WO2023047349A1 - Stabilized coronavirus spike protein fusion proteins - Google Patents

Abstract

The present invention relates to stabilized recombinant pre-fusion Coronavirus S proteins, and fragments thereof, as well nucleic acids molecules encoding such S proteins and fragments, and to uses thereof.

Description

STABILIZED CORONAVIRUS SPIKE PROTEIN FUSION PROTEINS

The present invention relates to the field of medicine. The invention, in particular, relates to stabilized recombinant pre-fusion coronavirus spike (S) proteins, in particular to SARS-CoV-2 S proteins, to nucleic acid molecules encoding said SARS-CoV-2 S proteins, and uses thereof, e.g. in vaccines.

BACKGROUND OF THE INVENTION

Coronaviruses (CoVs) are a large 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 (S) that mediate binding to host cell receptors as well as fusion of viral and host cell membranes. The Coronavirus family contains the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. These viruses cause a range of diseases including enteric and respiratory diseases. The host range is primarily determined by the viral spike protein (S) protein. Coronaviruses that can infect humans are found both in the genus Alphacoronavirus and the genus Betacoronavirus. Known coronaviruses of the genus Betacoronavirus that cause respiratory disease in humans include SARS-CoV, MERS-CoV, HCoV-OC43 and HCoV-HKUl, and the currently circulating SARS-CoV-2.

SARS-CoV-2 is a coronavirus that emerged in humans from an animal reservoir in 2019 and rapidly spreads globally. SARS-CoV-2, like MERS-CoV and SARS-CoV, is thought to have its origin in bats. The name of the disease caused by the virus is Coronavirus induced disease 2019, abbreviated as COVID-19. Symptoms of COVID-19 range from mild symptoms to severe illness and death for confirmed COVID-19 cases. In the case of SARS- CoV-2 the S protein is the major surface protein. The S protein forms homotrimers and is composed of an N-terminal SI subunit and a C-terminal S2 subunit, responsible for receptor binding and membrane fusion, respectively. Recent cryo-EM reconstructions of the CoV trimeric S structures of alpha-, beta-, and deltacoronaviruses revealed that the SI subunit comprises two distinct domains: an N-terminal domain (SI NTD) and a receptor-binding domain (SI RBD). SARS-CoV-2 makes use of its SI RBD to bind to human angiotensinconverting enzyme 2 (ACE2) (Hoffmann et. al. (2020) Cell 181, 271-280; Wrapp et. al. (2020) Science 367, 1260-1263).

Coronaviridae S proteins are classified as class I fusion proteins and are responsible for fusion of the viral and host cell membrane. The S protein fuses the viral and host cell membranes by irreversible protein refolding from the labile pre-fusion conformation to the stable post-fusion conformation. Like many other class I fusion proteins, the coronavirus S protein requires receptor binding and cleavage for the induction of the conformational change that is needed for fusion and entry (Belouzard et al. (2009) PNAS 106 (14) 5871-5876; Follis et al. (2006) Virology 5;350(2):358-69; Bosch et al. (2008) J Virol. 82(17): 8887-8890; Madu et al. (2009) Virology 393(2):265-71; Walls et al. (2016) Nature 531(7592): 114-117). Priming of SARS-CoV-2 involves cleavage of the S protein by furin at a furin cleavage site at the boundary between the SI and S2 subunits (S1/S2), and by TMPRSS2 at a conserved site upstream of the fusion peptide (S2’) (Bestle et al. (2020) Life Sci Alliance 3; Hoffmann et. al. (2020) Cell 181, 271-280).

In order to refold from the pre-fusion to the post-fusion conformation, two regions of the protein need to refold, which are referred to as the refolding region 1 (RR1) and refolding region 2 (RR2) (FIG. 1). In all class I fusion proteins, the RR1 includes the fusion protein (FP) and heptad repeat 1 (HR1) (Wrapp et al., Science 2020 Mar 13;367(6483): 1260-1263). After cleavage and receptor binding the stretch of helices, loops and strands of all three protomers in the trimer transform to a long continuous trimeric helical coiled coil. The FP, located at the N-terminal segment of RR1, is then able to extend away from the viral membrane and inserts in the proximal membrane of the target cell. Next, the refolding region 2 (RR2), which is located C-terminal to RR1, and closer to the transmembrane region (TM) and which includes the heptad repeat 2 (HR2), relocates to the other side of the fusion protein and binds the HR1 coiled-coil trimer with the HR2 domain to form the six-helix bundle (6HB).

When viral fusion proteins, like the SARS-CoV-2 S protein, are used as vaccine components, the fusogenic function of the proteins is not important. In fact, only the mimicry of the vaccine component to the virus is important to induce reactive antibodies that can bind and preferably neutralize the virus. Therefore, for development of robust efficacious vaccine components it is desirable that the meta-stable fusion proteins are maintained in their prefusion conformation. It is believed that a stabilized fusion protein, such as a SARS-CoV-2 S protein, in the pre-fusion conformation can induce an efficacious immune response.

In recent years several attempts have been made to stabilize various class I fusion proteins, including coronavirus S proteins. A particularly successful approach was shown to be the stabilization of the so-called hinge loop at the end of RR1 preceding the base helix (WO2017/037196; Krarup et al. (2015) Nat Comm 6, 8143; Rutten et al. (2020) Cell Rep 30, 4540-4550; Hastie et al. (2017) Science 356, 923-928). This approach has also proved successful for coronavirus S proteins, 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). However, although the proline mutations in the hinge loop indeed increase the expression of the coronavirus S protein, the S protein may still suffer from instability (Wrapp et al, (2020) Science 367, 1260-1263; Juraszek et al., Nature Comm (2021) 12, 244). Thus, for improved vaccines or in order to obtain S proteins which can for example be used as tools, e.g. as a bait for monoclonal antibody isolation, further stabilization may be desired.

Since the novel SARS-CoV-2 virus was first observed in humans in late 2019 over 600 million of people have been infected and more than 6 million have died as a result of COVID-19, in particular because SARS-CoV-2, and coronaviruses more generally, lack effective treatment. Several vaccines have recently become available to prevent coronavirus induced disease (COVID-19), however, since COVID-19 continues to present a major threat to public health and economic systems and new variants of SARS-CoV-2 emerge, there is an urgent need for novel components that can be used e.g. in vaccines to prevent Coronavirus induced respiratory disease.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides recombinant pre-fusion Coronavirus S proteins, comprising an SI and an S2 domain, or fragments thereof, and comprising a mutation of the amino acid residue corresponding to the amino acid residue at position 1072 in a SARS-CoV-2 S protein into P and/or a mutation of the amino acid residue corresponding to the amino acid residue at position 1203 in a SARS-CoV-2 S protein into K, wherein the numbering of the amino acid positions in the SARS-CoV-2 S protein is according to the numbering of the amino acid positions in SEQ ID NO: 1.

The invention in particular provides recombinant pre-fusion SARS-CoV-2 S proteins, or fragments thereof, comprising an SI and an S2 domain, and comprising a deletion of the furin cleavage site; and a mutation of the amino acid residue at position 1072 into P and/or a mutation of the amino acid residue at position 1203 into K, wherein the numbering of the amino acid positions is according to the numbering of the amino acid positions in SEQ ID NO: 1.

The present invention thus provides stabilized, recombinant, pre-fusion Coronavirus S proteins, such as SARS-CoV-2 S proteins that are stabilized in the pre-fusion conformation, that have improved percentage of trimer formation and/or improved trimer yields and/or improved (thermal) stability as compared to previously described Coronavirus S proteins.

The invention also provides nucleic acid molecules encoding the pre-fusion Coronavirus S proteins, such as SARS-CoV-2 S proteins, and fragments thereof, such as DNA, RNA, mRNA, as well as vectors comprising such nucleic acid molecules.

The invention moreover provides compositions, preferably vaccine compositions, comprising a Coronavirus S protein, such as a SARS-CoV-2 S proteins, or a fragment thereof, a nucleic acid molecule and/or a vector, as described herein.

The invention also provides compositions for use in inducing an immune response against a Coronavirus S protein, and in particular to the use thereof as a vaccine against a Coronavirus associated disease, such as COVID-19.

The invention also relates to methods for inducing an immune response against a Coronavirus in a subject, comprising administering to the subject an effective amount of a pre-fusion Coronavirus S protein or a fragment thereof, a nucleic acid molecule encoding said Coronavirus S protein, and/or a vector comprising said nucleic acid molecule, as described herein. Preferably, the induced immune response is characterized by the induction of neutralizing antibodies to the Coronavirus and/or protective immunity against the Coronavirus.

In particular aspects, the invention relates to methods for inducing anti-Coronavirus S protein antibodies in a subject, comprising administering to the subject an effective amount of an immunogenic composition comprising a pre-fusion Coronavirus S protein, or a fragment thereof, a nucleic acid molecule encoding said Coronavirus S protein, and/or a vector comprising said nucleic acid molecule, as described herein.

The invention also relates to the use of the Coronavirus S proteins or fragments thereof, as described herein, for isolating monoclonal antibodies against a Coronavirus S protein from infected humans.

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

Another general aspect relates to a host cell comprising the isolated nucleic acid molecule or vector encoding the recombinant Coronavirus S protein of the invention. Such host cells can be used for recombinant protein production, recombinant protein expression, or the production of protein particles or viral particles.

Another general aspect relates to methods of producing a recombinant Coronavirus S protein, comprising growing a host cell comprising an isolated nucleic acid molecule or vector encoding the recombinant Coronavirus S protein of the invention under conditions suitable for production of the recombinant Coronavirus S protein.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG.l Schematic representation of the conserved elements of the fusion domain of a SARS-

CoV-2 S protein. The head domain contains an N-terminal (NTD) domain, the receptor binding domain (RBD) and domains SD1 and SD2. The fusion domain contains the fusion peptide (FP), refolding region 1 (RR1), refolding region 2 (RR2), transmembrane region

(TM) and cytoplasmic tail. Cleavage site between SI and S2 and the S2’ cleavage sites are indicated with arrow.

FIG.2: Analytical SEC with purified foldon-less spike with the HexaPro substitutions (Hsieh et. al., (2020) Science 369(6510): 1501 -1505) after storage for indicated time at 4°C (A) or

37°C (B).

FIG. 3: Analytical SEC with Expi293F cell culture supernatants after transfection with plasmids coding for COR211185 protein (dashed line) and the COR211185 with the amino acid substitutions indicated at the top of each graph (solid line). The T indicates the trimer peak and the M indicates the monomer peak.

FIG. 4: Analytical SEC with Expi293F cell culture supernatants after transfection with plasmids coding for COR210613 protein (dashed line) and the COR210613 with the amino acid substitutions indicated at the top of each graph (solid line). The T indicates the trimer peak and the M indicates the monomer peak.

FIG. 5: A) Overview of mutations that delete the furin cleavage site (furin KO). B) Overview of stabilizing mutations in tested constructs in C. C) Analytical SEC with Expi293F cell culture supernatants after transfection with plasmids coding for COR210730 protein (light grey line) and COR210730 with the amino acid substitutions shown in B) (darker grey lines).

The T indicates the trimer peak, and the M indicates the monomer peak.

FIG. 6: A) Overview of mutations that delete furin cleavage site (furin KO) that were introduced in the background of the B.1.617.2 (delta) variant of concern (VoC). B) Overview of stabilizing substitutions in tested constructs in C. C) Analytical SEC with Expi293F cell culture supernatants after transfection with plasmids coding for COR210733 protein (light grey line) and COR210733 with the amino acid substitutions shown in B) (darker grey lines). The T indicates the trimer peak, and the M indicates the monomer peak. D), E), F) Analytical

SEC with Expi293F cell culture supernatants after transfection with plasmids directly after harvesting (solid lines) and after overnight slow freezing to -20°C (dashed lines). The T indicates the trimer peak, and the M indicates the monomer peak.

FIG. 7: A) Overview of mutations that abrogate furin cleavage (furin KO) that were introduced in the background of the B. 1.617.2 (delta) variant of concern (VoC); B, D)

Overview of stabilizing substitutions in tested constructs in C and E; C, E) Analytical SEC with Expi293F cell culture supernatants after transfection with plasmids coding for the construct without E1072P and the construct with the E1072P directly after harvesting (grey and black solid lines, respectively) and after overnight slow freezing to -20°C (dashed lines).

The T indicates the trimer peak, and the M indicates the monomer peak.

FIG. 8: A) Analytical SEC with S protein COR210613 that was purified with lentil lectin chromatography followed by preparative SEC (dashed curve) and the COR210613 with the amino acid substitution E1072P (COR210647) (solid curve). B) Analytical SEC with purified S protein COR210613 (dashed curve) and the COR210613 with the amino acid substitution L1203K (solid curve). The T indicates the trimer peak and the M indicates the monomer peak. The dashed vertical lines indicate the pooled fractions that were tested in slow freezing (see Fig. 9).

FIG. 9: A) Overview of mutations that abrogate furin cleavage (furin KO); B) Overview of stabilizing substitutions in tested constructs in C; C) Analytical SEC with purified

COR210613 and with COR210613 including E1072P (COR210647) directly after harvesting

(grey and black solid lines, respectively) and after overnight slow freezing to -20°C (dashed lines). The T indicates the trimer peak and M indicates the monomer peak.

FIG. 10: Analytical SEC with Expi293F cell culture supernatants after transfection. A)

Construct coding for COR220628 protein with both the E1072P and L1203K (solid line) is compared with COR220627 protein (dashed line) that does not have the E1072P amino acid substitution. B) Construct coding for COR220628 protein with both the E1072P and L1203K

(solid line) is compared with COR220626 protein (dashed line) that does not have the

L1203K amino acid substitution. The T indicates the trimer peak, and the M indicates the monomer peak.

FIG. 11 : Analytical SEC with Expi293F cell culture supernatants after transfection with

COR220341, an OC43 S construct with I1293K (equivalent to L1203K in Wuhan S) (solid line) compared to COR201332 that does not have I1293K. The T indicates the trimer peak.

FIG. 12: Analytical SEC with Expi293F cell culture supernatants after transfection with

COR220740, an NL63 S construct with L1286K (equivalent to L1203K in Wuhan S) (solid line) compared to COR220741 that does not have L1286K. The T indicates the trimer peak, and the M indicates the monomer peak.

FIG. 13: Analytical SEC with Expi293F cell culture supernatants after transfection with

COR220711, a MERS-CoV S construct with L1286K (equivalent to L1203K in Wuhan S)

(solid line) compared to COR220710 that does not have L1286K. The T indicates the trimer peak, and the M indicates the monomer peak.

DETAILED DESCRIPTION OF THE INVENTION

As explained above, the spike protein (S) of e.g. SARS-CoV-2 and of other Coronaviruses is involved in fusion of the viral membrane with a host cell membrane, which is required for infection. SARS-CoV-2 S RNA is translated into a 1273 amino acid precursor protein, which contains a signal peptide sequence at the N-terminus (e.g. amino acid residues 1-13 of SEQ ID NO: 1) 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 SARS-CoV- 2), and at a conserved site upstream of the fusion peptide (S2’) in all known Coronaviruses. For SARS-CoV-2, furin cleaves at S1/S2 between residues 685 and 686, and subsequently the S protein is cleaved within S2 at the S2’ site between residues at position 815 and 816 by TMPRSS2. C-terminal to the S2’ site the proposed fusion peptide is located at the N-terminus of the refolding region 1. Spike proteins assemble into trimers on the virion surface. Coronavirus S protein thus is a trimeric protein, formed by three identical S protein monomers, each monomer comprising an SI and S2 domain, as shown in FIG. l.

Several vaccines against SARS-CoV-2 infection are currently available, which are based on different vaccine modalities, such as RNA-based or vector-based vaccines or subunit vaccines based on purified S protein. Since class I proteins are metastable proteins, increasing the stability of the pre-fusion conformation of fusion proteins increases the expression level of the protein, because less protein will be misfolded and more protein will successfully transport through the secretory pathway. Therefore, if the stability of the prefusion conformation of the class I fusion protein, like SARS-CoV-2 S protein is increased, the immunogenic properties of a protein-based, RNA or vector-based vaccine will be improved since the expression of the S protein is higher and the conformation of the immunogen more closely resembles the pre-fusion conformation that is recognized by potent neutralizing and protective antibodies.

In addition, it is important that the stabilized S proteins have improved trimer yields as compared to previously described Coronavirus S protein trimers. Besides the importance of high expression, which is needed to manufacture a vaccine successfully, maintenance of the trimeric pre-fusion conformation during the manufacturing process and during storage over time is critical for protein-based vaccines. For a soluble, subunit-based vaccine, the Coronavirus S protein needs to be truncated by deletion of the transmembrane (TM) and the cytoplasmic region to create a soluble secreted S protein (sS). Because the TM region is responsible for membrane anchoring and increases stability, the anchorless soluble S protein is considerably more labile than the full-length protein and will even more readily refolds into the post-fusion end-state. In order to obtain soluble trimeric S protein in the stable pre-fusion conformation that shows high expression levels and high stability, the pre-fusion conformation thus needs to be stabilized. Because also the full length (membrane-bound) Coronavirus S protein is metastable, the stabilization of the pre-fusion conformation is also desirable for the full-length Coronavirus S protein, i.e. including the TM and cytoplasmic region, e.g. for any DNA, RNA, live attenuated or vector-based vaccine approach.

The present invention provides stabilized recombinant pre-fusion Coronavirus S proteins, comprising an SI and an S2 domain, and comprising a mutation of the amino acid residue corresponding to the amino acid residue at position 1072 of a SARS-CoV-2 S protein into P and/or a mutation of the amino acid residue corresponding to the amino acid residue at position 1203 of a SARS-CoV-2 S protein into K, wherein the numbering of the amino acid positions of the SARS-CoV-2 S protein is according to the numbering of the amino acid positions in SEQ ID NO: 1.

The present invention also provides fragments of the stabilized Coronavirus S proteins.

In certain embodiments, the invention provides stabilized recombinant pre-fusion SARS-CoV-2 S proteins, comprising an SI and an S2 domain, or fragments thereof, and comprising a mutation of the amino acid residue corresponding to the amino acid residue at position 1072 of a SARS-CoV-2 S protein into P and/or a mutation of the amino acid residue corresponding to the amino acid residue at position 1203 of a SARS-CoV-2 S protein into K, wherein the numbering of the amino acid positions of the SARS-CoV-2 S protein is according to the numbering of the amino acid positions in SEQ ID NO: 1. In certain embodiments, the proteins do not comprise a furin cleavage site. Thus, in certain embodiments, the furin cleavage site has been deleted.

The invention in particular provides recombinant pre-fusion SARS-CoV-2 S proteins, comprising an SI and an S2 domain, or fragments thereof, and comprising a deletion of the furin cleavage site; and a mutation of the amino acid residue at position 1072 into P and/or a mutation of the amino acid residue at position 1203 into K, wherein the numbering of the amino acid positions is according to the numbering of the amino acid positions in SEQ ID NO: 1.

According to the present invention, mutation(s) are described that stabilize soluble Coronavirus S proteins, even without a heterologous trimerization domain (such as a foldon domain). According to the present invention it has thus been shown that the mutation(s) prevent S trimer dissociation and stabilize the S protein trimers without foldon. It has in particular been shown the SARS-CoV-2 S proteins according to the invention are more stable after slow freezing and thawing compared to S proteins without the mutation(s).

In certain embodiments, the Coronavirus is an Alphacoronavirus, Betacoronavirus, Gammacoronavirus, or Deltacoronavirus. In particular embodiments, the S protein is from a Coronavirus that can infect humans, such as an Alphacoronavirus or a Betacoronavirus. In further preferred embodiments, the S protein is from SARS-CoV, MERS-CoV, HCoV-OC43 or HCoV-NL63, or the currently circulating SARS-CoV-2.

In certain embodiment, the Coronavirus S proteins comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 19-35, or a fragment thereof (i.e. without signal sequence and/or transmembrane/cytoplasmic domain).

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, or at positions corresponding to these positions in SEQ ID NO: 1, increases the stability of the S proteins in the pre-fusion conformation and/or increases trimer yields. According to the invention, the proteins thus comprise one or more mutations (substitutions) in their amino acid sequence as compared to the amino acid sequence of a wild type S protein. The mutations according to the invention can be introduced in any wild-type Coronavirus S protein, such as a SARS-CoV-2 S protein, including the S protein of the original Wuhan SARS-CoV-2 strain (SEQ ID NO: 1), or in the S proteins of any SARS-COV-2 variants, such as, but not limited to the Bl.617.2 strain. The wording ‘the amino acid at position 1072” refers to the amino acid residue that is at position 1072 in SEQ ID NO: 1. It will be understood by the skilled person that equivalent amino acids in S proteins of other SARS-CoV-2 strains can be determined by sequence alignment.

In certain embodiments, the mutations are introduced in the amino acid sequence of an S protein of the Bl.617.2 (Delta) strain.

In certain embodiments, the multimeric Coronavirus S proteins, such as SARS-CoV-2 S proteins, are trimeric, i.e. comprise three monomers comprising identical amino acid sequences.

In certain embodiments, the Coronavirus S protein is a SARS-CoV-2 S protein. In certain embodiments, the SARS-CoV-2 S protein (monomer) comprises an amino acid sequence wherein the amino acid residue at position 614 is not aspartic acid (D). Preferably, the amino acid residue at position 614 is glycine (G).

In certain embodiments, the SARS-CoV-2 S protein (monomer) comprises an amino acid sequence wherein the amino acid residue at position 572 is not threonine (T). Preferably, the amino acid residue at position 572 is isoleucine (I). In certain preferred embodiments, the SARS-CoV-2 S protein (monomer) comprises an amino acid sequence wherein the amino acid residue at position 614 is glycine (G) and the amino acid residue at position 572 is isoleucine (I).

It is known that SARS-CoV-2 S protein contains a unique furin-like cleavage site (FCS), RARR, which is absent in other lineage B PCOVS, such as SARS-CoV. According to the present invention, the SARS-CoV-2 S protein (monomer) comprises a deletion of the furin cleavage site. A deletion of the furin cleavage site may comprise one or more mutations that abrogate cleavage of the furin-cleavage site. A deletion of the furin cleavage, e.g. by mutation of one or more amino acids in the furin cleavage site (such that the protein is not cleaved by furin), renders the protein uncleaved, which further increases its stability. Deleting the furin cleavage site can be achieved in any suitable way that is known to the skilled person. In certain embodiments, the deletion of the furin cleavage site comprises a mutation of the amino acid arginine (R) at position 682 into serine (S), a mutation of the amino acid R at position 683 into glycin (G) and/or a mutation of the amino acid R at position 685 into G.

In certain embodiments, the SARS-CoV-2 S protein comprises an amino acid sequence wherein the amino acid residue at position 682 is S and the amino acid at position 685 is G.

In certain embodiments, the SARS-CoV-2 S protein comprises an amino acid sequence wherein the amino acid residue at position 682 is S, the amino acid at position 683 is G and the amino acid at position 685 is G.

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 Coronavirus S protein, such as a SARS-CoV-2 S protein, for example, the full-length sequence of a Coronavirus S protein. It will be appreciated that for inducing an immune response and in general for vaccination purposes, a protein needs not 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 Coronavirus 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 Coronavirus S protein, such as the SARS-CoV-2 S protein. In certain embodiment, the fragment is the Coronavirus 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 1-1208 (or 14-1208 without signal peptide) of SEQ ID NO: 1.

The pre-fusion Coronavirus S proteins, such as the SARS-CoV-2 S proteins, according to the invention are trimeric and stable, i.e. do not readily change into the postfusion conformation upon processing of the proteins, such as e.g. upon purification, freezethaw cycles, and/or storage etc.

In addition, or alternatively, the recombinant prefusion Coronavirus S proteins, such as the SARS-CoV-2 S proteins, according to the invention preferably have an increased trimer : monomer ratio as compared to SARS-CoV-2 S proteins without the mutation(s) of the invention.

In certain embodiments, the pre-fusion SARS-CoV-2 S proteins have an increased trimer to monomer ratio 30 minutes after heating at 65°C as compared to SARS-CoV-2 S proteins without the mutation(s) of the invention.

In addition, or alternatively, the pre-fusion SARS-CoV-2 S proteins have an increased trimer to monomer ratio after storing at 4°C for at least 1 week, preferably at least 2 weeks, as compared to SARS-CoV-2 S proteins without the mutation(s) of the invention.

In addition, or alternatively, the pre-fusion SARS-CoV-2 S proteins have an increased trimer to monomer ratio after freezing and thawing as compared to SARS-CoV-2 S proteins without the mutation(s) of the invention.

The proteins according to the invention may comprise a signal peptide, also referred to as signal sequence or leader peptide, corresponding to amino acids 1-13 of SEQ ID NO: 1. 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. In certain embodiments, the SARS-CoV-2 S protein comprises an amino acid sequence wherein the amino acid residue at position 532 is not asparagine (N). Preferably, the amino acid residue at position 532 is proline (P).

In certain preferred embodiments, the SARS-CoV-2 S protein (monomer) comprises an amino acid sequence wherein the amino acid residue at position 614 is glycine (G), the amino acid residue at position 532 is P and the amino acid residue at position 572 is isoleucine (I).

In certain embodiments, the SARS-CoV-2 S proteins comprise an amino acid sequence, comprising one or more additional mutation(s) selected from the group consisting of: a mutation of the amino acid at position 892 into P, a mutation of the amino acid at position 942 into P, a mutation of the amino acid at position 944 into P, a mutation of the amino acid at position 987 into P, a mutation of the amino acid at position 1118 into H, an intraprotomeric disulfide bridge between the amino acid at position 880 and 888, and an interprotomeric disulfide bridge between amino acid residues 713 and 894.

According to the invention it is to be understood that “an interprotomeric disulfide bridge between residues 713 and 894” means that the amino acids at the positions 713 and 894 have been mutated into a cysteine (C) and a disulfide bridge is formed between the cysteine at position 547 of one monomer and the cysteine at position 987 of another monomer. An intraprotomeric disulfide bridge is formed between two cysteine residues within one mononer. Thus, in certain embodiments, the SARS-CoV-2 S proteins, or fragments thereof, comprising an interprotomeric disulfide bridge comprise at least a first and a second S protein monomer and comprising a disulfide bridge between the amino acid residue 713 of the first monomer and the amino acid residue 894 of the second monomer.

In certain embodiments, the SARS-CoV-2 S proteins of the invention comprise an SI and an S2 domain, optionally a deletion of the furin cleavage site, and a mutation of the amino acid residue at position 1072 into P and/or a mutation of the amino acid at position 1203 into K, and wherein the amino acid at position 614 is G.

In certain embodiments, the SARS-CoV-2 S proteins of the invention comprise an SI and an S2 domain, optionally a deletion of the furin cleavage site, a mutation of the amino acid residue at position 1072 into P and/or a mutation of the amino acid at position 1203 into K, wherein the amino acid at position 614 is G, the amino acid at position 892 is P, the amino acid at position 942 is P and the amino acid at position 987 is P.

In certain embodiments, the SARS-CoV-2 S proteins of the invention comprise an SI and an S2 domain, optionally a deletion of the furin cleavage site, and a mutation of the amino acid residue at position 1072 into P and/or a mutation of the amino acid at position 1203 into K, wherein the amino acid at position 532 is P, the amino acid at position 572 is I, the amino acid at position 614 is G, the amino acid at position 892 is P, the amino acid at position 944 is P, the amino acid at position 987 is P, the amino acid at position 1118 is H, and the amino acid at position 1072 is P, and further comprising an interprotomeric disulfide bridge between residues 713 and 894.

In certain embodiments, the SARS-CoV-2 S proteins of the invention comprise an SI and an S2 domain, optionally a deletion of the furin cleavage site; and a mutation of the amino acid residue at position 1072 into P and/or a mutation of the amino acid at position 1203 into K, and wherein the amino acid at position 532 is P, the amino acid at position 572 is I, the amino acid at position 614 is G, the amino acid at position 892 is P, the amino acid at position 944 is P, the amino acid at position 987 is P, the amino acid at position 1118 is H, the amino acid at position 880 is C and the amino acid at position 888 is C.

In certain embodiments, the SARS-CoV-2 S protein comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 6-8, 10, 11, 13 and 14-18. The present invention further provides nucleic acid molecules encoding the Coronavirus S proteins, such as the SARS-CoV-2 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), 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 Coronavirus S proteins, such as the SARS-CoV-2 S proteins, also form part of the invention. The prefusion Coronavirus S proteins, such as the SARS-CoV-2 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 SARS-CoV-2 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, such as the pre-fusion SARS-CoV-2 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 polyA 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 Coronavirus S protein, such as a SARS-CoV-2 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 Coronavirus S protein, e.g. SARS-CoV-2 S protein. The invention further provides immunogenic compositions comprising a pre-fusion Coronavirus S protein, such as a SARS-CoV-2 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 Coronavirus S protein, such as a SARS-CoV-2 S protein, a nucleic acid molecule, and/or a vector, according to the invention, for inducing an immune response against a Coronavirus S protein, such as a SARS-CoV-2 S protein in a subject. Further provided are methods for inducing an immune response against Coronavirus S protein, such as a SARS- CoV-2 S protein in a subject, comprising administering to the subject a pre-fusion Coronavirus S protein, such as a SARS-CoV-2 S protein, and/or a nucleic acid molecule, and/or a vector according to the invention. Also provided are pre-fusion Coronavirus S proteins, such as SARS-CoV-2 S proteins, nucleic acid molecules, and/or vectors, according to the invention for use in inducing an immune response against Coronavirus S protein, such as a SARS-CoV-2 S protein in a subject. Further provided is the use of the pre-fusion Coronavirus S proteins, such as SARS-CoV-2 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 Coronavirus S protein, such as a SARS-CoV-2 S protein in a subject. In certain embodiments, the nucleic acid molecule is DNA and/or an RNA molecule.

The pre-fusion Coronavirus S proteins, such as SARS-CoV-2 S proteins, nucleic acid molecules, or vectors of the invention may be used for prevention (prophylaxis, including post-exposure prophylaxis) of Coronavirus infections, such as SARS-CoV-2 infections. In certain embodiments, the prevention may be targeted at patient groups that are susceptible for and/or at risk of Coronavirus infection, such as a SARS-CoV-2 infection or have been diagnosed with a SARS-CoV-2 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 Coronavirus S proteins, such as SARS-CoV-2 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 Coronavirus, such as SARS-CoV-2, 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 Coronavirus S infection, such as a SARS-CoV-2 infection, in a subject utilizing the pre-fusion Coronavirus S proteins, such as SARS-CoV-2 S proteins, nucleic acid molecules and/or vectors according to the invention. In a specific embodiment, a method for preventing and/or treating Coronavirus infection, such as SARS-CoV-2 infection, in a subject comprises administering to a subject in need thereof an effective amount of a pre-fusion Coronavirus S protein, such as a 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 Coronavirus, such as SARS-CoV-2. Prevention encompasses inhibiting or reducing the spread of Coronavirus, such as SARS-CoV-2, or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection by Coronavirus, e.g. 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 Coronavirus infection, such as SARS-CoV-2 infection. For administering to subjects, such as humans, the invention may employ pharmaceutical compositions comprising a pre-fusion Coronavirus S protein, such as a SARS- CoV-2 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 SARS-CoV-2 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. coll 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 Coronavirus S proteins, such as SARS-CoV-2 S proteins may also be administered in combination with or conjugated to nanoparticles, such as e.g. polymers, liposomes, virosomes, virus-like particles. The Coronavirus S proteins, such as SARS-CoV-2 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 Coronavirus S proteins, such as SARS-CoV-2 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 Coronavirus, such as a SARS-CoV-2 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 Coronavirus S protein, such as a SARS-CoV-2 S protein and/or a nucleic acid molecule encoding a pre-fusion Coronavirus S protein, such as a SARS-CoV-2 S protein, and/or a vector comprising said nucleic acid molecule, which results in an immune response against the S protein of Coronavirus S protein, such as a SARS-CoV-2 . 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 Coronavirus infection and replication, such as a SARS-CoV-2 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 Coronavirus, such as SARS-CoV-2, e.g. against other antigenic proteins of SARS- CoV-2, 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, HMPV, PIV and/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.

Compositions may be administered to a subject, e.g. a human subject. The total dose of the SARS-CoV-2 S proteins in a composition for a single administration can for instance be about 0.01 pg to about 10 mg, e.g. 1 pg - 1 mg, e.g. 10 pg - 100 pg. Determining the recommended dose will be carried out by experimentation and is routine for those skilled in the art.

Administration of the compositions according to the invention can be performed using standard routes of administration. Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transcutaneous, or mucosal administration, e.g. intranasal, oral, and the like. In one embodiment a composition is administered by intramuscular injection. The skilled person knows the various possibilities to administer a composition, e.g. a vaccine in order to induce an immune response to the antigen(s) in the vaccine.

A subject as used herein preferably is a mammal, for instance a rodent, e.g. a mouse, a cotton rat, or a non-human-primate, or a human. Preferably, the subject is a human subject.

The proteins, nucleic acid molecules, vectors, and/or compositions may also be administered, either as primary vaccination, or as a boost, in a homologous or heterologous prime-boost regimen. If a boosting vaccination is performed, typically, such a boosting vaccination will be administered to the same subject at a time between one week and one year, preferably between two weeks and four months, after administering the composition to the subject for the first time (which is in such cases referred to as ‘primary vaccination’).

The Coronavirus S proteins, such as SARS-CoV-2 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 Coronavirus S protein, such as a SARS-CoV-2 protein, as bait for isolating monoclonal antibodies.

Also provided is the use of the pre-fusion Coronavirus S proteins, such as SARS-CoV-2 S proteins, of the invention in methods of screening for candidate Coronavirus, such as a SARS-CoV-2, antiviral agents, including but not limited to antibodies against Coronavirus, such as a SARS-CoV-2

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 CoV 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.

EXAMPLES

EXAMPLE 1 S protein with HexaPro substitutions and no foldon is instable, introduction of stabilizing mutations

Current SARS-CoV-2 S protein subunit vaccines may not be as stable as desired for optimal developability, storage and efficacy. Thus, the previously described double proline substitutions (Wrapp et. al., (2020) Science 367(6483): 1260-1263) and even the HexaPro designs (Hsieh et. al., (2020) Science 369(6510): 1501 -1505) have been shown to suffer from cold denaturation (Olmedillas et al (2021) bioRxiv, doi.org/10.1101/2021.05.06.441046), wherein in a few weeks at 4°C (Fig. 2), S proteins containing a foldon open up, losing the closed prefusion conformation, and S proteins without foldon dissociate. Although a foldon trimerization domain prevents trimers from dissociating into monomers, it is desired to not having such additional domain, as an immune response against the foldon could arise when used as a vaccine.

In this Example two novel stabilizing mutations are described that stabilize S proteins, even without a heterologous trimerization domain (such as a foldon domain).

Materials and Methods

SEC analysis

An ultra-high-performance liquid chromatography system (Vanquish, Thermo Scientific) and pDAWN Light Scattering Detector (Wyatt) coupled to an pT-rEX Refractive Index Detector (Wyatt), in combination with an in-line Nanostar DLS reader (Wyatt), was used for performing the analytical SEC experiment. The cleared crude cell culture supernatants either freshly after harvest or after ~24 hours slow freezing of the 96-well plate in a Styrofoam box in a -20°C freezer were applied to an SRT-10C SEC-500 15 cm column (Sepax Cat. #235500-4615) with the corresponding guard column (Sepax) equilibrated in running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0) at 0.35 ml/min. When analyzing supernatant samples, pMALS detectors were offline and analytical SEC data were analyzed using Chromeleon 7.2.8.0 software package. The signal of supernatants of nontransfected cells was subtracted from the signal of supernatants of S transfected cells. When purified proteins were analyzed using SEC-MALS, pMALS detectors were inline and data were analyzed using Astra 7.3.2 software package. Results:

HexaPro without foldon disintegrates into monomers over time

SARS-CoV-2 S trimers containing the HexaPro substitutions (Hsieh at al. (2020) Science 369(6510): 1501-1505), but without foldon for trimerization, were purified. The SEC pattern of the freshly purified proteins shows only trimers. However, after storing the protein at 4°C and 37°C for 2 and 8 weeks, the trimer dissociated into monomers. After 8 weeks at 4°C most of the trimers have disintegrated into monomers (Fig. 2).

E1072P and L1203K increase trimer yield and trimer/monomer ratio

Amino acid substitutions E1072P in the inter-subunit beta-sheet and L1203K in the stem resulted in increased trimer formation and relative reduction of monomer formation when compared with the COR211185 backbone, i.e. an S protein without these mutations (Fig. 3). COR211185 is an S ectodomain with the Wuhan S sequence with now dominant D614G mutation, furin cleavage site knock out substitutions (R682S, R685G) and without a C-terminal foldon domain. E1072P and L1203K also increased trimer yield and reduce monomer yield in a somewhat further stabilized S protein ectodomain, i.e. the COR210613 backbone (Fig. 4). COR210613 is an S ectodomain with four stabilizing amino acid substitutions (D614G, A892P, A942P and V987P) and furin cleavage site knock out substitutions (R682S, R685G) and without a C-terminal foldon domain.

E1072P and/or L1203K also increase trimer yield and trimer/monomer ratio in stable S variants

Next, the substitutions were evaluated in a more stable S variant (COR210730) that contained additional stabilizing substitutions compared to COR210613 (COR210730: N532P, T572I, DI 118H, A713C and L894C in addition to D614G, A892P and V987P and A944P instead of A942P). Comparison of the stable COR210730 and variants with the additional substitution E1072P (COR211046), L1203K (COR211047) and E1072P plus L1203K (COR211048) is shown in Fig. 5. In the COR210730 backbone the L1203K and the E1072P separately and in combination increased trimer yield and decreased monomer yield resulting in a higher trimer/monomer ratio.

Besides the effect of the stabilizing substitutions in the Wuhan lineage (Fig. 5) the stabilizing substitutions were also tested in a similarly stabilized S based on the amino acid sequence of the S protein of the B.1.617.2 lineage (Fig. 6) confirming the trimer yield enhancing and trimer/monomer ratio enhancing effects of these substitutions in another backbone.

E1072P and L1203K protect S protein from dissociating into monomers after slow freezing E1072P and L1203K protected the spike protein to dissociate into monomers after the cell culture supernatants were slowly frozen to -20°C, showing that they stabilize the spike protein (Fig. 6 D, E and F). The stabilizing effect of E1072P was also confirmed in two Delta variant spikes with fewer stabilizing substitutions (Fig. 7).

Purification of S proteins

The S proteins (COR210613 with E1072P (i.e. COR210647) and COR210613 with L1203K (i.e. COR210675)) were purified using Lentil Lectin Agarose followed by size exclusion chromatography (SEC). The SEC pattern confirms that the mutation increased the trimer yield and trimer/monomer ratio (Fig. 8). Also after slow freezing of the purified trimers, E1072P was shown to protect the protein from dissociating into monomers (Fig. 9). Conclusion:

According to the present invention it has been shown that the introduction of one or both of the mutations E1072P and L1203K increases the trimer/monomer ratio of soluble S trimer.

EXAMPLE 2 E1072P and L1203K increase trimer yield and trimer/monomer ratio in SARS-

CoV S proteins

A construct was made of a SARS-CoV S protein with the double proline substitutions described by Pallesen et al. 2017 (supra) but without the foldon domain and including the E1072P and L1203K mutations, according to the Wuhan S numbering, i.e. according to the numbering of SEQ ID NO: 1 (COR220628). The constructs without E1072P (COR220627) and L1203K (COR220626) both showed lower trimer expressions and lower trimer/monomer ratios, showing that both E1072P and L1203K both improve the trimer yield and trimer/monomer ratio in SARS-CoV S (Fig. 10).

1203K increases trimer yield in other coronavirus S proteins (e.g. OC43, NL63 and MERS- CoV)

The 1203K mutation (numbering of SEQ ID NO: 1) was also tested in S proteins (without foldon trimerization domains) of other Coronaviruses, e.g. of OC43, NL63 and MERS-CoV. OC43 S protein stabilized with only double proline substitutions described by Pallessen et al., 2017 (supra) (COR201332) hardly produces any trimers, whereas a clear trimer peak becomes visible when the mutation corresponding to 1203K (i.e. I1293K in OC43 S) is introduced (COR220341) (Fig. 11). Trimer expression ofNL63 S protein stabilized with double proline substitutions (COR220741) is increased by the introduction of the of the 1203K equivalent (L1286K in NL63 S) (COR220740) (Fig. 12). MERS-CoV S with only the double proline substitutions and with the furin cleavage site knock out described by Pallesen et al. 2017 (supra) hardly produced trimers (COR220710), but when the mutation corresponding to the 1203K mutation (L1286K in MERS-CoV S) was introduced (COR220711) a trimer expression was enhanced substantially (Fig. 13).

Alignment of S proteins across coronaviruses, including genera Alphacoronavirus (a- CoV), Betacoronavirus (P-CoV), Gammacoronavirus (y-CoV), and Deltacoronavirus (6-CoV) is shown below (Wuhan S numbering, SEQ ID NO: 1 : aa 1163-1213, corresponding to the HR2 region until TMD; Li et al. (2022) Int J Mol Sci, 23: 1040), showing strong sequence conservation at amino acid position corresponding to position 1203 (Wuhan S numbering, SEQ ID NO: 1), with the presence of leucine (L) for genera a-CoVs (i.e., NL63, 229E, FIPV, PEDV, TGEV, CCoV-HuPn-2018), p-CoVs (lineages B (i.e., SARS-CoV, SARS-CoV-2), C (i.e., MERS, HKU4), D (i.e., HKU9)), y -CoVs (i.e., IBV, HKU22), and 8-CoVs (i.e., HKU16, HKU19, PDCoV, hu-PDCoV), and isoleucine (I) for P-CoVs of A lineage (i.e., OC43, HKU1) (L and I at position 1203 highlighted in black). Mutation of this conserved amino acid into lysine (K) thus provides a universal approach towards increasing coronavirus S trimer expression.

Alignment of S proteins across coronaviruses

Table 1. Standard amino acids, abbreviations and properties

SEQUENCES

SEQ ID NO: 1 full length S protein (underline signal peptide, double underline TM and cytoplasmic domain that is deleted in the soluble version)

SEQ ID NO: 2 COR210613

SEQ ID NO: 3 COR210647 (=COR210613+E1072P)

Claims

1. Recombinant pre-fusion Coronavirus S protein, comprising an SI and an S2 domain, or a fragment thereof, and comprising a mutation of the amino acid residue corresponding to the amino acid at position 1072 in a SARS-CoV-2 S protein into P and/or a mutation of the amino acid residue corresponding to the amino acid at position 1203 in a SARS-CoV-2 S protein into K, wherein the numbering of the amino acid positions in said SARS-CoV-2 S protein is according to the numbering of the amino acid positions in SEQ ID NO: 1.

2. The protein according to claim 1, wherein the Coronavirus S protein is from an a- or P- coronavirus.

3. The protein according to claim 1 or 2, wherein the Coronavirus S protein is a SARS-CoV-2 S protein.

4. The protein according to claim 1, 2 or 3, comprising a deletion of the furin cleavage site.

5. The protein, or fragment thereof, according to claim 3 or 4, comprising an amino acid sequence wherein the amino acid residue at position 614 is not aspartic acid (D).

6. The protein, or fragment thereof, according to claim 3, 4 or 5, wherein the amino acid residue at position 614 is glycine (G).

7. The protein, or fragment thereof, according to claim 3 or 4, wherein the amino acid residue at position 572 is not threonine (T).

8. The protein, or fragment thereof, according to any one of the claims 3-7, wherein the amino acid residue at position 572 is isoleucine (I).

9. The protein, or fragment thereof, according to any one of the preceding claims 4-7, wherein the deletion of the furin cleavage site comprises a mutation of the amino acid at position 682 into S, a mutation of the amino acid at position 683 into G and/or a mutation of the amino acid at position 685 into G.

10. The protein, or fragment thereof, according to any one of the preceding claims 3-9, wherein the amino acid at position 532 is P.

11. The protein, or fragment thereof, according to any one of the preceding claims 3-10, further comprising one or more mutations selected from the group consisting of: a mutation of the amino acid at position 892 into P, a mutation of the amino acid at position 942 into P, a mutation of the amino acid at position 944 into P, a mutation of the amino acid at position 987 into P, a mutation of the amino acid at position 1118 into H, an intraprotomeric disulfide bridge between the amino acid at position 880 and 888, and an interprotomeric disulfide bridge between amino acid residues 713 and 894.

12. The protein, or fragment thereof, according to any one of the preceding claims, comprising a truncated S2 domain.

13. The protein, or fragment thereof, according to claim 12, wherein the transmembrane and cytoplasmic domain have been removed.

14. Nucleic acid molecule encoding a protein, or fragment thereof, according to any one of the preceding claims 1-12.

15. Nucleic acid according to claim 14, wherein the nucleic acid molecule is DNA or RNA.

16. Vector comprising a nucleic acid according to claim 14 or 15.

17. Vector according to claim 16, wherein the vector is a human recombinant adenoviral vector.

18. A composition comprising a protein according to any one of the claims 1-13, a nucleic acid according to claim 14 or 15 and/or vector according to claim 16 or 17.

19. A method for vaccinating a subject against COVID-19, the method comprising administering to the subject a composition according to claim 18.

20. A method for reducing infection and/or replication of SARS-CoV-2 in a subject, comprising administering to the subject a composition according to claim 18.

21. An isolated host cell comprising a nucleic acid according to claim 14 or 15.

22. An isolated host cell comprising a recombinant human adenovirus of serotype 26 comprising a nucleic acid according to claim 14 or 15.