WO2023047348A1

WO2023047348A1 - Stabilized corona virus spike protein fusion proteins

Info

Publication number: WO2023047348A1
Application number: PCT/IB2022/059015
Authority: WO
Inventors: Johannes Petrus Maria Langedijk; Lucy RUTTEN; Jaroslaw JURASZEK
Original assignee: Janssen Pharmaceuticals, Inc.
Priority date: 2021-09-24
Filing date: 2022-09-23
Publication date: 2023-03-30

Abstract

The present invention relates to stabilized recombinant pre-fusion SARS CoV-2 S proteins, and fragments thereof, as well as nucleic acids molecules encoding the SARS CoV-2 S proteins or fragments thereof, and to uses thereof.

Description

STABILIZED CORONA VIRUS 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 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 SARS-CoV-2 S proteins, comprising an SI and an S2 domain, or fragments thereof, and comprising at least one interprotomeric disulfide bridge selected from the group consisting of a disulfide bridge between amino acid residues 547 and 978, a disulfide bridge between amino acid residues 547 and 982, and a disulfide bridge between amino acid residues 713 and 894, 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 invention, it has been shown that the disulfide bridges stabilize

SARS-CoV-2 S protein trimers, both truncated soluble variants and full-length membranebound variants. The invention also provides nucleic acid molecules encoding the pre-fusion SARS-

CoV-2 S proteins and fragments thereof, such as DNA, RNA, mRNA, as well as vectors, e.g. DNA vectors, adenovectors, comprising such nucleic acid molecules.

The invention moreover provides compositions, preferably vaccine compositions, comprising a SARS-CoV-2 S protein, 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 SARS-CoV-2 S protein, and in particular to the use thereof as a vaccine against SARS-CoV-2 associated disease, such as COVID-19.

The invention also relates to methods for inducing an immune response against SARS-CoV-2 in a subject, comprising administering to the subject an effective amount of a pre-fusion SARS-CoV-2 S protein or a fragment thereof, a nucleic acid molecule encoding said SARS-CoV-2 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 SARS-CoV-2 virus and/or protective immunity against the SARS-CoV-2 virus.

In particular aspects, the invention relates to methods for inducing anti-SARS-CoV-2 S protein antibodies in a subject, comprising administering to the subject an effective amount of an immunogenic composition comprising a pre-fusion SARS-CoV-2 S protein, or a fragment thereof, a nucleic acid molecule encoding said SARS-CoV-2 S protein, and/or a vector comprising said nucleic acid molecule, as described herein.

The invention also relates to the use of the SARS-CoV-2 S proteins or fragments thereof, as described herein, for isolating monoclonal antibodies against a SARS-CoV-2 S protein from infected humans. Also provided is the use of the pre-fusion SARS-CoV-2 S proteins of the invention in methods of screening for candidate SARS-CoV-2 antiviral agents, including but not limited to antibodies against SARS-CoV-2.

Another general aspect relates to a host cell comprising the isolated nucleic acid molecule or vector encoding the recombinant SARS-CoV-2 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 SARS-CoV-2 S protein, comprising growing a host cell comprising an isolated nucleic acid molecule or vector encoding the recombinant SARS-CoV-2 S protein of the invention under conditions suitable for production of the recombinant SARS-CoV-2 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. Trimers elute at approximately 5.3 minutes and monomers elute at approximately 6 minutes. 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) and at 37°C (B).

FIG. 3: Analytical SEC with Expi293F cell culture supernatants after transfection with plasmids coding for S-2P (COR200017) protein (dashed line) and the S-2P with the disulfides indicated at the top of each graph (solid line). Trimers elute at approximately 5 minutes and monomers elute at approximately 6 minutes.

FIG. 4: Analytical SEC with Expi293F cell culture supernatants after transfection with plasmids coding for S with only furin knock-out and foldon (COR200151) protein (dashed line) and the COR200151 with the disulfides indicated at the top of each graph (solid line).

Trimers elute at approximately 5 minutes.

FIG. 5: Analytical SEC with Expi293F cell culture supernatants after transfection with plasmids coding for S protein without the X2 disulfide (dashed line) and the same S protein with the X2 (solid line). Trimers elute at approximately 5.2 minutes and monomers elute at approximately 5.8 minutes.

FIG. 6: Disulfides that results in trimer formation. Analytical SEC with Expi293F cell culture supernatants after transfection with plasmids coding for COR210284 S protein (dashed line) and the COR210284 with the disulfides indicated at the top of each graph (solid line). One of the graphs of a duplicate experiment is shown. T indicates where the trimer eluted and M indicates where the monomers eluted.

FIG. 7: Western blot of cell culture supernatant under reducing conditions. The trimers, dimers and monomers are indicated by the arrows.

FIG. 8: Analytical SEC with Expi293F cell culture supernatants stored at 4°C (dashed line) and heated for 30 minutes at 65°C (solid line). The vertical dashed line shows the retention time of the trimer of the backbone COR210284. FIG. 9 Characterization of S protein containing T547C-N978C. A) Analytical SEC with

Expi293F cell culture supernatants after transfection with plasmids coding for COR201291 S protein (dashed line) and the same protein containing T547C-N978C (COR210118) (solid line). B) SEC pattern of purified proteins stored at 4°C and 37°C for 2 and 8 weeks. C) Main melting events measured with differential scanning fluorimetry (DSF). D) Biolayer interferometry showing the initial slope calculated over 300 seconds. Antibodies used are indicated on the X-axis.

FIG. 10: Characterization of purified S proteins with S982C-T547C and A713C-L894C. A)

SEC pattern of purified proteins stored at 4°C and 37°C for 0, 2 days and 1 and 2 weeks. B)

A zoom in on the SEC patterns of figure A. C) Reducing and non-reducing SDS-PAGE D)

Main melting events measured with differential scanning fluorimetry (DSF). E) First derivative curves measured from differential scanning fluorimetry (DSF). F) Biolayer interferometry showing the initial slope calculated over 300 seconds and G) showing the R equilibrium. Antibodies used are indicated on the X-axis.

FIG. 11 : Disulfides protect S protein from dissociation into monomers after slow freezing of the protein. 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), F)

Overview of stabilizing substitutions in tested constructs in C), E) and G). C), E), G)

Analytical SEC with Expi293F cell culture supernatants after transfection with plasmids coding for the construct without disulfide and the construct with the disulfide 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.

Right panel of C) shows a zoom in on the SEC patterns of the left panel.

FIG. 12: Disulfides protect S protein from dissociation into monomers after slow freezing of the protein even in the absence of V987P. 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), F) Overview of stabilizing substitutions in tested constructs in C), E) and G). C), E), G) Analytical SEC with Expi293F cell culture supernatants after transfection with plasmids coding for the construct without V987P and the construct with V987P 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.

FIG. 13 T547C-N978C, T547C-S892C and A713C-L894C stabilize S with only furin cleavage site knock out mutations and the naturally occurring D614G (COR211185).

Analytical SEC with Expi293F cell culture supernatants after transfection with plasmids coding for the construct without disulfide (COR211185 in left upper corner) and the constructs with the disulfides, indicated on top of the chromatograms, directly after harvesting (black 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. 14 FACS experiment with S with T547C-N978C disulfide. A-C) Median fluorescence intensities of some neutralizing and non-neutralizing antibodies. D-F) Same as A-C, but only the non-neutralizing antibodies are shown. G) Substitutions in the different constructs.

FIG. 15 Cell-based ELISA with S with T547C-N978C disulfide. A) Luminescence intensities measured by Cell-based ELISA (n=2) of two neutralizing (2-51 and S309) and two non-neutralizing (CR3046 and CR3022) antibodies. B) Ratio of luminescence intensities of neutralizing versus non-neutralizing antibodies. C) Substitutions in the different constructs.

FIG. 16 Cell-based ELISA with S with T547C-S982C disulfide. A) Luminescence intensities measured by Cell-based ELISA (n=2) of two neutralizing (2-51 and S309) and two nonneutralizing antibodies (CR3046 and CR3022). B) Ratio of luminescence intensities of neutralizing versus non-neutralizing antibodies. C) Substitutions in the different constructs. FIG. 17 Analytical SEC with Expi293F cell culture supernatants of S2 with (solid line) and without (dashed line) T547C-N978C. Trimer and monomer peaks are labeled.

DETAILED DESCRIPTION OF THE INVENTION

As explained above, the spike protein (S) of 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 protein 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. SARS- CoV-2 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 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 SARS-CoV-2 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 SARS-CoV-2 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 refold 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) SARS-CoV-2 S protein is metastable, the stabilization of the pre-fusion conformation is also desirable for the full-length SARS-CoV-2 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 SARS-CoV-2 S proteins, comprising an SI and an S2 domain, or fragments thereof, comprising at least one interprotomeric disulfide bridge selected from the group consisting of a disulfide bridge between amino acid residues 547 and 978, a disulfide bridge between amino acid residues 547 and 982, and a disulfide bridge between amino acid residues 713 and 894, wherein the numbering of the amino acid positions is according to the numbering of the amino acid positions in SEQ ID NO: 1.

In certain embodiments, the proteins, or fragments thereof do not comprise a furin cleavage site, i.e. the furin cleavage site has been deleted.

Thus, in certain embodiments, the present invention provides stabilized 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 at least one interprotomeric disulfide bridge selected from the group consisting of a disulfide bridge between amino acid residues 547 and 978, a disulfide bridge between amino acid residues 547 and 982, and a disulfide bridge between amino acid residues 713 and 894, 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, novel engineered (i.e. non-native) interprotomeric disulfide bridges (or disulfides) are described that stabilize both soluble and membranebound 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 novel interprotomeric disulfides linking the three monomers of the trimer, prevent S trimer dissociation and stabilize the S protein trimers without foldon. It has, in particular, been shown that the trimers in which the protomers are cross-linked by the novel disulfides are more stable upon heating to 65°C and do not fall apart in monomers, like the S variants without the disulfide. Furthermore, the SARS-CoV-2 S proteins according to the invention are more stable at 4°C and after slow freezing and thawing compared to S proteins without the disulfide.

According to the invention it is to be understood that “an interprotomeric disulfide bridge between residues 547 and 978” means that the amino acids at the positions 547 and 978 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.

The invention thus in particular relates to recombinant multimeric pre-fusion SARS- CoV-2 S proteins, or fragments thereof, comprising at least a first and a second S protein monomer, said monomers comprising an SI and an S2 domain and optionally comprising a deletion of the furin cleavage site; wherein the protein comprises at least one disulfide bridge selected from the group consisting of a disulfide bridge between the amino acid residue 547 of the first monomer and the amino acid residue 978 of the second monomer, a disulfide bridge between the amino acid residue 547 of the first monomer and the amino acid residue 982 of the second monomer, and a disulfide bridge between the amino acid residue 713 of the first monomer and the amino acid residue 894 of the second monomer, wherein the numbering of the amino acid positions is according to the numbering of the amino acid positions in SEQ ID NO: 1.

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 pre-fusion 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 mutations according to the invention can be introduced in any S wild-type S protein, including the S protein of the original Wuhan SARS-CoV-2 strain, 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 547” thus refers to the amino acid residue that is at position 547 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 SARS-CoV-2 S proteins are trimeric, i.e. comprise three monomers comprising identical amino acid sequences.

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, 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 (monomer) 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 (monomer) 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.

In certain embodiments, the amino acid residue at position 986 is not P.

In certain embodiments, the amino acid residue at position 986 and 987 is not P.

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 SARS-CoV-2 S protein, for example, the full-length sequence of a SARS-CoV-2 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 SARS-CoV-2 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 SARS-CoV-2 S protein. In certain embodiment, the fragment is the SARS-CoV2 ectodomain.

In certain embodiments, the fragment is the SARS-CoV-2 S2 domain. The present invention thus provides stable trimers of the S2 domain of SARS-CoV-2 (i.e. wherein the SI domain has been deleted) without the presence of a heterologous trimerization domain, such as a foldon. Since the S2 domain of the SARS-CoV-2 S protein is more conserved than the

51 domain, the S2 domain in pre-fusion conformation without heterologous trimerization domain, for example, is a suitable vaccine candidate to generate broadly neutralizing antibodies. In addition, the stabilized pre-fusion S2 domain could suitably be used as a tool to isolate broadly neutralizing antibodies.

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

52 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.

For the stabilization of soluble SARS-CoV-2 S proteins in the pre-fusion conformation, a heterologous trimerization domain, such as a fibritin - based trimerization domain, may be fused to the C-terminus of the Coronavirus S protein ectodomain. This fibritin domain or ‘Foldon’ is derived from T4 fibritin and was described earlier as an artificial natural trimerization domain (Letarov et al., 1993) Biochemistry Moscow 64: 817- 823; S-Guthe et al., (2004) J. Mol. Biol. 337: 905-915). Thus, in certain embodiments, the transmembrane region has been replaced by a heterologous trimerization domain. In certain embodiments, the heterologous trimerization domain is a foldon domain comprising the amino acid sequence of SEQ ID NO: 8. 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 SARS-CoV-2 S proteins do not comprise a heterologous trimerization domain.

The pre-fusion SARS-CoV-2 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.

In certain embodiments, the pre-fusion SARS-CoV-2 S proteins, or fragments thereof, have an increased thermal stability as compared to SARS-CoV-2 S proteins without the disulfide bridge of the invention, e.g. as indicated by an increased melting temperature (measured by e.g. differential scanning fluorimetry). In addition, or alternatively, the recombinant prefusion 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 disulfide bridge 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 disulfide bridge 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 disulfide bridge 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 disulfide bridge 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 (monomer) 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 protein (monomer) comprises one of more additional mutations selected from the group consisting of: a mutation of the amino acid at position 944 into P, a mutation of the amino acid at position 892 into P, a mutation of the amino acid 942 into P and a mutation of the amino acid at position 987 into P, a mutation of the amino acid at position 1072 into P, a mutation of the amino acid 1203 into K, and an intraprotomeric disulfide bridge between the amino acids at position 880 and 888.

In certain embodiments, the SARS-CoV-2 S proteins of the invention comprise an SI and an S2 domain, a deletion of the furin cleavage site; and at least one interprotomeric disulfide bridge selected from the group consisting of a disulfide bridge between amino acid residues 547 and 978, a disulfide bridge between amino acid residues 547 and 982, and a disulfide bridge between amino acid residues 713 and 894, 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 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 at least one interprotomeric disulfide bridge selected from the group consisting of a disulfide bridge between amino acid residues 547 and 978, a disulfide bridge between amino acid residues 547 and 982, and a disulfide bridge between amino acid residues 713 and 894, 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 1203 is K and the amino acid at position 1072 is P.

In certain embodiments, the SARS-CoV-2 S proteins of the invention comprise an SI and an S2 domain, a deletion of the furin cleavage site; and at least one interprotomeric disulfide bridge selected from the group consisting of a disulfide bridge between amino acid residues 547 and 978, a disulfide bridge between amino acid residues 547 and 982, and a disulfide bridge between amino acid residues 713 and 894, and wherein, the amino acid at position 572 is I, the amino acid at position 614 is G, the amino acid at position 892 is P and the amino acid at position 942 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 at least one interprotomeric disulfide bridge selected from the group consisting of a disulfide bridge between amino acid residues 547 and 978, a disulfide bridge between amino acid residues 547 and 982, and a disulfide bridge between amino acid residues 713 and 894, 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 and the amino acid at position 942 is P, and optionally 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 at least one interprotomeric disulfide bridge selected from the group consisting of a disulfide bridge between amino acid residues 547 and 978, a disulfide bridge between amino acid residues 547 and 982, and a disulfide bridge between amino acid residues 713 and 894, 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 880 is C, the amino acid at position 888 is C and the amino acid at position 944 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 at least one interprotomeric disulfide bridge selected from the group consisting of a disulfide bridge between amino acid residues 547 and 978, a disulfide bridge between amino acid residues 547 and 982, and a disulfide bridge between amino acid residues 713 and 894, 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 and the amino acid at position 944 is P.

In certain embodiments, the SARS-CoV-2 S protein of the invention comprises an SI and an S2 domain, optionally a deletion of the furin cleavage site; and at least one interprotomeric disulfide bridge selected from the group consisting of a disulfide bridge between amino acid residues 547 and 978, a disulfide bridge between amino acid residues 547 and 982, and a disulfide bridge between amino acid residues 713 and 894, 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 880 is C, the amino acid at position 888 is C and the amino acid at position 944 is P and the amino acid at position 987 is P.

In certain embodiments, the SARS-CoV-2 S protein comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 9-13 and 17 and 19-21, 23, 25-29 and 31- 33.

The present invention further provides nucleic acid molecules encoding 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. 1

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 SARS-CoV- 2 S proteins also form part of the invention. The pre-fusion 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, here 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 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 SARS-CoV-2 S protein. The invention further provides immunogenic compositions comprising a pre-fusion 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 pre-fusion SARS-CoV-2 S protein, a nucleic acid molecule, and/or a vector, according to the invention, for inducing an immune response against a SARS-CoV-2 S protein in a subject. Further provided are methods for inducing an immune response against SARS-CoV-2 S protein in a subject, comprising administering to the subject a pre-fusion SARS-CoV-2 S protein, and/or a nucleic acid molecule, and/or a vector according to the invention. Also provided are pre-fusion SARS-CoV-2 S proteins, nucleic acid molecules, and/or vectors, according to the invention for use in inducing an immune response against SARS-CoV-2 S protein in a subject. Further provided is the use of the pre-fusion 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 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 SARS-CoV-2 S proteins, nucleic acid molecules, or vectors of the invention may be used for prevention (prophylaxis, including post-exposure prophylaxis) of SARS-CoV-2 infections. In certain embodiments, the prevention may be targeted at patient groups that are susceptible for and/or at risk of 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 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 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 SARS-CoV-2 infection in a subject utilizing the pre-fusion 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 SARS-CoV-2 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 SARS-CoV-2. Prevention encompasses inhibiting or reducing the spread of SARS-CoV-2 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 SARS-CoV-2 infection.

For administering to subjects, such as humans, the invention may employ pharmaceutical compositions comprising a pre-fusion 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 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 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 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-Barnum, S. et al., Nature 592, 623-628, (2021)).

In certain embodiments, the invention provides methods for making a vaccine against 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 SARS-CoV-2 S protein and/or a nucleic acid molecule encoding a pre-fusion 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 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 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 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 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 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 SARS- CoV-2 protein as bait for isolating monoclonal antibodies.

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

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 several novel engineered interprotomeric disulfide bridges (or disulfides) are described that stabilize both soluble and membrane-bound S proteins, even without a heterologous trimerization domain (such as a foldon domain).

Materials and Methods

SDS-PAGE and Western Blotting

Cell lysates were harvested 48 hrs post transduction and, after heating for 10 min at 95°C, samples were loaded under non-reduced conditions on a precasted Bolt 4-12% Bis-Tris SDS-PAGE gel (Novex). Proteins were transferred to a PVDF membrane using an iBlot2 dry blotting system (Invitrogen), and membrane blocking was performed with Intercept Blocking Buffer (Li-COR) at RT for 1 hr. Following blocking, the membrane was incubated for 1 hr with 2 pg/ml 1 A9 in Intercept Blocking Buffer. 1 A9 is a mouse monoclonal antibody directed against SARS-CoV Spike and binds to the Spike S2 domain and also cross-reacts with SARS-CoV-2 Spike S2 (GenTex). After incubation, the membrane was washed three times with TBST for 5 min and subsequently incubated for 1 hr with 1 : 10,000 IRDye 800CW conjugated goat anti-mouse secondary antibody (Li-COR) in Intercept Blocking Buffer. Finally, the PVDF membrane was washed three times with TBST for 5 min and once with IxPBS (Gibco) for 15 min, and immediately thereafter developed using an ODYSSEY® CLx Infrared Imaging System (Li-COR). Cell-Based ELISA

HEK293 cells were seeded at 2 * 10⁵ cells/ml in appropriate medium in a flat- bottomed 96-well microtiter plate (Corning). The plate was incubated overnight at 37 °C in 10% CO2. After 24 hrs, transfection of the cells was performed with 300 ng DNA for each well and the plate was incubated for 48 hrs at 37 °C in 5% CO2. Two days post transfection, cells were washed with 100 pl/well of blocking buffer containing 1% (w/v) BSA (Sigma), 1 mM MgC12, 1.8 mM CaC12 and 5 mM Tris pH 8.0 in lx PBS (GIBCO). After washing, nonspecific binding was blocked, using 100 pl/well of blocking solution for 20 min at 4 °C. Subsequently, cells were incubated in 50 pl/well blocking buffer containing primary antibodies 2-51 (1 pg//ml), S309 (1 pg/ml), CR3022 (5 pg/ml) and CR3046 (5 pg/ml) for 1 hr at 4 °C. The plate was washed three times with 100 pl/well of the blocking buffer, three times with 100 pl/well of washing buffer containing 1 mM MgC12, 1.8 mM CaC12 in lx PBS and then incubated with 100 pl/well of the blocking buffer for 5 min at 4 °C. After blocking, the cells were incubated with 50 pl/well of secondary antibodies HRP conjugated mouse antihuman IgG (Jackson, 1 :2500) or HRP conjugated goat anti-mouse IgG (Jackson, 1 :2500) then incubated 40 min at 4 °C. The plate was washed 3 times with 100 pl/well of the blocking buffer, 3 times with 100 pl/well washing buffer. 30 pl/well of BM Chemiluminescence ELISA substrate (Roche, 1 :50) was added to the plate, and the luminosity was immediately measured using the Ensight Plate Reader.

Flow-cytometry (FACS)

HEK293 cells (0.4x 106 cells/well) were seeded in 6-well plates and after overnight growth transfected with 2 pg SARS-CoV-2 and 0.5 pg eGFP DNA construct according to manufacturer’s instructions (TransIT-LTl, MirusBio) and cultured for 48 hrs. Cells were detached with 5 mM EDTA, washed with PBS, and stained with LIVE/DEADTM Fixable Violet Dead Cell Stain Kit (Invitrogen). For SARS-CoV-2 surface staining, cells were washed twice with PBS and then incubated with mAbs S309, S2M11, CR3022 and CR3046 (1 pg/ml) for 30 min in FACS buffer (PBS with 1% FBS). Cells were washed twice with FACS buffer and stained with goat anti -human IgG Alexa Fluor 647 (2 pg/ml) (Invitrogen) secondary antibody for 30 min in FACS buffer. Cells were washed twice with FACS buffer and fixed with 2% formaldehyde (ThermoSci entific) for 15 min. Cells were washed once with FACS buffer and resuspended in FACS buffer before analysis on a FACS Canto instrument (BD Biosciences). Data were analyzed with FlowJoTM Software version 10.7.1 (Becton, Dickinson and Company) and plotted as median fluorescence intensity of the GFP positive, live, single cell population.

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. Differential scanning fluorometry (DSF)

A total of 5 pg of purified protein in 50 pl PBS pH 7.4 (Gibco) was mixed with 16.7 pl of 20 times diluted SYPRO orange fluorescent dye (5000*stock, Invitrogen S6651), 20pl of this mix (per replicate) was added to a 96-well optical qPCR plate. The measurement was performed in a qPCR instrument (Applied Biosystems ViiA 7) using a temperature ramp from 25 to 95°C with a rate of 0.015 °C per second. Data were collected continuously. The negative first derivative was plotted as a function of temperature. The melting temperature corresponds to the lowest point in the curve. BioLayer Interferometry (BLI)

A solution of 1 A9 at a concentration of 5 pg/ml, SAD-S35 at a concentration of 0.5 pg/ml and 2-51, S2M11, C144, 2-43, S309, ACE2-Fc, 0304 3H3, CR3022, CR3015 and CR3046 at a concentration of 10 pg/ml was used to immobilize the ligand on anti- hlgG (AHC) sensors (ForteBio, cat. #18-5060) in I xkinetics buffer (ForteBio, cat. # 18- 1105) in 384-well black tilted-bottom polypropylene microplates (ForteBio, cat. # 18-5080). The experiment was performed on an Octet HTX instrument (Pall-ForteBio) at 30°C with a shaking speed of 1000 rpm. Activation was 600 s, immobilization of antibodies 600 s, followed by washing for 300 s, and then binding the S proteins for 300 s and dissociation for 300 s. Data analysis was performed using the ForteBio Data Analysis 12.0 software (ForteBio).

Results:

HexaPro without foldon disintegrates into monomers over time

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 dissociates into monomers. After 8 weeks at 4°C most of the trimers have disintegrated into monomers (Fig. 2).

One of the novel engineered disulfides, i.e. T547C-N978C, has been described before in the semi-stabilized S-2P variant (i.e. an S protein containing a proline (P) at positions 986 and 987), but failed as it did not show any trimer formation (Hsieh et al (2020) Science 369(6510): 1501-1505 and Fig. 3 second panel).

None of the disulfides tested according to the invention in the S-2P variant resulted in trimer formation (Fig. 3 and Fig. 4). In addition, recently two different interprotomeric disulfides were described (xl (383C-985C) and x2 (413C-987C)) (Xiong et al (2020) Nature Structural & Molecular Biology volume 27, pages 934-941; Camell et al (2021) Journal of Virology Vol. 95, No. 15). However, the problem with these disulfides is that the xl disulfide is not completely formed in purified trimers and x2 disulfide reduces trimer yields approximately by a factor of 10 (Fig. 5).

According to the present invention, the disulfides were introduced in an S variant without foldon and containing several stabilizing substitutions (referred to as COR210284, or COR210284 backbone): i.e. the furin cleavage site knockout R682S, R685G and the stabilizing substitutions N532P, T572I, D614G, A892P, A944P and V987P. For 24 of the screened interprotomeric disulfides introduced in the COR210284 backbone, ten resulted in reasonable trimer yields (Figure 6). Most of the tested interprotomeric disulfides however reduced the trimer yield substantially.

Some of the trimers with disulfides (e.g. S987C-A570C, A570C-L966C, A701C- Q787C and P862C-A647C) had longer retention times in analytical SEC, implying that the proteins with these disulfides have a lower hydrodynamic radius compared with the COR2 10284 trimer (Figure 6). Cell culture supernatants were analyzed on Western blot using 1 A9 as detection antibody and revealed that the S protein trimers that had longer retention times contained covalently linked dimers besides trimers, explaining the lowered hydrodynamic radius (Figure 7).

Stress testing by heating 30 minutes at 65 °C

To test the impact of the interprotomeric disulfides on trimer stability, the cell culture supernatants were heated for 30 minutes at 65°C, at which temperature the backbone COR2 10284 forms mainly aggregates and monomers. All the tested interprotomeric disulfides prevented dissociation to monomers (Figure 8).

T547C-N978C stabilizes the S protein

The disulfide T547C-N978C is relatively close to the V987P substitution in the hinge loop in the three-dimensional structure of S. Therefore, the T547C-N978C disulfide was also evaluated in a variant with the original valine at position 987 (COR201291 (SEQ ID NO: 3)), to test the impact on trimer stability (Figure 9). The variant with the T547C-N978C disulfide (COR210118; SEQ ID NO: 9) and the backbone without the 547-978 disulfide (COR201291; SEQ ID NO: 3) also contained the G880C-F888C, showing that the presence of G880C- F888C together with T547C-N978C did not result in aberrant interprotomeric disulfides. Although the trimer yield was a bit reduced by the T547C-N978C substitution, according to analytical SEC, the monomer yield was reduced even more, resulting in a preferred higher trimer/monomer ratio for COR210118. The purified protein was much more stable at 4°C and 37°C than the COR201291 backbone. The melting temperature (Tm50) was increased by more than 20°C upon the introduction of the disulfide. Furthermore, the antigenicity of COR210118 was better than that of the backbone, as the binding potencies with the neutralizing MAbs 4A8 and S2M11 were improved. S982C-T547C and A713C-L894C stabilize S protein

The S variant that contains the S982C-T547C disulfide (COR210445, SEQ ID NO: 10) and the one with A713C-L894C disulfide (COR210439, SEQ ID NO: 11) described in Figure 6 were purified and characterized. Although, the COR210284 backbone (SEQ ID NO: 2) is much more stable at 4°C than the COR201291 backbone (SEQ ID NO: 3), the stabilizing effect of the two disulfides can still be observed after storage for 1 and 2 weeks at 4°C. Some monomer starts to appear for the COR210284 protein, whereas this is not the case for the constructs with the additional disulfides. This shows that the disulfides are preventing the spike protein from dissociation into monomers at 4°C (Figure 10B). Non-reducing SDS- PAGE shows that the protein containing T547C-N978C contained a low level of monomers, but the proteins with S982C-T547C and A713C-L894C showed very little monomers, indicating a well-defined monomorphic character (Figure 10 C). The melting temperatures of the variants with the 713-894 and the 982-547 disulfides were not increased and even showed a slight decrease (Figure 10D and E). The antigenicity of the protein with A713C-L894C was not very different from that of the backbone, but the binding of the protein with S982C- T547C to ACE2 was completely abrogated, indicating that the structure is more closed (Figure 10F and G). A more closed conformation without ACE2 binding properties could be a favorable aspect of a safe immunogen (Lei et al (2021) Circulation Research. 128: 1323— 1326).

T547C-N978C, S982C-T547C and A713C-L894C protect S proteins from dissociation into monomers after slow freezing

Cell culture supernatant were slowly frozen to -20°C and thawed again after which the supernatants were subjected to analytical SEC (Fig. 11). Whereas the S proteins without any interprotomeric disulfide dissociate partly into monomers, all the S protein trimers with any of the three disulfides T547C-N978C, S982C-T547C or A713C-L894C stay intact, even when no V987P substitution is present in the protein (Fig. 12). T547C-N978C, S982C-T547C or A713C-L894C also keep trimers intact in an S protein ectodomain version with only the furin cleavage site knock out mutations and the naturally occurring D614G (COR211185), whereas the protein without the disulfide fully dissociates into monomers after slow freezing to -20°C (Fig. 13).

T547C-N978C and T547C-S982C stabilize membrane bound S

The disulfide T547C-N978C was also tested with FACS in two full length membranebound S proteins, i.e. COR210567 (SEQ ID NO: 12) and COR210571 (SEQ ID NO: 13), which contain the T547C-N978C disulfide, introduced into the COR210485 (SEQ ID NO: 14) and COR210569 (SEQ ID NO: 15) backbones, respectively). In both cases the binding of non-neutralizing antibody CR3046 was reduced, whereas only in COR210571, the CR3022 binding was reduced significantly as measured in two independent FACS experiments, indicating that the T547C-N978C disulfide also stabilizes the membrane bound S in combination with other stabilizing substitutions (Fig. 14).

The same constructs and COR210562 (SEQ ID NO: 16) (without T547C-N978C) and COR210572 (SEQ ID NO: 17) (with T547C-N978C) were also measured in cell-based ELISA. In all cases, the engineered disulfide T547C-N978C reduced the binding of the nonneutralizing antibodies CR3046 and CR3022 and increased the ratio of neutralizing antibody binding (2-51 and S309) versus non-neutralizing antibody binding (CR3046 and CR3022) (Fig. 15).

The T547C-S982C disulfide was introduced into two different backbones

(COR210485 (SEQ ID NO: 14) and COR210719 (SEQ ID NO: 18)) and tested with Cellbased ELISA, showing that this disulfide also reduced non-neutralizing antibody binding upon introduction (Fig. 16). Conclusion

The interprotomeric disulfides of the invention stabilize the soluble S trimer by preventing the trimers to dissociate into monomers in supernatants. A713C-L894C, T547C- N978C and T547C-S982C were shown to decrease cold-denaturation of purified S protein. T547C-N978C and T547C-S982C were also shown to stabilize the membrane bound S.

EXAMPLE 2: A713C-L894C stabilizes S2 trimers

An S2 protein was made based on the stabilized COR211039 S protein, by using only the S2 part of the sequence after the furin cleavage site and by adding the tPA signal peptide at the N-terminus followed by two glutamic acids. When expressed, this S2 protein (COR220744) was demonstrated to form predominantly trimers in cell culture supernatant as determined with analytical SEC (Figure 17). By removing the A713C-L894C disulfide (COR220745) the S2 trimers completely fell apart into monomers, indicating that the disulfide is critical for maintaining trimers.

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)

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTK

RFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWM

ESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIG

INITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKG

IYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLND

LCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER

DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNF

NGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEV

PVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTM

SLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQ.DK

NTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKF

NGLTVLPPLLTDEM IAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAI

GKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVT

QQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHD

GKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHT

SPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMT SCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO: 2

COR210284 S protein FurinKO N532P T572I D614G A892P A944P V987P (signal peptide in bold)

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTpLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADiTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQgV NCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPsR AgSVASQSHAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLL QYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLF NKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGp ALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASpLGKLQDWNQNAQALN TLVKQLSSNFGAISSVLNDILSRLDKpEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVS NGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD

LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ SEQ ID NO: 3

COR201291 S protein - no foldon R682S R685G N532P T572I D614G G880C F888C A944P

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTpLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADiTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQgV NCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPsR AgSVASQSIlAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLL QYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLF NKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAcTITSGWTcGAGAA LQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASpLGKLQDVVNQNAQALNTL VKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSEC VLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSN GTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDL

GDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

SEQ ID NO: 4

CGR200017 sWuhan-S R682S, R685G, K986P, V987P, foldon, 25GS-tag

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQD VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPs RAgSVASQSHAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLL LQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLL FNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAG AALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQAL NTLVKQLSSNFGAISSVLNDILSRLDppEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMS ECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFV SNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDV DLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYVRKDGEWVLL STFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSGGGGSGGGGSHHHHH H SEQ ID NO: 5

CGR200151 sWuhan-S R682S, R685G, foldon, 25GS-tag

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQD VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPs RAgSVASQSHAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLL LQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLL FNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAG AALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQAL NTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM SECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVF VSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPD VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYVRKDGEWV LLSTFLGRSLEVLFQGPGSLPETGGGSDYKDDDDKGGGGSGGGGSGGGGSGGGGSGGGGSHHH HHH

SEQ ID NO: 6

CGR200619 no_foldon_no_tags_R682S_R685G_A892P_A942P_D614N_V987P

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQN VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS RAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQA LNTLVKQLSSNFGAISSVLNDILSRLDKPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATK MSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGV FVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSP

DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

SEQ ID NO: 7

COR201225 S protein - no foldon R682S R685G N532P T572I D614G G880C F888C A944P V987P

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI

HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCN

DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY

SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR

TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTpLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADiTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQgV NCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPsR AgSVASQSHAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLL QYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLF NKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAcTITSGWTcGAGAA LQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASpLGKLQDVVNQNAQALNTL VKQLSSNFGAISSVLNDILSRLDKpEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSEC VLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSN GTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDL

GDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

SEQ ID NO: 8 foldon

GYIPEAPRDGQAYVRKDGEWVLLSTFL

SEQ ID NO: 9

COR210118 S protein - no foldon R682S R685G N532P T572I D614G G880C F888C A944P T547C- N978C

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTpLVKNKCVNFNFNG LcGTGVLTESNKKFLPFQQFGRDIADiTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQgV NCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPsR AgSVASQSHAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLL QYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLF NKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAcTITSGWTcGAGAA LQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASpLGKLQDVVNQNAQALNTL VKQLSSNFGAISSVLcDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSEC VLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSN GTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDL

GDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

SEQ ID NO: 10

COR210445 S protein - no foldon R682S R685G N532P T572I D614G A944P S982C T547C

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNG LCGTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG

VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS RAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL

LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA

GPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASPLGKLQDVVNQNAQA

LNTLVKQLSSNFGAISSVLNDILCRLDKPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATK

MSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGV

FVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSP

DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

SEQ ID NO: 11

COR210439 S protein - no foldon R682S R685G N532P T572I D614G A944P A713C L894C

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS RAGSVASQSIIAYTMSLGAENSVAYSNNSICIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GPACQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASPLGKLQDVVNQNAQA LNTLVKQLSSNFGAISSVLNDILSRLDKPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATK MSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGV FVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSP

DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

SEQ ID NO: 12

COR210567 A892P+D614G+R682S+R683G+R685G+A942P+N532P+T572I+T547C-N978O

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNG LCGTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS GAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQA

LNTLVKQLSSNFGAISSVLCDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATK MSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGV FVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSP DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIM LCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO: 13

COR210571 A892P+D614G+R682S+R683G+R685G+A942P+N532P+T572I+T547C-N978O+V987P

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNG LCGTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS GAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQA LNTLVKQLSSNFGAISSVLCDILSRLDKPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATK MSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGV FVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSP DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIM

LCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO: 14

COR210485 A892P+D614G+R682S+R683G+R685G+A942P+N532P+T572I

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS GAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQA LNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATK MSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGV FVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSP DVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIM

LCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO: 15

COR210569 A892P+D614G+R682S+R683G+R685G+A942P+N532P+T572I+V987P MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS GAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQA LNTLVKQLSSNFGAISSVLNDILSRLDKPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATK MSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGV FVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSP DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIM

LCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO: 16

COR210562: A892P+D614G+R682S+R683G+R685G+A942P+T572I

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS GAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDWNQNAQA LNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATK MSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGV FVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSP DVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIM

LCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO: 17

COR210572 A892P+D614G+R682S+R683G+R685G+A942P+T572I+T547C-N978O

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG LCGTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS GAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDWNQNAQA LNTLVKQLSSNFGAISSVLCDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATK MSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGV FVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSP DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIM LCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO: 18

COR210719 A892P+D614G+R682S+R683G+R685G+A942P+N532P+T572I+D1118H+V987P

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN

ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS

GAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDWNQNAQA LNTLVKQLSSNFGAISSVLNDILSRLDKpEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM

SECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVF VSNGTHWFVTQRNFYEPQIITThNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPD VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIML CCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO: 19:

COR211039

Stabilized_soluble_B.1.617.2_vl_R682S_R683G_R685G_N532P_T572l_D614G_A944P_V987P_A892P_L1203K_ E1072P A713C L894C

MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTK RFDNPVLPFNDGVYFASiEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWME SGVYSSANNCTFEYVSQPFLM DLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINI TRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLC

FTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDI STEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFN GLTGTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPV AlHADQLTPTWRVYSTGSNVFQTRAGCUGAEHVNNSYECDIPIGAGICASYQTQTNSPSgAGSVASQSHAYTMSL GAENSVAYSNNSICIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNT

QEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNG LTVLPPLLTDEM IAQYTSALLAGTITSGWTFGAGPACQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGK IQDSLSSTASPLGKLQNVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKPEAEVQIDRLITGRLQSLQTYVTQQ LIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQPKNFTTAPAICHDGKA HFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDV DLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEKGKYEQ

SEQ ID NO: 20

COR210713 A892P+D614G+R682S+R683G+R685G+A942P+N532P+T572I+T547C-S982O

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNG LcGTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS GAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDWNQNAQA LNTLVKQLSSNFGAISSVLNDILcRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM SECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVF VSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPD VDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIML CCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO: 21

COR210718 A892P+D614G+R682S+R683G+R685G+A942P+N532P+T572I+T547C-

S982C+D1118H+V987P

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNG LcGTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS GAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQA LNTLVKQLSSNFGAISSVLNDILcRLDKpEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM SECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVF VSNGTHWFVTQRNFYEPQIITThNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPD

VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIML CCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO: 22

COR211032

Stabilized_soluble_B.1.617.2_R682S_R683G_R685G_N532P_T572l_D614G_A944P_V987P_A892P

_L1203K_E1072P

MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASiEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSK HTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTF LLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNAT RFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPG QTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLT GTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVN CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPSgA GSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQ YGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFN KVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGPA LQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASPLGKLQNWNQNAQALNT LVKQLSSNFGAISSVLNDILSRLDKPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQPKNFTTAPAICHDGKAHFPREGVFVS NGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD

LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEKGKYEQ SEQ ID NO: 23

COR211041

Stabilized_soluble_B.1.617.2_R682S_R683G_R685G_N532P_T572l_D614G_A944P_A892P_T547C

_N978C_L1203K_E1072P_V987P

MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASiEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSK HTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTF LLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNAT RFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPG QTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLC GTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVN CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPSgA GSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQ YGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFN KVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGPA LQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASPLGKLQNVVNQNAQALNT LVKQLSSNFGAISSVLCDILSRLDKPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQPKNFTTAPAICHDGKAHFPREGVFVS NGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD

LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEKGKYEQ

SEQ ID NO: 24

COR210733

Stabilized_soluble_B.1.617.2_R682S_R683G_R685G_N532P_T572l_D614G_G880C_F888C_A944

P_V987P_A892P_D1118H

MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSK HTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTF LLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNAT RFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPG QTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLT GTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVN CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPSRA GSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQ YGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFN KVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLACTITSGWTCGAGPA LQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASPLGKLQNVVNQNAQALNT

LVKQLSSNFGAISSVLNDILSRLDKPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVS NGTHWFVTQRNFYEPQIITTHNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

SEQ ID NO: 25

COR211028

P_A892P_D1118H_T547C_S982C_V987P

MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI

HVSGTNGTKRFDNPVLPFNDGVYFASiEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN

DPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSK

HTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTF

LLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNAT

RFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPG

QTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCN

GVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLC

GTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVN

CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPSgA

GSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQ

YGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFN

KVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLACTITSGWTCGAGPA

LQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASPLGKLQNWNQNAQALNT

LVKQLSSNFGAISSVLNDILCRLDKPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE

CVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVS

NGTHWFVTQRNFYEPQIITTHNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD

LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

SEQ ID NO: 26

COR211183

Stabilized_soluble_B.1.617.2_R682S_R683G_R685G_N532P_T572l_D614G_A944P_A892P_L1203

K_E1072P_A713C_L894C

MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI

HVSGTNGTKRFDNPVLPFNDGVYFASiEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN

DPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSK

HTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTF

LLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNAT

RFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPG

QTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCN

GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLT

GTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVN

CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPSgA

GSVASQSIIAYTMSLGAENSVAYSNNSICIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLL

QYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLF

NKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGP

ACQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASPLGKLQNVVNQNAQALN

TLVKQLSSNFGAISSVLNDILSRLDKvEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE

CVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQPKNFTTAPAICHDGKAHFPREGVFVS

NGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD

LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEKGKYEQ

SEQ ID NO: 27

COR211036

P_A892P_T547C_N978C_V987P_L1203K_E1072P

MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI

HVSGTNGTKRFDNPVLPFNDGVYFASiEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN

DPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSK

HTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTF

LLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNAT

RFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPG

QTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCN

GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLC

GTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVN CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPSgA GSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQ YGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFN KVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLACTITSGWTCGAGPA LQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASPLGKLQNVVNQNAQALNT LVKQLSSNFGAISSVLCDILSRLDKPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQPKNFTTAPAICHDGKAHFPREGVFVS NGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEKGKYEQ

SEQ ID NO: 28

COR211037

P_A892P_T547C_N978C_L1203K_E1072P

MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASiEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSK HTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTF LLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNAT RFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPG QTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLC GTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVN

CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPSgA GSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQ YGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFN KVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLACTITSGWTCGAGPA LQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASPLGKLQNVVNQNAQALNT LVKQLSSNFGAISSVLCDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQPKNFTTAPAICHDGKAHFPREGVFVS NGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEKGKYEQ

SEQ ID NO: 29

COR210734

P_A892P_D1118H_T547C_S982C

MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSK HTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTF LLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNAT RFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPG QTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRWVLSFELLHAPATVCGPKKSTPLVKNKCVNFNFNGLC GTGVLTESNKKFLPFQQFGRDIADITDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVN

CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPSRA GSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQ YGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFN KVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLACTITSGWTCGAGPA LQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASPLGKLQNWNQNAQALNT LVKQLSSNFGAISSVLNDILCRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE CVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVS NGTHWFVTQRNFYEPQIITTHNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

SEQ ID NO: 30

COR211185 S ectodomain R682S, R685G, D614G

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS RAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQA LNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATK MSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGV FVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSP DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

SEQ ID NO: 31

COR211189 S ectodomain R682S, R685G, D614G + A713C-L894C

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS RAGSVASQSIlAYTMSLGAENSVAYSNNSIcIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLL LQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLL FNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAG AAcQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQAL NTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM SECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVF VSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPD

VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

SEQ ID NO: 32

COR211190 S ectodomain R682S, R685G, D614G + T547C-N978C

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG LcGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS RAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDWNQNAQA LNTLVKQLSSNFGAISSVLcDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM SECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVF VSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPD

VDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ SEQ ID NO: 33

COR211191 S ectodomain R682S, R685G, D614G + T547C-S982C

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCN DPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG LcGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQG VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPS RAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNL LLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDL LFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGA GAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDWNQNAQA LNTLVKQLSSNFGAISSVLNDILcRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM SECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVF

VSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPD VDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

SEQ ID NO: 34

COR220744 S2 of B.1 .617.2 with the following stabilizing substitutions

R682S_R683G_R685G_N532P_T572l_D614G_A944P_V987P_A892P_L1203K_E1072P_A713C_L8 94C

MDAMKRGLCCVLLLCGAVFVSAQQMSLGAENSVAYSNNSICIPTNFTISVTTEILPVSMTKTSVDCTM YICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDP SKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLA GTITSGWTFGAGPACQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASPLGK LQNVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKPEAEVQIDRLITGRLQSLQTYVTQQLIRAA EIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQPKNFTTAPAICH DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEE LDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEKGKYEQ tPA signal sequence underlined

SEQ ID NO: 35

COR220745 S2 of B.1 .617.2 with the following stabilizing substitutions

R682S_R683G_R685G_N532P_T572l_D614G_A944P_V987P_A892P_L1203K_E1072P

MDAMKRGLCCVLLLCGAVFVSAQQMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTM YICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDP SKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLA GTITSGWTFGAGPALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASPLGKL QNVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEI RASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQPKNFTTAPAICHD GKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEEL DKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEKGKYEQ tPA signal sequence underlined

Claims

1. Recombinant pre-fusion SARS-CoV-2 S protein, comprising an SI and an S2 domain, or a fragment thereof, and comprising at least one interprotomeric disulfide bridge selected from the group consisting of a disulfide bridge between amino acid residues 547 and 978, a disulfide bridge between amino acid residues 547 and 982, and a disulfide bridge between amino acid residues 713 and 894, wherein the numbering of the amino acid positions is according to the numbering of the amino acid positions in SEQ ID NO: 1.

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

3. The protein, or fragment thereof, according to claim 1 or 2, comprising an amino acid sequence wherein the amino acid residue at position 614 is not aspartic acid (D).

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

5. The protein, or fragment thereof, according to any one of the claims 1-4, wherein the amino acid residue at position 572 is not threonine (T).

6. The protein, or fragment thereof, according to any one of the claims 1-5, wherein the amino acid residue at position 572 is isoleucine (I).

7. The protein, or fragment thereof, according to any one of the preceding claims 2-6, 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.

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

9. The protein, or fragment thereof, according to any one of the preceding claims, comprising one of more mutations selected from the group consisting of: a mutation of the amino acid at position 944 into P, a mutation of the amino acid at position 892 into P, a mutation of the amino acid 942 into P and a mutation of the amino acid at position 987 into P, a mutation of the amino acid at position 1072 into P, a mutation of the amino acid 1203 into K, and a intraprotomeric disulfide bridge between the amino acid at position 880 and 888.

10. The protein, or fragment thereof, according to any one of the preceding claims, wherein the amino acid residue at position 986 is not P.

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

12. The protein, or fragment thereof, according to claim 11, wherein the transmembrane and cytoplasmic domain have been removed.

13. The protein, or fragment thereof, according to claim 11 or 12, wherein a heterologous trimerization domain has been linked to the truncated S2 domain.

14. The protein, or fragment thereof, according to claim 13, wherein the heterologous trimerization domain is a foldon domain comprising the amino acid sequence of SEQ ID NO:8.

15. The protein, or fragment thereof, according to any one of the claims 1-10, wherein the fragment is the S2 domain.

16. Nucleic acid molecule encoding a protein, or fragment thereof, according to any one of the preceding claims 1-15.

17. Nucleic acid according to claim 16, wherein the nucleic acid molecule is DNA or RNA.

18. Vector comprising a nucleic acid according to claim 16 or 17.

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

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

21. 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 19.

22. An isolated host cell comprising a nucleic acid according to claim 15 or 17.

23. An isolated host cell comprising a recombinant human adenovirus of serotype 26 comprising a nucleic acid according to claim 16 or 17.