA, circular mRNA), and synthetic forms and mixed polymers of the above.

In preferred embodiments, the nucleic acid molecules encoding the proteins according to the invention have been codon-optimized for expression in mammalian cells, preferably human cells, or insect cells. Methods of codon-optimization are known and have been described previously (e.g. WO 96/09378 for mammalian cells). A sequence is considered codon-optimized if at least one non-preferred codon as compared to a wild type sequence is replaced by a codon that is more preferred. Herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non-preferred codon. The frequency of codon usage for a specific organism can be found in codon frequency tables, such as in http://www.kazusa.or.jp/codon. Preferably more than one non-preferred codon, preferably most or all non-preferred codons, are replaced by codons that are more preferred. Preferably the most frequently used codons in an organism are used in a codon-optimized sequence. Replacement by preferred codons generally leads to higher expression.

It will be understood by a skilled person that numerous different polynucleotides and nucleic acid molecules can encode the same protein as a result of the degeneracy of the genetic code. It is also understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the protein sequence encoded by the nucleic acid molecules to reflect the codon usage of any particular host organism in which the proteins are to be expressed. Therefore, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may or may not include introns.

Nucleic acid sequences can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g. GeneArt, GenScript, Invitrogen, Eurofins).

The invention also provides vectors comprising a nucleic acid molecule as described above. In certain embodiments, a nucleic acid molecule according to the invention thus is part of a vector. Such vectors can easily be manipulated by methods well known to the person skilled in the art and can for instance be designed for being capable of replication in prokaryotic and/or eukaryotic cells. In addition, many vectors can be used for transformation of eukaryotic cells and will integrate in whole or in part into the genome of such cells, resulting in stable host cells comprising the desired nucleic acid in their genome. The vector used can be any vector that is suitable for cloning DNA and that can be used for transcription of a nucleic acid of interest.

In certain embodiments of the invention, the vector is an adenovirus vector. An adenovirus according to the invention belongs to the family of the Adenoviridae, and preferably is one that belongs to the genus Mastadenovirus. It can be a human adenovirus, but also an adenovirus that infects other species, including but not limited to a bovine adenovirus (e.g. bovine adenovirus 3, BAdV3), a canine adenovirus (e.g. CAdV2), a porcine adenovirus (e.g. PAdV3 or 5), or a simian adenovirus (which includes a monkey adenovirus and an ape adenovirus, such as a chimpanzee adenovirus or a gorilla adenovirus). Preferably, the adenovirus is a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV), or a rhesus monkey adenovirus (RhAd). In the invention, a human adenovirus is meant if referred to as Ad without indication of species, e.g. the brief notation “Ad26” means the same as HAdV26, which is human adenovirus serotype 26. Also as used herein, the notation “rAd” means recombinant adenovirus, e.g., “rAd26” refers to recombinant human adenovirus 26.

Most advanced studies have been performed using human adenoviruses, and human adenoviruses are preferred according to certain aspects of the invention. In certain preferred embodiments, a recombinant adenovirus according to the invention is based upon a human adenovirus. In preferred embodiments, the recombinant adenovirus is based upon a human adenovirus serotype 5, 11, 26, 34, 35, 48, 49, 50, 52, etc. According to a particularly preferred embodiment of the invention, an adenovirus is a human adenovirus of serotype 26. Advantages of these serotypes include a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and experience with use in human subjects in clinical trials.

Simian adenoviruses generally also have a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and a significant amount of work has been reported using chimpanzee adenovirus vectors (e.g. US6083716; WO 2005/071093; WO 2010/086189; WO 2010085984; Farina et al, 2001, J Virol 75: 11603-13; Cohen et al,

2002, J Gen Virol 83: 151-55; Kobinger et al, 2006, Virology 346: 394-401; Tatsis et al.,

2007, Molecular Therapy 15: 608-17; see also review by Bangari and Mittal, 2006, Vaccine

24: 849-62; and review by Lasaro and Ertl, 2009, Mol Ther 17: 1333-39). Hence, in other embodiments, the recombinant adenovirus according to the invention is based upon a simian adenovirus, e.g. a chimpanzee adenovirus. In certain embodiments, the recombinant adenovirus is based upon simian adenovirus type 1, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33, 34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50 or SA7P. In certain embodiments, the recombinant adenovirus is based upon a chimpanzee adenovirus such as ChAdOx 1 (see e.g. WO 2012/172277), or ChAdOx 2 (see e.g. WO 2018/215766). In certain embodiments, the recombinant adenovirus is based upon a chimpanzee adenovirus such as BZ28 (see e.g. WO 2019/086466). In certain embodiments, the recombinant adenovirus is based upon a gorilla adenovirus such as BLY6 (see e.g. WO 2019/086456), or BZ1 (see e.g. WO 2019/086466).

Preferably, the adenovirus vector is a replication deficient recombinant viral vector, such as rAd26, rAd35, rAd48, rAd5HVR48, etc.

In a preferred embodiment of the invention, the adenoviral vectors comprise capsid proteins from rare serotypes, e.g. including Ad26. In the typical embodiment, the vector is an rAd26 virus. An “adenovirus capsid protein” refers to a protein on the capsid of an adenovirus (e.g., Ad26, Ad35, rAd48, rAd5HVR48 vectors) that is involved in determining the serotype and/or tropism of a particular adenovirus. Adenoviral capsid proteins typically include the fiber, penton and/or hexon proteins. As used herein a “capsid protein” for a particular adenovirus, such as an “Ad26 capsid protein” can be, for example, a chimeric capsid protein that includes at least a part of an Ad26 capsid protein. In certain embodiments, the capsid protein is an entire capsid protein of Ad26. In certain embodiments, the hexon, penton and fiber are of Ad26.

One of ordinary skill in the art will recognize that elements derived from multiple serotypes can be combined in a single recombinant adenovirus vector. Thus, a chimeric adenovirus that combines desirable properties from different serotypes can be produced. Thus, in some embodiments, a chimeric adenovirus of the invention could combine the absence of pre-existing immunity of a first serotype with characteristics such as temperature stability, assembly, anchoring, production yield, redirected or improved infection, stability of the DNA in the target cell, and the like. See for example WO 2006/040330 for chimeric adenovirus Ad5HVR48, that includes an Ad5 backbone having partial capsids from Ad48, and also e.g. WO 2019/086461 for chimeric adenoviruses Ad26HVRPtrl, Ad26HVRPtrl2, and Ad26HVRPtrl3, that include an Ad26 virus backbone having partial capsid proteins of Ptrl, Ptrl2, and Ptrl3, respectively)

In certain embodiments the recombinant adenovirus vector useful in the invention is derived mainly or entirely from Ad26 (i.e., the vector is rAd26). In some embodiments, the adenovirus is replication deficient, e.g., because it contains a deletion in the El region of the genome. For adenoviruses being derived from non-group C adenovirus, such as Ad26 or Ad35, it is typical to exchange the E4-orf6 coding sequence of the adenovirus with the E4- orf6 of an adenovirus of human subgroup C such as Ad5. This allows propagation of such adenoviruses in well-known complementing cell lines that express the El genes of Ad5, such as for example 293 cells, PER.C6 cells, and the like (see, e.g. Havenga, et al., 2006, J Gen Virol 87: 2135-43; WO 03/104467). However, such adenoviruses will not be capable of replicating in non-complementing cells that do not express the El genes of Ad5.

The preparation of recombinant adenoviral vectors is well known in the art. Preparation of rAd26 vectors is described, for example, in WO 2007/104792 and in Abbink et al., (2007) Virol 81(9): 4654-63. Exemplary genome sequences of Ad26 are found in GenBank Accession EF 153474 and in SEQ ID NO: 1 of WO 2007/104792. Examples of vectors useful for the invention for instance include those described in WO2012/082918, the disclosure of which is incorporated herein by reference in its entirety. Typically, a vector useful in the invention is produced using a nucleic acid comprising the entire recombinant adenoviral genome (e.g., a plasmid, cosmid, or baculovirus vector). Thus, the invention also provides isolated nucleic acid molecules that encode the adenoviral vectors of the invention. The nucleic acid molecules of the invention can be in the form of RNA or in the form of DNA obtained by cloning or produced synthetically. The DNA can be double-stranded or single-stranded.

The adenovirus vectors useful in the invention are typically replication deficient. In these embodiments, the virus is rendered replication deficient by deletion or inactivation of regions critical to replication of the virus, such as the El region. The regions can be substantially deleted or inactivated by, for example, inserting a gene of interest, such as a gene encoding the SARS-CoV2 S protein (usually linked to a promoter) within the region. In some embodiments, the vectors of the invention can contain deletions in other regions, such as the E2, E3 or E4 regions, or insertions of heterologous genes linked to a promoter within one or more of these regions. For E2- and/or E4-mutated adenoviruses, generally E2- and/or E4-complementing cell lines are used to generate recombinant adenoviruses. Mutations in the E3 region of the adenovirus need not be complemented by the cell line, since E3 is not required for replication.

A packaging cell line is typically used to produce sufficient amounts of adenovirus vectors for use in the invention. A packaging cell is a cell that comprises those genes that have been deleted or inactivated in a replication deficient vector, thus allowing the virus to replicate in the cell. Suitable packaging cell lines for adenoviruses with a deletion in the El region include, for example, PER.C6, 911, 293, and El A549.

In a preferred embodiment of the invention, the vector is an adenovirus vector, and more preferably a rAd26 vector, most preferably a rAd26 vector with at least a deletion in the El region of the adenoviral genome, e.g. such as that described in Abbink, J Virol, 2007. 81(9): p. 4654-63, which is incorporated herein by reference. Typically, the nucleic acid sequence encoding the stabilized SARS-CoV2 S protein is cloned into the El and/or the E3 region of the adenoviral genome.

Host cells comprising the nucleic acid molecules encoding the pre-fusion HCoV- NL63 S proteins also form part of the invention. The pre-fusion HCoV-NL63 S proteins may be produced through recombinant DNA technology involving expression of the molecules in host cells, e.g. Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6 cells, or yeast, fungi, insect cells, and the like, or transgenic animals or plants. In certain embodiments, the cells are from a multicellular organism, in certain embodiments they are of vertebrate or invertebrate origin. In certain embodiments, the cells are mammalian cells, such as human cells, or insect cells. In general, the production of a recombinant proteins, such the pre-fusion HCoV-NL63 S proteins of the invention, in a host cell comprises the introduction of a heterologous nucleic acid molecule encoding the protein in expressible format into the host cell, culturing the cells under conditions conducive to expression of the nucleic acid molecule and allowing expression of the protein in said cell. The nucleic acid molecule encoding a protein in expressible format may be in the form of an expression cassette, and usually requires sequences capable of bringing about expression of the nucleic acid, such as enhancer(s), promoter, polyadenylation signal, and the like. The person skilled in the art is aware that various promoters can be used to obtain expression of a gene in host cells. Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed. Cell culture media are available from various vendors, and a suitable medium can be routinely chosen for a host cell to express the protein of interest, here the pre-fusion HCoV- NL63 S proteins. The suitable medium may or may not contain serum.

A “heterologous nucleic acid molecule” (also referred to herein as ‘transgene’) is a nucleic acid molecule that is not naturally present in the host cell. It is introduced into for instance a vector by standard molecular biology techniques. A transgene is generally operably linked to expression control sequences. This can for instance be done by placing the nucleic acid encoding the transgene(s) under the control of a promoter. Further regulatory sequences may be added. Many promoters can be used for expression of a transgene(s), and are known to the skilled person, e.g. these may comprise viral, mammalian, synthetic promoters, and the like. A non-limiting example of a suitable promoter for obtaining expression in eukaryotic cells is a CMV-promoter (US 5,385,839), e.g. the CMV immediate early promoter, for instance comprising nt. -735 to +95 from the CMV immediate early gene enhancer/promoter. A polyadenylation signal, for example the bovine growth hormone poly A signal (US 5,122,458), may be present behind the transgene(s). Alternatively, several widely used expression vectors are available in the art and from commercial sources, e.g. the pcDNA and pEF vector series of Invitrogen, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc, which can be used to recombinantly express the protein of interest, or to obtain suitable promoters and/or transcription terminator sequences, polyA sequences, and the like.

The cell culture can be any type of cell culture, including adherent cell culture, e.g. cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture. Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. Nowadays, continuous processes based on perfusion principles are becoming more common and are also suitable. Suitable culture media are also well known to the skilled person and can generally be obtained from commercial sources in large quantities, or custom-made according to standard protocols. Culturing can be done for instance in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems and the like. Suitable conditions for culturing cells are known (see e.g. Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and R.I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley -Liss Inc., 2000, ISBN 0-471-34889-9)).

The invention further provides compositions comprising a pre-fusion HCoV-NL63 S protein and/or a nucleic acid molecule, and/or a vector, as described above. The invention also provides compositions comprising a nucleic acid molecule and/or a vector, encoding such pre-fusion HCoV-NL63 S protein. The invention further provides immunogenic compositions comprising a pre-fusion HCoV-NL63 S protein, and/or a nucleic acid molecule, and/or a vector, as described above. The invention also provides the use of a stabilized prefusion HCoV-NL63 S protein, a nucleic acid molecule, and/or a vector, according to the invention, for inducing an immune response against a HCoV-NL63 S protein in a subject. Further provided are methods for inducing an immune response against HCoV-NL63 S protein in a subject, comprising administering to the subject a pre-fusion HCoV-NL63 S protein, and/or a nucleic acid molecule, and/or a vector according to the invention. Also provided are pre-fusion HCoV-NL63 S proteins, nucleic acid molecules, and/or vectors, according to the invention for use in inducing an immune response against HCoV-NL63 S protein in a subject. Further provided is the use of the pre-fusion HCoV-NL63 S proteins, and/or nucleic acid molecules, and/or vectors according to the invention for the manufacture of a medicament for use in inducing an immune response against HCoV-NL63 S protein in a subject. In certain embodiments, the nucleic acid molecule is DNA and/or an RNA molecule. The pre-fusion HCoV-NL63 S proteins, nucleic acid molecules, or vectors of the invention may be used for prevention (prophylaxis, including post-exposure prophylaxis) of HCoV-NL63 infections. In certain embodiments, the prevention may be targeted at patient groups that are susceptible for and/or at risk of HCoV-NL63 infection or have been diagnosed with a HCoV-NL63 infection. Such target groups include, but are not limited to e.g., the elderly (e.g. > 50 years old, > 60 years old, and preferably > 65 years old), hospitalized patients and patients who have been treated with an antiviral compound but have shown an inadequate antiviral response. In certain embodiments, the target population comprises human subjects from 2 months of age.

The pre-fusion HCoV-NL63 S proteins, nucleic acid molecules and/or vectors according to the invention may be used e.g. in stand-alone treatment and/or prophylaxis of a disease or condition caused by HCoV-NL63, or in combination with other prophylactic and/or therapeutic treatments, such as (existing or future) vaccines, antiviral agents and/or monoclonal antibodies.

The invention further provides methods for preventing and/or treating HCoV-NL63 infection in a subject utilizing the pre-fusion HCoV-NL63 S proteins, nucleic acid molecules and/or vectors according to the invention. In a specific embodiment, a method for preventing and/or treating HCoV-NL63 infection in a subject comprises administering to a subject in need thereof an effective amount of a pre-fusion-SARS CoV-2 S protein, nucleic acid molecule and/or a vector, as described above. A therapeutically effective amount refers to an amount of a protein, nucleic acid molecule or vector, that is effective for preventing, ameliorating and/or treating a disease or condition resulting from infection by HCoV-NL63. Prevention encompasses inhibiting or reducing the spread of HCoV-NL63 or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection by SARS CoV-2. Amelioration as used in herein may refer to the reduction of visible or perceptible disease symptoms, viremia, or any other measurable manifestation of HCoV-NL63 infection.

For administering to subjects, such as humans, the invention may employ pharmaceutical compositions comprising a pre-fusion HCoV-NL63 S protein, a nucleic acid molecule and/or a vector as described herein, and a pharmaceutically acceptable carrier or excipient. In the present context, the term "pharmaceutically acceptable" means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]). The CoV S proteins, or nucleic acid molecules, preferably are formulated and administered as a sterile solution although it may also be possible to utilize lyophilized preparations. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, e.g. pH 5.0 to 7.5. The CoV S proteins typically are in a solution having a suitable pharmaceutically acceptable buffer, and the composition may also contain a salt. Optionally stabilizing agent may be present, such as albumin. In certain embodiments, detergent is added. In certain embodiments, the CoV S proteins may be formulated into an injectable preparation.

In certain embodiments, a composition according to the invention further comprises one or more adjuvants. Adjuvants are known in the art to further increase the immune response to an applied antigenic determinant. The terms “adjuvant” and "immune stimulant' are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to the HCoV-NL63 S proteins of the invention. Examples of suitable adjuvants include aluminium salts such as aluminium hydroxide and/or aluminium phosphate; oilemulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see e.g. US 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the like; eukaryotic proteins (e.g. antibodies or fragments thereof (e.g. directed against the antigen itself or CDla, CD3, CD7, CD80) and ligands to receptors (e.g. CD40L, GMCSF, GCSF, etc), which stimulate immune response upon interaction with recipient cells. In certain embodiments the compositions of the invention comprise aluminium as an adjuvant, e.g. in the form of aluminium hydroxide, aluminium phosphate, aluminium potassium phosphate, or combinations thereof, in concentrations of 0.05 - 5 mg, e.g. from 0.075-1.0 mg, of aluminium content per dose.

In other embodiments, the compositions do not comprise adjuvants.

The pre-fusion HCoV-NL63 S proteins may also be administered in combination with or conjugated to nanoparticles, such as e.g. polymers, liposomes, virosomes, virus-like particles. The HCoV-NL63 S proteins may be combined with or encapsidated in or conjugated to the nanoparticles with or without adjuvant. Encapsulation within liposomes is described, e.g. in US 4,235,877. Conjugation to macromolecules is disclosed, for example in

US 4,372,945 or US 4,474,757. Alternatively, the HCoV-NL63 S proteins may be fused to a self-assembling protein domain (e.g. I53_dn5 trimerization domains ) that can self-assemble into 2-component particles by addition of pentamers (Boyoglu-Bamum, S. et al., Nature 592, 623-628, (2021)).

In certain embodiments, the invention provides methods for making a vaccine against a HCoV-NL63 virus, comprising providing a composition according to the invention and formulating it into a pharmaceutically acceptable composition.

The term "vaccine" refers to an agent or composition containing an active component effective to induce a certain degree of immunity in a subject against a certain pathogen or disease, which will result in at least a decrease (up to complete absence) of the severity, duration or other manifestation of symptoms associated with infection by the pathogen or the disease. In the present invention, the vaccine comprises an effective amount of a pre-fusion HCoV-NL63 S protein and/or a nucleic acid molecule encoding a pre-fusion HCoV-NL63 S protein, and/or a vector comprising said nucleic acid molecule, which results in an immune response against the S protein of HCoV-NL63. This provides a method of preventing serious lower respiratory tract disease leading to hospitalization and the decrease in frequency of complications such as pneumonia and bronchiolitis due to HCoV-NL63 infection and replication in a subject. The term “vaccine” according to the invention implies that it is a pharmaceutical composition, and thus typically includes a pharmaceutically acceptable diluent, carrier or excipient. It may or may not comprise further active ingredients. In certain embodiments it may be a combination vaccine that further comprises additional components that induce an immune response against HCoV-NL63, e.g. against other antigenic proteins of HCoV-NL63, or may comprise different forms of the same antigenic component. A combination product may also comprise immunogenic components against other infectious agents, e.g. other respiratory viruses including but not limited to influenza virus or RSV. The administration of the additional active components may for instance be done by separate, e.g. concurrent administration, or in a prime-boost setting, or by administering combination products of the vaccines of the invention and the additional active components.

The HCoV-NL63 S proteins may also be used to isolate monoclonal antibodies from a biological sample, e.g. a biological sample (such as blood, plasma, or cells) obtained from an immunized animal or infected human. The invention thus also relates to the use of the HCoV- NL63 protein as bait for isolating monoclonal antibodies.

Also provided is the use of the pre-fusion HCoV-NL63 S proteins of the invention in methods of screening for candidate HCoV-NL63 antiviral agents, including but not limited to antibodies against HCoV-NL63.

In addition, the proteins of the invention may be used as diagnostic tool, for example to test the immune status of an individual by establishing whether there are antibodies in the serum of such individual capable of binding to the protein of the invention. The invention thus also relates to an in vitro diagnostic method for detecting the presence of an ongoing or past HCoV-NL63 infection in a subject said method comprising the steps of a) contacting a biological sample obtained from said subject with a protein according to the invention; and b) detecting the presence of antibody -protein complexes.

The invention is further illustrated in the following example. The example does not limit the invention in any way. It merely serves to clarify the invention.

EXAMPLES

EXAMPLE 1, Stabilizing proline substitutions in the HR1 region ofNL63 increase expression of soluble trimeric NL63 S ectodomain.

To increase NL63 S ectodomain expression, amino acid residues in the al3al4 loop of HR1 at positions 993, 996, 997, 998, and 1000, and amino acid residues in the hinge loop region at the C-terminus ofHRl region at positions 1048, 1049, 1050, 1051, 1052, 1053, or 1054, were mutated to prolines. Plasmids encoding NL63 S protein ectodomain in which the transmembrane and cytoplasmic tail were replaced with a GCN4 trimerization domain and a C-tag were synthesized and codon-optimized at Genscript. The constructs were cloned into pCDNA2004 by standard methods widely known within the field involving site-directed mutagenesis and PCR and sequenced. Proteins were expressed in the Expi293F cell system. Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 3 days at 37°C and 10% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 10 minutes at 600 g, followed by sterile filtration using a 0.22 pm filter. The cell culture supernatants of the different NL63 S variants were analyzed by quantitative Octet (qOctet) using biolayer interferometry with biotinylated anti-C-tag antibody immobilized to streptavidin sensors. Streptavidin-conjugated sensors (ForteBio) were hydrated in kinetic buffer (ForteBio) for 10 minutes prior to nanobody loading. Biotinylated C-tag nanobody was loaded onto the corresponding sensors at 5 pg/mL for 30 minutes. Sensors were blocked for 10 minutes in supernatant of mock-transfected cells in 96-well black flat-bottom microplates (Corning). Association of supernatant samples of transfected cells was measured on an Octet Red384 (Pall-ForteBio) for 300 seconds. All steps were performed at 30°C with shaking at 1000 rpm. Initial binding rate was calculated per for each sample with the corresponding mock as a reference using the ForteBio Data Analysis software vlO.

Introduction of a proline at either position 993, 996, 997, 998, or 1000 enhanced expression of NL63 S ectodomain, as measured by an increase in initial binding rate to anti- C-tag nanobody in qOctet, compared to wildtype NL63 S (Figure 2B). Proline screening at positions 1048-1054 demonstrated an increase in NL63 S expression by introducing a proline substitution at positions 1051, 1052, 1053, and 1054, but not at positions 1048, 1049, and 1050 (Figure 3B).

A proline substitution in both the al3al4 loop and in the hinge loop region of HR1 was successfully combined in COR210427 (A996P+S1052P) (Figure 4B). To characterize A996P and S1052P variants in more detail, single (A996P; COR210047 and S1052P; COR201308) and double proline (COR210427) substitution constructs were transiently expressed in Expi293 cells using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 5 days at 37 °C and 10% CO2. NL63 S trimer was purified from sterile-filtered crude cell culture supernatant using a two-step purification protocol including CaptureSelect™ C-tagXL affinity column (Thermofisher Scientific), followed by size-exclusion chromatography using a Superose 6 10/300 GL column (Cytiva). The trimeric fraction was pooled and further characterized by analytical SEC using an ultra- high-performance liquid chromatography system (Vanquish, Thermofisher Scientific). Protein was loaded onto an SRT-10C SEC-500 15 cm column with the corresponding guard column (Sepax Technologies) in 150 mM sodium phosphate, 50 mM NaCl, pH 7.0 buffer. Molecular weight analysis was performed using pDAWN TREOS instrument (Wyatt) coupled to an Optilab pT-rEX Refractive Index Detector (Wyatt), in combination with an inline Nanostar DLS reader (Wyatt). SEC chromatograms were analyzed using Chromeleon 7.2.7 software package and showed a single peak (Figure 4C). MALS analysis was performed using Astra software version 7.3.2 and compared to the calculated weight. Conjugate analysis of glycosylation was done using a dn/dc of 0.1850 for protein, and 0.1410 for the contribution of glycans. All three purified NL63 S variants were trimeric proteins of the expected molecular weight, with a glycan mass of approximately 26%, and with comparable retention time and hydrodynamic radius (Figure 4D). The melting temperature (Tm50) of purified NL63 S trimers was determined by Differential Scanning Fluorimetry (DSF). To this end, the fluorescent emission of Sypro Orange Dye (Thermofisher Scientific) added to NL63 S protein in solution was monitored. The measurement was performed with a starting temperature of 25 °C and a final temperature of 95 °C (54 °C increase per hour). Melting curves were measured using a ViiA7 real-time PCR machine (Applied Biosystems), and Tm50 values were derived from the negative first derivative as described previously (Rutten et al. (2020) Cell Rep 30, 4540-4550). All three purified NL63 S showed two melting events with a lower melting temperature around 57 °C and a higher melting temperature around 64 °C (Figure 4E).

Table 1. Standard amino acids, abbreviations and properties

SEQUENCES

SEP ID NO: 1 - FL NL63 (Full length NL63 accession: ARB07399.1)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF S ANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF NKAINNIVASFSSVNDAITQTAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQ AISNSIQAIYDRLDSIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT VELQGLIDQINSTYVDLKLLNRFENYIKWPWWVWLIISVVFVVLLSLLVFCCLSTGCC GCCNCLTSSMRGCCDCGSTKLPYYEFEKVHVQ

SEQ ID NO: 2 - GCN4 trimerization domain

EDKIEEILSKIYHIENEIARIKKLIGEA

SEQ ID NO: 3 - COR201206 (NL63 Ectodomain +GCN4)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF SANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF NKAINNIVASFSSVNDAITQTAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQ AISNSIQAIYDRLDSIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI

YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT VELQGLIDQINSTYVDLEDKIEEILSKIYHIENEIARIKKLIGEAGSGEPEA

SEP ID NO: 4 - COR210046 (V993P)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF S ANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF NKAINNIVASFSSPNDAITQTAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQ AISNSIQAIYDRLDSIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT VELQGLIDQINSTYVDLEDKIEEILSKIYHIENEIARIKKLIGEAGSGEPEA SEP ID NO: 5 COR210047 (A996P)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF S ANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF NKAINNIVASFSSVNDPITQTAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQ AISNSIQAIYDRLDSIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT VELQGLIDQINSTYVDLEDKIEEILSKIYHIENEIARIKKLIGEAGSGEPEA

SEP ID NO: 6 - COR210048 (I997P)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF SANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHF SIRYIYNRVKSGSPGDS SWHIYLKSGTCPF SFSKLNNFQKFKTICF ST VAVPGS CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA

ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF

NKAINNIVASFSSVNDAPTQTAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQ

AISNSIQAIYDRLDSIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI

NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI

YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT

VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT

VELQGLIDQINSTYVDLEDKIEEILSKIYHIENEIARIKKLIGEAGSGEPEA

SEP ID NO: 7 - COR210049 (T998P)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF S ANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF NKAINNIVASFSSVNDAIPQTAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQ AISNSIQAIYDRLDSIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT VELQGLIDQINSTYVDLEDKIEEILSKIYHIENEIARIKKLIGEAGSGEPEA

SEP ID NO: 8 COR210050 (T1000P)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY

SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF S ANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF NKAINNIVASFSSVNDAITQPAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQ AISNSIQAIYDRLDSIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT VELQGLIDQINSTYVDLEDKIEEILSKIYHIENEIARIKKLIGEAGSGEPEA

SEP ID NO: 9 - COR201304 (D1048P)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF SANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF NKAINNIVASFSSVNDAITQTAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQ AISNSIQAIYPRLDSIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI

YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT

VELQGLIDQINSTYVDLEDKIEEILSKIYHIENEIARIKKLIGEAGSGEPEA

SEP ID NO: 10 - COR201305 (R1049P)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF S ANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF NKAINNIVASF S S VND AITQTAEAIHT VTIALNKIQD VVNQQGSALNHLTSQLRHNFQ AISNSIQAIYDPLDSIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT VELQGLIDQINSTYVDLEDKIEEILSKIYHIENEIARIKKLIGEAGSGEPEA

SEP ID NO: 11 - COR201306 (L1050P)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF SANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS

CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF NKAINNIVASFSS VND AITQTAEAIHT VTIALNKIQD VVNQQGSALNHLTSQLRHNFQ AISNSIQAIYDRPDSIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT

VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT VELQGLIDQINSTYVDLEDKIEEILSKIYHIENEIARIKKLIGEAGSGEPEA

SEP ID NO: 12 - COR201307 (D1051P)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF S ANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF NKAINNIVASF S S VND AITQTAEAIHT VTIALNKIQD VVNQQGSALNHLTSQLRHNFQ AISNSIQAIYDRLPSIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT VELQGLIDQINSTYVDLEDKIEEILSKIYHIENEIARIKKLIGEAGSGEPEA SEP ID NO: 13 - COR201308 (S1052P)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF S ANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF NKAINNIVASF S S VND AITQTAEAIHT VTIALNKIQD VVNQQGSALNHLTSQLRHNFQ AISNSIQAIYDRLDPIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT VELQGLIDQINSTYVDLEDKIEEILSKIYHIENEIARIKKLIGEAGSGEPEA

SEP ID NO: 14 - COR201309 (I1053P)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF SANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT

SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA

ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF

NKAINNIVASFSSVNDAITQTAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQ

AISNSIQAIYDRLDSPQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI

NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI

YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT

VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT

VELQGLIDQINSTYVDLEDKIEEILSKIYHIENEIARIKKLIGEAGSGEPEA

SEP ID NO: 15 - COR201310 (Q1054P)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF S ANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF NKAINNIVASF S S VND AITQTAEAIHT VTIALNKIQD VVNQQGSALNHLTSQLRHNFQ AISNSIQAIYDRLDSIPADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI YGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHT VIPDYVDVNKTLQEFAQNLPKYVKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTT VELQGLIDQINSTYVDLEDKIEEILSKIYHIENEIARIKKLIGEAGSGEPEA

SEP ID NO: 16 - COR210427 (A996P+S1052P)

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVY

SANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDF LSNSSSSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC YFNYSCVF S VVNAT VTVNVTTHNGRVVNYTVCDDCNGYTDNIF S VQQDGRIPNGFP FNNWFLLTNDSTLVDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGY QHNS VAD VMRYNLNF S ANS VDNLKSGVIVFKTLQ YDVLF YC SNSS SGVLDTTIPFGP SSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFN VTTASATDFWTVAFATFVDVLVNVSATKIQNLLYCDSPFEKLQCEHLQFGLQDGFYS ANFLDDNVLPETYVALPIYYQHTDINFTATASFGGSCYVCKPHQVNISLNGNTSVCV RTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSFSKLNNFQKFKTICFSTVAVPGS

CNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGIREFSNLVLNNCTKYNIYD YVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCNQPDQVAVYQQSIIG AMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVRPRN SSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYT SACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSSRI

AGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADA

ERMAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASF NKAINNIVASFSSVNDPITQTAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQ AISNSIQAIYDRLDPIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKI NECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGI

Claims

1. HCoV-NL63 S protein, or fragment thereof, comprising a proline substitution in the al3al4 loop (amino acid residues 992-1001) and/or in the hinge loop region (amino acid residues 1049-1054) of HR1, wherein the numbering of the amino acid positions in said NL63 S protein is according to the numbering of the amino acid positions in SEQ ID NO: 1.

2. Protein or fragment according to claim 1, comprising a proline substitution in the al3al4 loop (amino acid residues 992-1001) and in the hinge loop region (amino acid residues 1049-1054) of HR1.

3. Protein or fragment according to claim 1 or 2, wherein the proline is introduced in the al3al4 loop at position 993, 996, 997, 998, and/or 1000, wherein the numbering of the amino acid positions in said NL63 S protein is according to the numbering of the amino acid positions in SEQ ID NO: 1.

4. Protein or fragment according to claim 3, wherein the proline in the al3al4 loop is introduced at position 996.

5. Protein or fragment according to claim 1, 2 or 3, wherein a proline is introduced in the hinge loop region at position 1051, 1052, 1053, and/or 1054, wherein the numbering of the amino acid positions in said NL63 S protein is according to the numbering of the amino acid positions in SEQ ID NO: 1.

6. Protein or fragment according to claim 5, wherein the proline hinge loop region is introduced at position 1052.

7. Protein or fragment according to any one of the preceding claims, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3-8 and 12-

8. Protein, or fragment, according to any one of the preceding claims, comprising a truncated S2 domain.

9. Protein, or fragment, according to claim 8, wherein the transmembrane and cytoplasmic domain have been removed.

10. Nucleic acid molecule encoding a protein, or fragment thereof, according to any one of the preceding claims 1-9.

11. Nucleic acid according to claim 10, wherein the nucleic acid molecule is DNA or RNA.

12. Vector comprising a nucleic acid according to claim 10 or 11.

13. Vector according to claim 12, wherein the vector is a human recombinant adenoviral vector.

14. Composition comprising a protein according to any one of the claims 1-9, a nucleic acid according to claim 10 or 11 and/or vector according to claim 12 or 13.

15. Method for vaccinating a subject against a disease associated with infection by HCoV-NL63, the method comprising administering to the subject a composition according to claim 14.

16. Method for reducing infection and/or replication of HCoV-NL63 in a subject, comprising administering to the subject a composition according to claim 14.

17. Isolated host cell comprising a nucleic acid according to claim 10 or 11.