CN116745408A - Stabilized coronavirus spike protein fusion proteins - Google Patents

Abstract

The present invention provides stable recombinant pre-fusion SARS CoV-2S protein, nucleic acid molecule encoding the SARS-CoV-2S protein and the use of these proteins and nucleic acid molecules.

Description

Stabilized coronavirus spike protein fusion proteins

The present invention relates to the field of medicine. The invention relates in particular to stable recombinant pre-fusion coronavirus spike (S) proteins, in particular SARS-CoV-2S proteins, to nucleic acid molecules encoding said SARS-CoV-2S proteins, and to the use of these proteins and nucleic acid molecules, for example in vaccines.

Background

Coronaviruses (covs) are viruses that cause mild respiratory infections and atypical pneumonia in humans. CoV is a large family of enveloped single stranded sense RNA viruses belonging to the order of the mantle viruses (Nidovirales) that can infect a wide range of mammalian species including cat, dog, cow, bat and human, as well as avian species. Coronaviruses have large trimeric spike glycoproteins (S) that mediate binding to host cell receptors and fusion of the viral and host cell membranes. The coronaviridae family includes genus a coronavirus (alphacoronovir), genus b coronavirus (betacoronovir), genus c coronavirus (Gammacoronavirus), and genus t coronavirus (deltacoronovir). These viruses cause a range of diseases including intestinal and respiratory diseases. The host range is largely determined by the viral spike protein (S protein), which mediates viral entry into the host cell. Coronaviruses that can infect humans are found in both the genus a and the genus b. Coronaviruses of the genus B coronavirus known to cause human respiratory disease include SARS-CoV, MERS-CoV, HCoV-OC43 and HCoV-HKU1, and SARS-CoV-2 currently being transmitted.

SARS-CoV-2 is a coronavirus that has emerged from animal hosts into humans in 2019 and is rapidly spread worldwide. SARS-CoV-2 is a genus of coronaviruses such as MERS-CoV and SARS-CoV, all of which are derived from bats. The name of the disease caused by this virus is 2019 coronavirus disease, abbreviated as covd-19. In the diagnosed cases of COVID-19, the symptoms of COVID-19 varied from mild to severe illness and death.

It is well known that viruses are constantly changing by mutation, and new viral variants are expected to occur over time. Sometimes new variants appear and disappear. While at other times new variants persist after appearance. During this global pandemic, a number of viral variants have been identified worldwide that cause covd-19. Scientists constantly monitor changes in viruses, including changes in spike proteins on the surface of the virus. CDC in cooperation with the SARS-CoV-2 trans-sector group (SIG) established 3 classifications of SARS-CoV-2 variants under monitoring: variants of interest (VOIs), alarming Variants (VOCs), and variants that cause serious consequences (VOHC). Several VOCs are currently identified, including:

b.1.1.7: this variant was originally detected in the uk.

B.1.351: this variant was originally detected in south africa at 12 months 2020.

P.1: this variant was originally found for passengers from brazil who received detection during routine screening at japan airport at the beginning of 1 month.

B.1.427 and b.1.429: these two variants were first found in california at month 2 of 2021 and classified as VOCs at month 3 of 2021.

B.1.526, b.1.526.1, b.1.525, b.1.617, b.1.617.1, b.1.617.2, b.1.617.3, and p.2 variants propagated in the united states are classified as variants of interest.

The b.1.1.7, b.1.351, p.1, b.1.427 and b.1.429 variants propagated in the united states were classified as alarming variants.

In the case of SARS-CoV-2, the S protein is the major surface protein. The S protein forms homotrimers and consists of an N-terminal S1 subunit and a C-terminal S2 subunit, responsible for receptor binding and membrane fusion, respectively. Recent electron cryo-microscopic (cryo-EM) reconstruction of CoV trimer S structures from the genera coronavirus a, coronavirus b and coronavirus d reveals that the S1 subunit comprises two distinct domains: an N-terminal domain (S1 NTD) and a receptor binding domain (S1 RBD). SARS-CoV-2 binds to human angiotensin converting enzyme 2 (ACE 2) using its S1 RBD (Hoffmann et al (2020); wrapp et al (2020)).

Coronaviridae S proteins are classified as class I fusion proteins and are responsible for fusion. The S protein fuses the viral membrane and the host cell membrane by irreversible protein refolding from an unstable pre-fusion conformation to a stable post-fusion conformation. Like many other class I fusion proteins, coronaviridae S proteins require receptor binding and cleavage to induce the conformational changes required for fusion and entry (Belouzard et al (2009); follis et al (2006); bosch et al (2008); madu et al (2009); walls et al (2016)). Initiation of SARS-CoV2 involves cleavage of the S protein by furin at the furin cleavage site at the boundary between the S1 and S2 subunits (S1/S2), and cleavage of the S protein by TMPRSS2 at a conserved site upstream of the fusion peptide (S2') (Bestle et al (2020); hoffmann et al (2020)).

To refold from the pre-fusion conformation to the post-fusion conformation, there are two regions that need refolding, designated refolding region 1 (RR 1) and refolding region 2 (RR 2) (fig. 1). For all class I fusion proteins, RR1 includes Fusion Peptide (FP) and heptad repeat 1 (HR 1). After cleavage and receptor binding, stretching of all three original helices, loops and chains in the trimer translates into a long, continuous trimeric helical coiled-coil. The FP located at the N-terminal segment of RR1 can then extend away from the viral membrane and insert into the proximal membrane of the target cell. Next, refolding region 2 (RR 2), which is located at the C-terminal end of RR1 and closer to transmembrane region (TM) and includes heptad repeat 2 (HR 2), is repositioned to the other side of the fusion protein, and the HR1 coiled-coil trimer is combined with the HR2 domain to form a six-helix bundle (6 HB).

When viral fusion proteins such as SARS-CoV-2S protein are used as vaccine components, the fusion promoting function of the protein is not important. Indeed, only the mimicking of the virus by the vaccine component is important for the induction of reactive antibodies that bind to the virus. Thus, to develop a robust and effective vaccine component, it is desirable to maintain the metastable fusion protein in its pre-fusion conformation. Stable fusion proteins in the pre-fusion conformation, such as SARS-CoV-2S protein, are believed to induce an effective immune response.

In recent years, there have been many attempts to stabilize various class I fusion proteins including coronavirus S proteins. One approach that has proved particularly successful is to stabilize the so-called hinge loop at the RR1 terminus before the base helix (WO 2017/037196, krarop et al (2015); rutten et al (2020), hastin et al (2017)). The method has also proven successful for coronavirus S proteins, as shown for SARS-CoV, MERS-CoV and SARS-CoV2 (Pallesen et al (2016); wrapp et al (2020)). Although the proline mutation in the hinge loops does increase the expression of the coronavirus S protein, instability of the S protein may still exist. Thus, further stabilization is needed for improved vaccine designs or S proteins (which may be used, for example, as tools, e.g., as baits for monoclonal antibody isolation).

Since the first observation of the novel SARS-CoV-2 virus in humans in the 2019 year, the number of infection with COVID-19 was over 1.54 hundred million, resulting in over 300 thousands of deaths, especially because of the lack of effective treatment of SARS-CoV-2 and, more generally, coronaviruses. Furthermore, there is currently no vaccine available for the prevention of coronavirus-induced disease (covd-19), which results in a large number of medical needs being unmet. Since emerging infectious diseases such as covd-19 pose a major threat to public health and economic systems, there is a great need for new components that can be used, for example, in vaccines to prevent coronavirus-induced respiratory diseases.

Disclosure of Invention

The present invention provides recombinant SARS-CoV-2S proteins having increased trimer production and/or increased (thermal) stability as compared to the SARS-CoV-2S proteins previously described.

The invention also provides stable recombinant pre-fusion SARS-CoV-2S protein (i.e., pre-fusion conformationally stable SARS-CoV-2S protein), and fragments thereof.

In certain embodiments, the pre-fusion SARS-CoV-2S protein is a soluble protein, preferably a trimeric soluble protein.

The resulting stabilized pre-fusion SARS-CoV-2S protein trimers can be used for immunization (vaccination) purposes, e.g., to increase the chance of inducing broadly neutralizing antibodies and reducing the induction of non-neutralizing and weakly neutralizing antibodies after administration of recombinant stabilized SARS-CoV-2S protein trimers or nucleic acids encoding these stabilized SARS-CoV-2S protein trimers.

The invention also provides nucleic acid molecules encoding the pre-fusion SARS-CoV-2S protein and fragments thereof, as well as vectors, e.g., adenoviral vectors, comprising such nucleic acid molecules.

The invention further provides methods of stabilizing SARS-CoV 2S protein in a pre-fusion conformation, and pre-fusion SARS-CoV 2S proteins obtainable by said methods.

The invention also provides compositions, preferably immunogenic compositions, comprising SARS-CoV-2S protein or fragment thereof, a nucleic acid molecule and/or a vector as described herein.

The invention also provides compositions for inducing an immune response against SARS-CoV-2S protein, and in particular the use of these compositions as vaccines against SARS-CoV-2 related 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 a subject an effective amount of a pre-fusion SARS-CoV-2S protein or fragment thereof described herein, a nucleic acid molecule encoding said SARS-CoV-2S protein, and/or a vector comprising said nucleic acid molecule. Preferably, the immune response induced is characterized by induction of neutralizing antibodies against SARS-CoV-2 virus and/or protective immunity against SARS-CoV-2 virus.

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

The invention also relates to the use of the SARS-CoV-2S protein or fragment thereof described herein for isolating a monoclonal antibody directed against the SARS-CoV-2S protein from an infected human.

Also provided is the use of the pre-fusion SARS-CoV-2S protein of the invention in a method of screening for a candidate SARS-CoV-2 antiviral agent (including but not limited to antibodies to SARS-CoV-2).

In another general aspect, the present invention relates to isolated nucleic acid molecules encoding the recombinant SARS-CoV-2S proteins of the invention and vectors comprising the isolated nucleic acid molecules operably linked to a promoter. In one embodiment, the vector is a viral vector. In another embodiment, the vector is an expression vector. In a preferred embodiment, the viral vector is an adenovirus vector.

Another general aspect relates to a host cell comprising an isolated nucleic acid molecule or vector encoding a recombinant SARS-CoV-2S protein of the invention. Such host cells may be used for recombinant protein production, recombinant protein expression or viral particle production.

Another general aspect relates to methods of producing recombinant SARS-CoV-2S protein, comprising growing a host cell comprising an isolated nucleic acid molecule or vector encoding a recombinant SARS-CoV-2S protein of the invention under conditions suitable for production of the recombinant SARS-CoV-2S protein.

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. 1:schematic representation of conserved elements of fusion domains of SARS-CoV-2S protein. The head domain contains an N-terminal (NTD) domain, a Receptor Binding Domain (RBD), and domains SD1 and SD2. The fusion domain contains a Fusion Peptide (FP), refolding region 1 (RR 1), refolding region 2 (RR 2), transmembrane region (TM) and cytoplasmic tail. The cleavage sites between S1 and S2' cleavage sites are indicated by arrows.

Fig. 2:analytical SEC samples of semi-stable SARS-CoV-2S trimeric protein after freeze-thaw cycling. S-trimer protein (A) according to SEQ ID NO:3 and the same protein (B) in which the tag was replaced with a C tag after flash-freezing and thawing 1 time (dark solid line) and 5 times (light solid line) in liquid nitrogen, compared to the unfrozen S-protein (dotted line). The peak at 5 minutes corresponds to S trimer.

Fig. 3:the following comparison of ACE2-Fc binding measurements in the AlphaLISA assay: percentage of S trimer expression of the S protein with the indicated mutation versus control, unstabilized, uncleaved SARS-CoV-2S (with furin site mutation) (SEQ ID NO: 2). The recombinant S protein tested contained a single amino acid substitution, which was introduced into the backbone of the extracellular domain of the labile uncleaved SARS-CoV-2S (SEQ ID NO: 2) (furin KO, left panel) and into the backbone of the semi-stable uncleaved SARS-CoV-2S (SEQ ID NO: 3) with a biproline mutation at positions 986 and 987 (furin KO+PP, right panel), as shown. The crude cell culture supernatant was analyzed.

FIG. 4: analytical SEC profiles of semi-stable uncleaved SARS-CoV-2S (SEQ ID NO: 3) (a-C) and of the unstable uncleaved SARS-CoV-2S protein (SEQ ID NO: 2) (D-F) (dashed line) with two proline stability mutations (+pp) in the hinge loops, compared to variants with the indicated point mutations a892P (A, D), a942P (B, E), D614N (C, F, shown in black), D614M (shown in dark grey) and D614L (shown in light grey, solid line). The crude cell culture supernatant was analyzed. The peak at 5 minutes corresponds to S trimer. G) SEC-MALS of purified stable S protein with A942P mutation (SEQ ID NO: 5). The SEC signal is shown in gray bold lines and corresponds to the left axis. The black thin lines show the molar mass trace (right y-axis). The dn/dc value used was 0.185.

Fig. 5:the following comparison of ACE2-Fc binding measurements in the AlphaLISA assay: percentage of S trimer expression of the S protein with the indicated mutation versus control, unstabilized, uncleaved SARS-CoV-2S (with furin site mutation) (SEQ ID NO: 2). The recombinant S protein tested contained a single amino acid substitution or disulfide bridge, which was introduced into the backbone of the extracellular domain of the labile uncleaved SARS-CoV 2S (SEQ ID NO: 2) (furin KO, left panel) and into the backbone of the semi-stable uncleaved SARS-CoV 2S (SEQ ID NO: 3) with biproline at positions 986 and 987 (furin KO+PP, right panel), as shown. The crude cell culture supernatant was analyzed.

FIG. 6: analytical SEC plots of semi-stable uncleaved SARS-CoV2S+PP (SEQ ID NO: 3) (A-D) and unstable uncleaved SARS-CoV 2S protein (SEQ ID NO: 2) (E-H) (dotted line) compared to variants with indicated point mutations or disulfide bridges (solid line). The crude cell culture supernatant was analyzed. The peak at 5 minutes corresponds to S trimer.

Fig. 7:the following comparison of ACE2-Fc binding measurements in the AlphaLISA assay: percentage of S trimer expression of the S protein with the indicated mutation versus control, unstabilized, uncleaved SARS-CoV-2S (with furin site mutation) (SEQ ID NO: 2). The recombinant S proteins tested contained single amino acid substitutions as shown, which Is introduced into the backbone of the extracellular domain of the labile uncleaved SARS-CoV 2S (SEQ ID NO: 2) (A). Analytical SEC profile of unstabilized uncleaved SARS-CoV 2S (SEQ ID NO:2 (dashed line)) compared to variants with indicated point mutations (solid line). The crude cell culture supernatant was analyzed. The peak at 5 minutes corresponds to S trimer. The crude cell culture supernatant was analyzed.

Fig. 8:temperature stability of purified S trimer as measured by DSC. Two melting events are represented by Tm1 and Tm 2. Corresponding to SEQ ID NO. 2 (comprising furin KO), there is one stable proline mutation in the hinge loop (furin KO K986P or furin KO V987P), and an uncleaved SARS2-S variant (A) with two proline mutations in the hinge loop (SEQ ID NO. 3, S-2P). An uncleaved variant (B) having the indicated mutation in S1 and an uncleaved variant (C) having the indicated mutation in S2.

FIG. 9: cell-cell fusion assay. Full-length wild-type SARS-CoV-2 spike protein and variants thereof (shown within the frame of the image), human ACE2, human TMPRSS2 and GFP are co-expressed in HEK293 cells. Redistribution of GFP signal was used for visual compound cell formation. Furin KO, PP mutations and two cystines completely abrogate fusions, while all single point mutants still allow fusion to occur. This means that the single point mutant still allows the S protein to sample all possible conformational intermediates between pre-and post-fusion states.

FIG. 10: analytical SEC profile (A) of uncleaved SARS-CoV 2S (SEQ ID NO:2 (dashed line)) compared to variants with indicated point mutations (solid line). The recombinant S proteins tested contained single amino acid substitutions or multiple mutations as shown. The peak at 5 minutes corresponds to S trimer. The following comparisons, measured by AlphaLISA assay: amount of ACE2-Fc binding of S protein with indicated mutation to control unstable uncleaved SARS-CoV-2S (with furin site mutation) (SEQ ID NO: 2) (B). The crude cell culture supernatant was analyzed. Analytical SEC comparing the semi-stable S trimer (K986P+V987P, SEQ ID NO: 3) with the four stabilities indicated by the presence or absence of the (DF) foldonMutant variant (C). Temperature stability of purified, uncleaved S trimer with the indicated stability mutation as measured by DSC. Variants without a folded daughter trimerization domain are denoted as Fd (D) free.

FIG. 11: the freeze-thaw stability of the purified uncleaved S trimer with the indicated stability mutations as measured by analytical SEC. Chromatograms of unfrozen, 1 Xfrozen and 3 Xfrozen are shown. The right panel is the S trimer without the fold trimerization site (delta fold).

FIG. 12: binding of SAD-S35, ACE2-Fc and CR3022 to 986p+987p variants, new single point mutations and disulfide bridges, and variants with combinations of mutations measured using biofilm interferometry showed an initial slope (a-C) at the beginning of binding. After 2 weeks of storage at 4 degrees, S309, ACE2 and CR3022 bind in equilibrium with the semi-stable variant (furin KO+PP, SEQ ID NO: 3) and the stable variant with 4 stability mutations without a folin (D).

FIG. 13: western blot stained with 1A9 (GeneTex) antibody against S2, which detects SARS-CoV-2 spike protein, and anti-beta actin (AC-15) primary antibody (Ai Bokang, abcam, ab 6276). Since all four full length spikes 660, 662, 007 and 664 knocked out the furin cleavage site, the complete S protein was detected. Cell culture supernatants, which may contain exosomes produced after the membranes of the Expi293F cells are saturated with incorporated spikes, are loaded. Molecular weight markers are loaded into lane 1. Lanes indicated with pcDNA show the supernatant of cells transfected with empty pcDNA vector.

Fig. 14:luminescence intensity measured with cell-based ELISA (CBE). Luminescence was calculated as the average of two equal parts.

FIG. 15: binding of a set of antibodies (on the X-axis) to the three proteins used to immunize the mice was measured using biofilm interferometry, showing an initial slope V0 at the onset of binding.

Fig. 16:A. SARS-CoV-2L-0008 (lineage b.1) isolate neutralizing antibody titers were measured by wild-type VNA (wtVNA) on day 27. Sample due to wtVNAThe number is limited so not all samples of the 0.5 μg COR201225 (n=5), COR200619 (n=5) and COR200627 (n=6) groups are measured. The SARS-CoV-2b.1 spike protein neutralizing antibody titers induced by the present invention, as measured by lentiviral pseudoparticle neutralization assay (psVNA) on days b.27 and c.41. 2 mice were excluded because there was insufficient serum to perform psVNA on these samples on day 27. The value on each x-axis is the group geometric mean titer. The horizontal bars of each group represent the group geometric mean. The horizontal dashed line represents the lower detection limit (LLOD) and the upper detection limit.

Fig. 17:analytical SEC plots with two proline stability mutations (+PP) and semi-stable uncleaved SARS-CoV-2S with a folder in the hinge loops shown in dashed lines (SEQ ID NO: 3) and more stable PP-free and folder-free S protein shown in solid lines (COR 201291).

Fig. 18:and (5) a data processing workflow of the frozen electron microscope. A typical micrograph representing approximately 75% of 9760 micrographs and a representative class 2D are shown. 3D classification is performed to distinguish heterogeneity in the sample and optimize the class that shows the highest resolution.

FIG. 19: resolution evaluation of the cryo-electron-microscope structure: a) partial resolution map of closed structure (full, slide through, top view) B) "partial resolution map of one up" structure (full, slide, top view) C) overall resolution evaluation by fourier shell correlation under 0.143 standard (Fourier shell correlation) D) correlation of the map by fourier shell correlation model under 0.5 standard.

Detailed Description

As described above, SARS-CoV-2 and other coronaviruses spike protein (S) is involved in fusion of the viral membrane with the host cell membrane, which is required for infection. SARS-CoV-2S RNA is translated into 1273 amino acid precursor proteins that contain a signal peptide sequence (e.g., amino acid residues 1-13 of SEQ ID NO: 1) at the N-terminus that is removed by a signal peptidase in the endoplasmic reticulum. The initiation of the S protein typically involves cleavage of the host protease at the boundary (S1/S2) between the S1 and S2 subunits in the coronavirus subgroup (including SARS-CoV-2) and at a conserved site (S2') upstream of the fusion peptide in all known coronaviruses. For SARS-CoV-2, furin cleaves at S1/S2 between residues 685 and 686, followed by cleavage of the S2' site between residues at 815 and 816 within S2 by TMPRSS 2. The proposed fusion peptide is located at the C-terminal end of the S2' site, at the N-terminal end of refolding region 1 (FIG. 1).

Several vaccines against SARS-CoV-2 infection are currently available. Several different vaccine forms are possible, such as RNA or vector based vaccines, and/or subunit vaccines based on purified S protein. Since class I proteins are metastable proteins, increasing the stability of the pre-fusion conformation of the fusion protein may increase the expression level of the protein, as fewer proteins will be misfolded and more will be successfully transported through the secretory pathway. Thus, if the stability of the pre-fusion conformation of a class I fusion protein, such as the SARS-CoV-2S protein, is increased, the immunogenic properties of a vector-based vaccine will be improved because the expression of the S protein is higher and the conformation of the immunogen is similar to the pre-fusion conformation recognized by the effective neutralizing and protective antibodies. For subunit-based vaccines, it is even more important to stabilize the pre-fusion S conformation. Furthermore, it is important that the trimer yield of stable S protein is increased compared to the previously described SARS-CoV-2S protein trimer. In addition to the importance of high expression required for successful vaccine preparation, maintaining the pre-trimeric fusion conformation over time during preparation and during storage is critical for protein-based vaccines. In addition, for soluble subunit-based vaccines, the SARS-CoV-2S protein needs to be truncated by deletion of the Transmembrane (TM) and cytoplasmic regions to produce a soluble secreted S protein (sS). Because this TM region is responsible for membrane anchoring and increased stability, the non-anchored soluble S protein is significantly more labile than the full-length protein and will refold into post-fusion final state even more easily. In order to obtain a soluble trimeric S protein in a stable pre-fusion conformation and exhibiting high expression levels and high stability, it is therefore necessary to stabilize the pre-fusion conformation. Also because the full length (membrane bound) SARS-CoV-2S protein is metastable, it is desirable to stabilize the pre-fusion conformation for the full length SARS-CoV-2S protein (i.e., comprising the TM region and cytoplasmic region), e.g., for any DNA, RNA, inactivating or vector-based vaccine approach.

The present invention provides recombinant SARS-CoV-2S proteins having increased trimer production and/or increased (thermal) stability as compared to the SARS-CoV-2S proteins previously described.

Accordingly, the present invention provides a stable recombinant pre-fusion SARS-CoV-2S protein and fragments thereof comprising S1 and S2 domains and comprising at least one mutation selected from the group consisting of: mutations corresponding to at least one amino acid in the loop region of amino acid residues 941-945 to P, mutations at amino acid position 892, mutations at amino acid position 614, mutations at position 572, mutations at position 532, disulfide bridges between residues 880 and 888, and disulfide bridges between residues 884 and 893, wherein the numbering of amino acid positions is according to the numbering of amino acid positions in SEQ ID NO: 1. According to the present invention, the presence of specific amino acids and/or disulfide bridges at specified positions has been demonstrated to increase the stability of proteins in the pre-fusion conformation. According to the invention, a specific amino acid or disulfide bridge is introduced by substitution (mutation) of the amino acid at this position to a specific amino acid according to the invention. According to the invention, the protein thus comprises one or more mutations in its amino acid sequence, i.e. the naturally occurring amino acids at these positions have been substituted with another amino acid. In certain embodiments, the protein comprises an amino acid sequence wherein the amino acid at position 892 is not alanine (a), the amino acid at position 614 is not aspartic acid (D), the amino acid at position 532 is not asparagine (N) and/or the amino acid at position 572 is not threonine (T).

In certain embodiments, the protein comprises at least two mutations comprising a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 to P, and a mutation selected from the group consisting of: mutation of amino acid 892, mutation of amino acid 614, mutation of amino acid 572, mutation of amino acid 532, disulfide bridge between residues 880 and 888 and disulfide bridge between residues 884 and 893.

In certain embodiments, the protein comprises at least two mutations comprising a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 to P, and a mutation selected from the group consisting of: mutations at amino acid 892, at amino acid 614, at 572 and at 532, disulfide bridges between residues 880 and 888 and between residues 884 and 893, provided that these proteins do not contain both disulfide bridges between residues 880 and 888 and between residues 884 and 893.

In certain embodiments, the protein thus comprises a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 to P, a mutation of amino acid 892, a mutation of amino acid 614, a mutation of amino acid 572 and/or a mutation at 532, and/or a disulfide bridge between residues 880 and 888 or between residues 884 and 893.

In a preferred embodiment, the disulfide bridge is a disulfide bridge between residues 880 and 888. According to the present invention, it is understood that "disulfide bridge between residues 880 and 880" means that the amino acids at positions 880 and 888 have been mutated to C. Similarly, it is understood that "disulfide bridge between residues 884 and 893" means that the amino acids at positions 884 and 893 have been mutated to C.

In certain embodiments, at least one mutation in the loop region corresponding to amino acid residues 941-945 is an amino acid mutation at position 942 to P.

Alternatively or additionally, at least one mutation in the loop region corresponding to amino acid residues 941-945 is a mutation of the amino acid at position 941 to P.

Alternatively or additionally, at least one mutation in the loop region corresponding to amino acid residues 941-945 is a mutation of amino acid position 944 to P.

Alternatively or additionally, the mutation at position 892 is a mutation to P.

Alternatively or additionally, the mutation at position 614 is a mutation to N or G.

Alternatively or additionally, the mutation at position 532 is a mutation to P.

Alternatively or additionally, the mutation at position 572 is a mutation to I.

Thus, the invention also provides a stable recombinant pre-fusion SARS-CoV-2S protein comprising S1 and S2 domains, wherein the amino acid at position 941, 942 or 944 is P, the amino acid at position 892 is P, the amino acid at position 614 is N or G, the amino acid at position 572 is I, and/or the amino acid at position 532 is P; and/or comprises a disulfide bridge between residues 880 and 888, and/or a disulfide bridge between residues 884 and 893, wherein the numbering of amino acid positions is according to the numbering of amino acid positions in SEQ ID NO. 1.

In a preferred embodiment, the amino acid at position 892 is proline (P), the amino acid at position 614 is asparagine (N) or glycine (G), the amino acid at position 942 is proline (P) or the amino acid at position 944 is proline (P).

Amino acids according to the invention may be any of the twenty naturally occurring (or "standard") amino acids or variants thereof, such as for example D-amino acids (D-enantiomers of amino acids with chiral centers), or any variants that do not naturally occur in proteins, such as for example norleucine. Each known natural amino acid has a full name, an abbreviated one-letter code, and an abbreviated three-letter code, all of which are well known to those of ordinary skill in the art. For example, the abbreviated codes for three and one letter for twenty naturally occurring amino acids are as follows: alanine (Ala; A), arginine (Arg; R), aspartic acid (Asp; D), asparagine (Asn; N), cysteine (Cys; C), glycine (Gly; G), glutamic acid (Glu; E), glutamine (Gln; Q), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V). Amino acids may be referred to by their full name, single letter abbreviation code or three letter abbreviation code. Standard amino acids can be grouped based on their characteristics. 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 cysteines, which can form covalent disulfide bonds (or disulfide bridges) with other cysteine residues; proline, which induces rotation of the polypeptide backbone; and glycine, which is more flexible than other amino acids. Table 1 shows abbreviations and properties of standard amino acids.

Those skilled in the art will appreciate that the protein may be mutated by conventional molecular biological procedures.

In certain embodiments, the invention provides recombinant SARS-CoV-2S proteins and fragments thereof, wherein the amino acid at position 942 is P, the amino acid at position 892 is P, the amino acid at position 614 is N or G, the amino acid at position 532 is P and/or the amino acid at position 572 is I; and/or the recombinant protein and fragments thereof comprises a disulfide bridge between residues 880 and 888 or a disulfide bridge between residues 884 and 893, wherein the numbering of amino acid positions is according to the numbering of amino acid positions in SEQ ID NO. 1.

In a preferred embodiment, the invention provides SARS-CoV-2 protein or a fragment thereof comprising a disulfide bridge between residues 880 and 888 wherein the amino acid at position 942 is P, the amino acid at position 614 is N or G, 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 SARS-CoV-2S protein further lacks a furin cleavage site. Deletion of 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), would leave the protein uncleaved, further increasing its stability. Deletion of the furin cleavage site can be accomplished in any suitable manner known to those skilled in the art. In certain embodiments, the deletion of the furin cleavage site comprises a mutation of the amino acid at position 682 to S and/or a mutation of the amino acid at position 685 to G.

In certain embodiments, the protein further comprises a mutation of amino acid 986 and/or 987 to proline. In certain embodiments, the amino acid at position 986 is not proline. In certain embodiments, the amino acid at position 986 is K and the amino acid at position 987 is P.

In a preferred embodiment, the invention provides recombinant SARS-CoV-2S protein and fragments thereof that lacks a furin cleavage site, wherein the amino acid at position 942 is P, the amino acid at position 892 is P, the amino acid at position 987 is P, and wherein the numbering of the amino acid positions is according to the numbering of the amino acid positions in SEQ ID NO. 1.

In another preferred embodiment, the invention provides recombinant SARS-CoV-2S protein and fragments thereof that lacks a furin cleavage site, wherein the amino acid at position 942 is P, the amino acid at position 892 is P, the amino acid at position 614 is N or G, and the amino acid at position 987 is P, wherein the numbering of the amino acid positions is according to the numbering of the amino acid positions in SEQ ID NO. 1.

In another preferred embodiment, SARS-CoV-2S protein lacks a furin cleavage site and the amino acid at position 942 is P, the amino acid at position 892 is P, the amino acid at position 614 is N, and the amino acid at position 987 is P.

In another preferred embodiment, the SARS-CoV-2S protein lacks a furin cleavage site and comprises a disulfide bridge between residues 880 and 888; and the amino acid at position 944 is P, the amino acid at position 614 is G, the amino acid at position 572 is I, the amino acid at position 532 is P, and wherein the amino acid at position 987 is P.

In certain embodiments, the invention provides SARS-CoV 2 proteins, or fragments thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID NOS 5-194 and 197-418, SEQ ID NOS 420 and 421. In a preferred embodiment, the SARS-CoV 2 protein comprises the amino acid sequence of SEQ ID NO. 417 or SEQ ID NO. 418. In certain embodiments, the protein according to the invention does not comprise a signal peptide sequence or a tag sequence.

As used herein, the term "fragment" refers to a peptide having an amino-terminal and/or carboxy-terminal and/or internal deletion, but wherein the remaining amino acid sequence is identical to the corresponding position in the SARS-CoV-2S protein sequence (e.g., the full length sequence of the SARS-CoV-2S protein). It will be appreciated that in order to induce an immune response and typically for vaccination purposes, the protein need not be full length nor need it have all of its wild type function, and fragments of the protein are equally useful. Fragments according to the invention are immunologically active fragments and typically comprise at least 15 amino acids, or at least 30 amino acids, of the SARS-CoV-2S 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-2S protein. In some documents, the fragment is the SARS-CoV-2S extracellular domain.

In certain embodiments, the protein according to the invention is a soluble (trimeric) protein, e.g. an S protein extracellular domain, and comprises a truncated S2 domain. As used herein, a "truncated" S2 domain refers to an S2 domain that is not a full-length S2 domain, i.e., wherein one or more amino acid residues have been deleted at the N-terminus or at the C-terminus. According to the invention, at least the transmembrane and cytoplasmic domains (corresponding to amino acids 1-1208 of SEQ ID NO: 1) are deleted to allow expression as a soluble extracellular domain (corresponding to amino acids 1-1208 of SEQ ID NO: 1). To stabilize this pre-fusion conformation of the soluble trimeric SARS-CoV-2S protein, a heterotrimeric domain (e.g., a fibrin-based trimerization domain) can be fused to the C-terminus of the coronavirus S protein extracellular domain. Such fibrin domains or "folmers" are derived from T4 fibrin and were described earlier as artificial natural trimerization domains (Letarov et al, (1993); S-Guche et al, (2004)). Thus, in certain embodiments, the transmembrane region is replaced by a heterotrimeric domain. In a preferred embodiment, the heterotrimeric domain is a folding subdomain comprising the amino acid sequence of SEQ ID NO. 4. However, it should be understood that other trimerization domains are possible, or that no heterotrimeric structure is added to the S ectodomain, according to the invention.

In a preferred embodiment, the soluble trimeric SARS-CoV 2S protein of the invention does not comprise a heterotrimeric domain.

In a preferred embodiment, the invention provides a recombinant SARS-CoV-2S protein comprising a truncated S2 domain that lacks a furin cleavage site, wherein the amino acid at position 942 is P, the amino acid at position 892 is P, the amino acid at position 987 is P, wherein the protein does not comprise a heterotrimeric domain, and wherein the numbering of the amino acid positions is according to the numbering of the amino acid positions in SEQ ID NO. 1.

In a preferred embodiment, the invention provides a recombinant SARS-CoV-2S protein comprising a truncated S2 domain that lacks a furin cleavage site, wherein the amino acid at position 942 is P, the amino acid at position 892 is P, the amino acid at position 614 is N or G, the amino acid at position 987 is P, wherein the protein does not comprise a heterotrimeric domain, and wherein the numbering of the amino acid positions is according to the numbering of the amino acid positions in SEQ ID NO. 1.

The recombinant pre-fusion SARS-CoV-2S protein according to the invention preferably has increased trimer production and/or increased (thermal) stability.

Alternatively or additionally, the pre-fusion SARS-CoV-2S protein according to the invention induces increased neutralizing antibody titers compared to SARS-CoV-2S protein without the stability mutation of the invention.

In certain embodiments, the pre-fusion SARS-CoV-2S protein according to the invention is stable, i.e., does not readily change to a post-fusion conformation upon processing of the protein, such as, for example, upon purification, freeze-thaw cycling, and/or storage, and the like. In certain embodiments, the pre-fusion SARS-CoV-2S protein has increased stability, as indicated by increased melting temperature (as measured by, for example, differential scanning fluorometry), as compared to the SARS-CoV-2S protein without the mutation of the invention.

The protein according to the invention may comprise a signal peptide (also known as signal sequence or leader peptide) corresponding to amino acids 1-13 of SEQ ID NO. 1. The signal peptide is a short peptide (typically 5-30 amino acids long) that is present at the N-terminus of most newly synthesized proteins that ultimately go to the secretory pathway. In certain embodiments, the protein according to the invention does not comprise a signal peptide.

In certain embodiments, the protein comprises a tag sequence, such as a HIS tag or a C tag. His tag (or polyhistidine tag) is an amino acid motif in proteins consisting of at least five histidine (H) residues, preferably at the N-or C-terminus of the protein, typically used for purification purposes. Alternatively, other tags, such as C-tags, may be used for these purposes. In certain embodiments, the protein according to the invention does not comprise a tag sequence.

The present invention also provides a method for stabilizing SARS-CoV-2S protein, said method comprising introducing at least one mutation in the amino acid sequence of SARS-CoV-2S protein, the at least one mutation selected from the group consisting of: mutations corresponding to at least one amino acid in the loop region of amino acid residues 941-945 to P, mutations at amino acid 892, mutations at amino acid 614, mutations at 572, mutations at 532, disulfide bridges between residues 880 and 888, and disulfide bridges between residues 884 and 893, wherein the numbering of these amino acid positions is according to the numbering of these amino acid positions in SEQ ID NO: 1.

In certain embodiments, the methods comprise introducing at least two mutations comprising a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 to P, and a mutation selected from the group consisting of: mutation of amino acid 892, mutation of amino acid 614, mutation of amino acid 572, mutation of amino acid 532, disulfide bridge between residues 880 and 888 and disulfide bridge between residues 884 and 893.

In certain embodiments, the methods comprise introducing at least two mutations comprising a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 to P, and a mutation selected from the group consisting of: mutation of amino acid 892, mutation of amino acid 614, mutation of amino acid 572, mutation of 532, disulfide bridge between residues 880 and 888 and disulfide bridge between residues 884 and 893, provided that these proteins do not contain both disulfide bridges between residues 880 and 888 and between residues 884 and 893.

In certain embodiments, at least one mutation in the loop region corresponding to amino acid residues 941-945 is an amino acid mutation at position 942 to P.

Alternatively or additionally, at least one mutation in the loop region corresponding to amino acid residues 941-945 is a mutation of the amino acid at position 941 to P.

Alternatively or additionally, at least one mutation in the loop region corresponding to amino acid residues 941-945 is a mutation of amino acid position 944 to P.

Alternatively or additionally, the mutation at position 892 is a mutation to P.

Alternatively or additionally, the mutation at position 614 is a mutation to N or G.

Alternatively or additionally, the mutation at position 532 is a mutation to P.

Alternatively or additionally, the mutation at position 572 is a mutation to I.

Alternatively or additionally, the methods further comprise deleting the furin cleavage site. Deletion of the furin cleavage site can be accomplished in any manner known in the art.

In certain embodiments, the deletion of the furin cleavage site includes a mutation that introduces amino acid at position 682 to S and/or a mutation that introduces amino acid at position 685 to G.

Alternatively or additionally, the methods further comprise introducing mutations of amino acids at positions 986 and/or 987 to proline. In a preferred embodiment, these methods comprise introducing a mutation of the amino acid at position 987 to proline.

The invention further provides nucleic acid molecules encoding SARS-CoV-2S protein according to the invention. The term "nucleic acid molecule" as used herein refers to a polymeric form of nucleotides (i.e., polynucleotides) and includes DNA (e.g., cDNA, genomic DNA) and RNA, as well as synthetic forms and mixed polymers of the foregoing.

In a preferred embodiment, the nucleic acid molecule encoding a protein according to the invention is codon optimized for expression in mammalian cells (preferably human cells) or insect cells. Methods of codon optimisation 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 is replaced with a more preferred codon compared to the wild-type sequence. Herein, a non-preferred codon is a codon that is not used frequently in an organism as another codon encoding the same amino acid, and a more preferred codon is a codon that is used more frequently in an organism than a non-preferred codon. The codon usage frequency for a particular organism can be found in a codon frequency table, such as http:// www.kazusa.or.jp/codon. Preferably more than one non-preferred codon, preferably the most or all non-preferred codons, are replaced by more preferred codons. Preferably, codons most frequently used in organisms are used for codon optimized sequences. Substitution by preferred codons generally promotes higher expression.

The skilled artisan will appreciate that many different polynucleotides and nucleic acid molecules may encode the same protein due to the degeneracy of the genetic code. It will also be appreciated that the skilled artisan can use conventional techniques to make nucleotide substitutions that do not affect the sequence of the protein encoded by the nucleic acid molecule to reflect codon usage of any particular host organism in which the protein is to be expressed. Thus, unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. The nucleotide sequences encoding proteins and RNAs may or may not include introns.

The nucleic acid sequences may be cloned using conventional molecular biology techniques or generated by DNA synthesis from the head, which may be performed by service companies having business in the DNA synthesis and/or molecular cloning field, such as Gene technologies Co (GeneArt), gold Style (GenScript), engineer (Invitrogen), european company (Eurofins), using conventional procedures.

In certain embodiments, the nucleic acid sequence encodes a SARS-CoV 2 protein or fragment thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID NO 5-194 and SEQ ID NO 197-418, SEQ ID NO 420 and SEQ ID NO 421.

In a preferred embodiment, the nucleic acid sequence encodes a SARS-CoV 2 protein that comprises the amino acid sequence of SEQ ID NO. 417 or SEQ ID NO. 418.

The invention also provides vectors comprising the nucleic acid molecules as described above. In certain embodiments, a nucleic acid molecule according to the invention is thus part of a vector. Such vectors can be readily manipulated by methods well known to those skilled in the art and can, for example, be designed to replicate in prokaryotic and/or eukaryotic cells. In addition, a number of vectors can be used for transformation of eukaryotic cells and integration of all or part of the genome of these cells to produce stable host cells comprising the desired nucleic acid in their genome. The vector used may be any vector which is suitable for cloning DNA and which can be used for transcription of the nucleic acid of interest.

In certain embodiments of the invention, the vector is an adenovirus vector. Adenoviruses according to the invention belong to the family adenoviridae and preferably are one belonging to the genus mammalian adenoviruses (mastadenoviruses). It may be a human adenovirus, but may also be an adenovirus that infects other species, including but not limited to bovine adenovirus (e.g., bovine adenovirus 3, badv 3), canine adenovirus (e.g., CAdV 2), porcine adenovirus (e.g., PAdV3 or 5), or simian adenovirus (which includes simian adenovirus and simian adenovirus, such as chimpanzee adenovirus or gorilla adenovirus). Preferably, the adenovirus is a human adenovirus (HAdV or AdHu) or a simian adenovirus such as a chimpanzee or gorilla adenovirus (ChAd, adCh, or SAdV) or a rhesus adenovirus (RhAd). In the present invention, human adenovirus means that if referred to as Ad without specifying a species, for example the abbreviated symbol "Ad26" means the same as HAdV26, which HAdV26 is human adenovirus serotype 26. Also as used herein, the symbol "rAd" means a recombinant adenovirus, e.g., "rAd26" means a 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, the recombinant adenoviruses according to the invention are based on human adenoviruses. In preferred embodiments, the recombinant adenovirus is based on human adenovirus serotypes 5, 11, 26, 34, 35, 48, 49, 50, 52, and the like. According to a particularly preferred embodiment of the invention, the adenovirus is human adenovirus serotype 26. Advantages of these serotypes include low seropositive rates and/or low pre-existing neutralizing antibody titers in the human population, as well as experience for human subjects in clinical trials.

Simian adenoviruses also typically have low seropositive rates and/or low pre-existing neutralizing antibody titers in humans, and extensive work with chimpanzee adenovirus vectors has been reported (e.g., U.S. Pat. No. 3, 6083716; WO 2005/071093; WO 2010/086189;WO 2010085984;Farina et al, 2001, J Virol J Virol 75:11603-13; cohen et al, 2002, J Gen Virol J general Virol 83:151-55; kobinger et al,2006, virology J346:394-401; tatsis et al, 2007,Molecular Therapy [ molecular therapy ]15:608-17; see also Bangari and Mittal,2006, vaccine [ vaccine ] 24:849-62; and Lasaro and ErtlTher [ molecular therapy ] 17:1333-39). Thus, in other embodiments, the recombinant adenoviruses according to the invention are based on simian adenoviruses, such as chimpanzee adenoviruses. In certain embodiments, the recombinant adenovirus is based on 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 on 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 on a chimpanzee adenovirus, such as BZ28 (see, e.g., WO 2019/086466). In certain embodiments, the recombinant adenovirus is based on 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-defective recombinant virus vector, such as rAd26, rAd35, rAd48, rAd5HVR48, and the like.

In a preferred embodiment of the invention, these adenovirus vectors comprise capsid proteins from rare serotypes, including Ad26, for example. In typical embodiments, the vector is a rAd26 virus. By "adenovirus capsid protein" is meant a protein on the capsid of an adenovirus (e.g., ad26, ad35, rAd48, rAd5HVR48 vector) that is involved in determining the serotype and/or tropism of a particular adenovirus. Adenovirus capsid proteins typically comprise fiber, penton and/or hexon proteins. As used herein, a "capsid protein" (such as an "Ad26 capsid protein") for a particular adenovirus may be, for example, a chimeric capsid protein that includes at least a portion of an Ad26 capsid protein. In certain embodiments, the capsid protein is the entire capsid protein of Ad26. In certain embodiments, the hexon, penton, and fiber are all Ad26.

One of ordinary skill in the art will recognize that elements derived from multiple serotypes may be combined in a single recombinant adenovirus vector. Thus, chimeric adenoviruses can be produced that combine desirable properties from different serotypes. Thus, in some embodiments, the chimeric adenoviruses of the invention may combine the absence of pre-existing immunity of the first serotype with the following features: such as temperature stability, assembly, anchoring, yield, redirected or improved infection, stability of DNA in target cells, etc. See, e.g., WO 2006/040330, chimeric adenoviruses Ad5HVR48, which include an Ad5 backbone with a partial capsid from Ad48, and see, e.g., WO 2019/086461, chimeric adenoviruses Ad26HVRPtr1, ad26HVRPtr12, and Ad26HVRPtr13, which include an Ad26 viral backbone with partial capsid proteins of Ptr1, ptr12, and Ptr13, respectively.

In certain embodiments, the recombinant adenovirus vectors useful in the invention are derived predominantly or entirely from Ad26 (i.e., the vector is rAd 26). In some embodiments, the adenovirus is a replication defective adenovirus, e.g., because it comprises a deletion in the E1 region of the genome. For adenoviruses derived from non-group C adenoviruses (e.g., ad26 or Ad 35), the E4-orf6 coding sequence of the adenovirus is typically exchanged with the E4-orf6 coding sequence of an adenovirus of human subgroup C (e.g., ad 5). This allows propagation of such adenoviruses in well-known complementing cell lines expressing the E1 gene of Ad5, such as, for example, 293 cells, PER.C6 cells, etc. (see, for example, havenga, et al, 2006, J Gen Virol [ J.Gen.Virol. ]87:2135-43; WO 03/104467). However, such adenoviruses will not replicate in non-complementing cells that do not express the E1 gene of Ad 5.

The preparation of recombinant adenovirus vectors is well known in the art. The preparation of rAD26 vectors is described, for example, in WO 2007/104792 and in Abbink et al, (2007) Virol [ virology ]81 (9): 4654-63. Exemplary genomic sequences for Ad26 are found in GenBank accession No. EF 153474 and in SEQ ID NO:1 of WO 2007/104792. Examples of vectors useful in the present invention include, for example, those described in WO2012/082918, the disclosure of which is incorporated herein by reference in its entirety.

Typically, nucleic acids comprising the entire recombinant adenovirus genome are used to generate vectors (e.g., plasmids, cosmids, or baculovirus vectors) useful in the invention. Thus, the invention also provides isolated nucleic acid molecules encoding the adenoviral vectors of the invention. The nucleic acid molecules according to the invention can be in the form of RNA or in the form of DNA, which is obtained by cloning or is produced synthetically. The DNA may be double-stranded or single-stranded.

These adenovirus vectors useful in the present invention are typically replication defective vectors. In these embodiments, the virus is rendered replication-defective by deleting or inactivating regions critical for viral replication (e.g., the E1 region). These regions may be substantially deleted or inactivated by, for example, inserting a gene of interest, such as a gene encoding the SARS-CoV-2S protein, typically linked to a promoter. In some embodiments, the vectors of the invention may comprise deletions in other regions, such as the E2, E3, or E4 regions, or insertions of heterologous genes linked to the promoter within one or more of these regions. For E2-and/or E4-mutated adenoviruses, E2-and/or E4-complementing cell lines are typically used to produce recombinant adenoviruses. Mutations in the E3 region of the adenovirus need not be complemented by the cell line, as E3 is not required for replication.

Packaging cell lines are typically used to produce sufficient amounts of adenovirus vectors for use in the present invention. Packaging cells are cells that contain genes that are deleted or inactivated in replication defective vectors, thus allowing the virus to replicate in the cell. Suitable packaging cell lines for adenoviruses with deletions in the E1 region include, for example, PER.C6, 911, 293 and E1A 549.

In a preferred embodiment of the invention, the vector is an adenovirus vector, and more preferably a rAD26 vector, most preferably a rAD26 vector having at least one deletion in the E1 region of the adenovirus genome, as described, for example, in Abbink, J Virol [ J virology ],2007.81 (9): pages 4654-63, which is incorporated herein by reference. Typically, the nucleic acid sequence encoding the SARS-CoV-2S protein is cloned into the E1 and/or E3 region of the adenovirus genome.

Host cells comprising a nucleic acid molecule encoding a pre-fusion SARS-CoV-2S protein also form part of the invention. The pre-fusion SARS-CoV-2S protein can be produced by recombinant DNA techniques that involve expression of these molecules in host cells (e.g., chinese Hamster Ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines (e.g., HEK293 cells, PER.C6 cells) or yeast, fungi, insect cells, etc.), or transgenic animals or plants. In certain embodiments, the cells are from a multicellular organism, and in certain embodiments, they are derived from a vertebrate or invertebrate. In certain embodiments, the cell is a mammalian cell, such as a human cell or an insect cell. In general, the production of recombinant proteins (e.g., the pre-fusion SARS-CoV-2S protein of the invention) in a host cell involves introducing into the host cell a heterologous nucleic acid molecule encoding the protein in an expressible form, culturing the cells under conditions conducive to expression of the nucleic acid molecule, and allowing expression of the protein in the cells. The nucleic acid molecule encoding the protein in an expressible form may be in the form of an expression cassette and typically requires sequences capable of causing expression of the nucleic acid, e.g., one or more enhancers, promoters, polyadenylation signals, and the like. Those skilled in the art know that different promoters may be used to achieve expression of a gene in a host cell. Promoters may be constitutive or regulated, and may be obtained from different sources (including viral, prokaryotic, or eukaryotic sources), or may be designed artificially.

Cell culture media are available from different suppliers, and suitable media can be routinely selected for host cells to express the protein of interest, here the pre-fusion SARS-CoV-2S protein. Suitable media may or may not contain serum.

A "heterologous nucleic acid molecule" (also referred to herein as a "transgene") is a nucleic acid molecule that does not naturally occur in a host cell. It is introduced into the carrier, for example by standard molecular biology techniques. Typically, the transgene is operably linked to an expression control sequence. This can be accomplished, for example, by placing nucleic acids encoding one or more transgenes under the control of a promoter. Additional regulatory sequences may be added. Many promoters are available for expression of one or more transgenes and are known to the skilled artisan, for example, these may comprise viral promoters, mammalian promoters, synthetic promoters, and the like. Non-limiting examples of suitable promoters for achieving expression in eukaryotic cells are the CMV promoter (US 5,385,839), such as the CMV immediate early promoter, e.g. comprising nucleotides-735 to +95 from the CMV immediate early gene enhancer/promoter. Polyadenylation signals, such as bovine growth hormone poly a signal (US 5,122,458), may be present after one or more transgenes. Alternatively, several widely used expression vectors are available in the art and are available from commercial sources, such as the pcDNA and pEF vector series from invitrogen, pMSCV and pTK-Hyg from the company bi di science (BD Sciences), pCMV-Script from the company s Qu Jie (Stratagene), etc., which can be used for recombinant expression of the protein of interest, or for obtaining suitable promoter and/or transcription terminator sequences, poly a sequences, etc.

The cell culture may be any type of cell culture, including adherent cell cultures, e.g., cells attached to the surface of a culture vessel or microcarriers, as well as suspension cultures. Most large scale suspension cultures are operated as batch or fed-batch processes because their operation and scale up is most straightforward. Continuous processes based on the principle of perfusion are becoming more common and also suitable today. Suitable media are also well known to the skilled person and are generally available in large quantities from commercial sources or are custom made according to standard protocols. For example, the cultivation may be carried out in a petri dish, roller bottle or bioreactor using batch, fed-batch, continuous systems, etc. Suitable conditions for culturing cells are known (see, e.g., tissue Culture, academic Press, kruse and Paterson editions, (1973), and R.I. Freshney, culture of animal cells: amanual of basic technique, animal cell Culture: basic technical Manual, fourth edition (Wiley-List Inc. [ Weili S Co., 2000, ISBN 0-471-34889-9)).

The invention further provides compositions comprising a pre-fusion SARS-CoV-2S protein and/or nucleic acid molecule and/or vector as described above. The invention also provides compositions comprising nucleic acid molecules and/or vectors encoding such pre-fusion SARS-CoV-2S protein. The invention further provides immunogenic compositions comprising a pre-fusion SARS-CoV-2S 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-2S protein, nucleic acid molecule, and/or vector according to the invention for inducing an immune response against a SARS-CoV-2S protein in a subject. Further provided are methods for inducing an immune response against SARS-CoV-2S protein in a subject, comprising administering to the subject a pre-fusion SARS-CoV-2S protein, and/or a nucleic acid molecule, and/or a vector according to the invention. Also provided are pre-fusion SARS-CoV-2S proteins, nucleic acid molecules and/or vectors according to the invention for inducing an immune response against SARS-CoV-2S protein in a subject. Further provided is the use of a pre-fusion SARS-CoV-2S protein, and/or a nucleic acid molecule, and/or a vector according to the invention for the manufacture of a medicament for inducing an immune response against a SARS-CoV-2S protein in a subject. In certain embodiments, the nucleic acid molecule is a DNA and/or RNA molecule.

The pre-fusion SARS-CoV-2S protein, nucleic acid molecule or vector of the invention can be used to prevent (control, including post-exposure control) SARS-CoV-2 infection.

As used herein, SARS-CoV-2 can refer to the Wuhan-Hu-1 strain, or variants thereof, such as variants comprising one or more mutations in the S protein, including but not limited to b.1, B1.1.7, b.1.351, P1, b.1.427, b.1.429, b.1.526, b.1.526.1, b.1.525, b.1.617, b.1.617.1, b.1.617.2, b.1.617.3, and p.2 viral variants.

In certain embodiments, prophylaxis may be performed against a group of patients susceptible to SARS-CoV-2 infection and/or at risk of a corresponding infection or having been diagnosed with SARS-CoV-2 infection. Such target groups include, but are not limited to, for example, elderly (e.g.,. Gtoreq.50 years,. Gtoreq.60 years, preferably. Gtoreq.65 years), hospitalized patients, and patients who have been treated with antiviral compounds but have shown inadequate antiviral response. In certain embodiments, the target population comprises human subjects from 2 months of age.

The pre-fusion SARS-CoV-2S protein, nucleic acid molecule and/or vector according to the invention can be used, for example, for the independent treatment and/or prevention of a disease or disorder caused by SARS-CoV-2, or in combination with other prevention 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 using the pre-fusion SARS-CoV-2S protein, nucleic acid molecule and/or vector according to the invention. In particular embodiments, a method for preventing and/or treating a SARS-CoV-2 infection in a subject comprises administering to a subject in need thereof an effective amount of a pre-fusion SARS-CoV-2S protein, nucleic acid molecule and/or vector as described above. A therapeutically effective amount refers to an amount of a protein, nucleic acid molecule or vector effective for preventing, alleviating and/or treating a disease or condition caused by infection with SARS-CoV-2. Prevention encompasses inhibiting or reducing the transmission of SARS-CoV-2 or inhibiting or reducing the onset, development, or progression of one or more symptoms associated with SARS-CoV-2 infection. As used herein, alleviating may refer to reducing the visible or perceptible symptoms of disease, viremia, or any other measurable manifestation of SARS-CoV-2 infection.

For administration to a subject (e.g., a human), the invention can employ pharmaceutical compositions comprising a pre-fusion SARS-CoV-2S 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 does not cause any unnecessary or adverse effects in the subject to which they are administered at the dosages and concentrations employed. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences [ leimington pharmaceutical sciences ], 18 th edition, a.r. gennaro editions, mack Publishing Company [ mike publishing company ] [1990]; pharmaceutical Formulation Development of Peptides and Proteins [ pharmaceutical formulation development of peptides and proteins ], s.frokjaer and l.hoveard editions, taylor & Francis [ Taylor Francis company ] [2000]; and Handbook of Pharmaceutical Excipients [ pharmaceutical excipient handbook ], 3 rd edition, a.kibbe editions, pharmaceutical Press [ pharmaceutical publishing company ] [2000 ]). The CoV S protein or nucleic acid molecule is preferably formulated and administered as a sterile solution, although lyophilized formulations can also be employed. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. These solutions are then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution is typically in the range of pH 3.0 to 9.5 (e.g., pH 5.0 to 7.5). CoV S proteins are typically in solution with a suitable pharmaceutically acceptable buffer, and the composition may also contain salts. Optionally, a stabilizing agent (e.g., albumin) may be present. In certain embodiments, a detergent is added. In certain embodiments, coV S protein can be formulated into an injectable formulation.

In certain embodiments, the compositions according to the present invention further comprise one or more adjuvants. Adjuvants are known in the art to further enhance the immune response to an applied epitope. The terms "adjuvant" and "immunostimulant" are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, adjuvants are used to enhance the immune response to the SARS-CoV-2S protein of the invention. Examples of suitable adjuvants include aluminum salts, such as aluminum hydroxide and/or aluminum phosphate; an oil-emulsion composition (or oil-in-water composition) comprising a squalene-water emulsion, such as MF59 (see, for example, WO 90/14837); sapogenins formulations such as, for example, QS21 and immunostimulatory complexes (ISCOMS) (see, for example, 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 (3 dMPL), oligonucleotides containing CpG motifs, ADP-ribosylated bacterial toxins or mutants thereof, such as E.coli heat labile enterotoxin LT, cholera toxin CT, etc.; eukaryotic proteins (e.g., antibodies or fragments thereof (e.g., directed against the antigen itself or CD1a, CD3, CD7, CD 80) and ligands for the receptor (e.g., CD40L, GMCSF, GCSF, etc.)) that stimulate an immune response upon interaction with the receptor cells. In certain embodiments, the compositions of the present invention comprise aluminum as an adjuvant, e.g., in the form of aluminum hydroxide, aluminum phosphate, potassium aluminum phosphate, or a combination thereof, at a concentration of 0.05-5mg, e.g., 0.075-1.0mg per dose of aluminum.

The pre-fusion SARS-CoV-2S protein can also be administered in combination with or conjugated to nanoparticles (e.g., like polymers, liposomes, virosomes, virus-like particles). The SARS-CoV-2S protein can be combined with, or encapsulated in, or conjugated to the nanoparticle with or without an adjuvant. Encapsulation within liposomes is described, for example, in US 4,235,877. Conjugation with macromolecules is disclosed, for example, in US 4,372,945 or US 4,474,757.

In other embodiments, these compositions do not comprise an adjuvant.

In certain embodiments, the invention provides methods for preparing a vaccine against 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 comprising an active ingredient effective to induce a degree of immunity in a subject against a pathogen or disease that will at least cause a reduction (up to no) in the severity, duration or other manifestation of symptoms associated with infection by the pathogen or disease. In the present invention, the vaccine comprises an effective amount of a pre-fusion SARS-CoV-2S protein and/or a nucleic acid molecule encoding a pre-fusion SARS-CoV-2S protein and/or a vector comprising said nucleic acid molecule that elicits an immune response against the S protein of SARS-CoV-2. This provides a method of preventing severe lower respiratory disease causing hospitalization and reducing the frequency of complications (such as pneumonia and bronchiolitis) caused by SARS-CoV-2 infection and replication in a subject. The term "vaccine" according to the invention means that it is a pharmaceutical composition and thus typically comprises a pharmaceutically acceptable diluent, carrier or excipient. It may or may not contain additional 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 components. The combination 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 ingredient(s) may be performed, for example, by separate (e.g., simultaneous) administration or under a prime-boost setting or by administration of the combination product of the vaccine of the invention and the additional active ingredient(s).

The composition may be administered to a subject, such as a human subject. The total dose of SARS-CoV-2S protein in the composition for separate administration can be, for example, from about 0.01 μg to about 10mg, such as from 1 μg to 1mg, such as from 10 μg to 100 μg. Determining the recommended dose will be done experimentally and is routine to those skilled in the art.

Administration of the composition according to the invention may be performed using standard routes of administration. Non-limiting examples include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, or mucosal administration, e.g., intranasal, oral, and the like. In one embodiment, the composition is administered by intramuscular injection. The skilled person knows the different possibilities of administering the composition (e.g. vaccine) to induce an immune response to one or more antigens in the vaccine.

As used herein, a subject is preferably a mammal, e.g., a rodent, e.g., a mouse, a cotton mouse, 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 as a prime or as a boost in a homologous or heterologous prime-boost regimen. If booster vaccination is performed, typically such booster vaccination will be administered to a subject (in such cases referred to as "primary vaccination") between one week and one year, preferably at some point in time between two weeks and four months, after the first administration of the composition to the same subject. In certain embodiments, the administration comprises at least one initial administration and at least one booster administration.

The invention further provides host cells comprising an isolated nucleic acid molecule or vector encoding the recombinant SARS-CoV-2S protein of the invention. Such host cells may be used for recombinant protein production, recombinant protein expression or viral particle production.

Furthermore, the present invention relates to methods of producing recombinant SARS-CoV-2S protein comprising growing a host cell comprising an isolated nucleic acid molecule or vector encoding a recombinant SARS-CoV-2S protein of the invention under conditions suitable for production of the recombinant SARS-CoV-2S protein.

The SARS-CoV-2S protein of the invention can also be used to isolate monoclonal antibodies from biological samples, such as biological samples (e.g., blood, plasma or cells) obtained from immunized animals or infected humans. Thus, the present invention also relates to the use of SARS-CoV-2 protein as a bait for isolating monoclonal antibodies.

Also provided is the use of the pre-fusion SARS-CoV-2S protein of the invention in a method of screening for a candidate SARS-CoV-2 antiviral agent (including but not limited to antibodies to SARS-CoV-2).

In addition, the proteins of the invention may be used as diagnostic tools, for example to test an individual for immune status by determining whether antibodies capable of binding to the proteins of the invention are present in the serum of such an individual. Accordingly, the present invention 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 the subject with a protein according to the invention; and b) detecting the presence of the antibody-protein complex.

Examples

Example 1:instability of semi-stable SARS-CoV 2S protein

Gene Art (Gene Art) (Life technologies (Life Technologies), calif. Carlsbad, calif.) synthesis corresponds to (Wrapp et al, science [ Science ]]2020, furin KO+PP, semi-stable plasmid of SARS-CoV 2S protein as described in SEQ ID NO: 3) and codon optimized. Variants with HIS tag (based on SEQ ID NO: 3) and variants with C tag were purified. These constructs were cloned into pCDNA2004 or generated by standard methods well known in the art, including site-directed mutagenesis and PCR, and sequenced. The expression platform used was an Expi293F cell. Using an Expiectamine according to manufacturer's instructionsTransient transfection of cells with ne (Life technologies Co.) and at 37℃and 10% CO ₂ Culturing for 6 days. The culture supernatant was harvested and spun at 300g for 5 minutes to remove cells and cell debris. The spun supernatant was then sterile filtered using a 0.22um vacuum filter and stored at 4 ℃ until use.

SARS-CoV 2S trimer was purified using a two-step purification protocol, including CaptureStylelect for the C-tag protein ^TM C-tag affinity column or for HIS-tag proteins 5mL (Roche) by cOmplete His tag. Both proteins were further purified by size exclusion chromatography using a HiLoad Superdex 200 16/600 column (general electric medical Co., ltd.). The C-tag and HIS-tag S-trimer are unstable after repeated freeze/thaw cycles (fig. 2a, b). Purified HIS tag S trimer and C tag trimer showed decay after 1, especially 5 flash cycles with liquid nitrogen (fig. 2a, b).

Example 2:analysis of stability mutations with alpha lisa and analytical SEC

To stabilize the unstable pre-fusion conformation of SARS-CoV 2S protein, the amino acid residues at positions 614, 892 and 942 (numbered according to SEQ ID NO: 1) are mutated. Plasmids encoding the extracellular domain of recombinant SARS-CoV-2S protein fused at the C-terminus to the folder (SEQ ID NO: 4) were expressed in the Expi293F cells and the supernatant was assayed for binding to ACE2-Fc using the AlphaLISA 3 days after transfection (FIG. 3).

For the AlphaLISA assay, a preparation containing a linker, followed by a sortase a tag, followed by a Flag tag, followed by a flexible (G ₄ S) ₇ SARS-CoV 2S variant in pcDNA2004 vector which is ligated and ends with His tag (the sequence of the tag at C-terminus of S protein is provided in SEQ ID NO: 2). Three days after transfection, the crude supernatant was diluted 300-fold in AlphaLISA buffer (PBS+0.05% Tween-20+0.5mg/mL BSA). Then 10. Mu.L of each dilution was transferred to a 96-well plate and mixed with 40. Mu.L of acceptor beads, donor beads and ACE 2-Fc. Donor beads were conjugated to ProtA (catalog number: AS102M, perkin Elmer), which binds to ACE2Fc. Binding of receptor beads to His tag of construct Is conjugated to an anti-His antibody (catalog number: AL128M, perkin Elmer).

The mixture containing the supernatant of expressed S protein, ACE-2-Fc, donor and acceptor beads was incubated at room temperature for 2 hours without shaking. Subsequently, chemiluminescent signals were measured using an Ensight reader instrument (Perkin Elmer). The average background signal due to mock transfected cells was subtracted from the AlphaLISA counts measured for each SARS-CoV-2S variant. The entire dataset was then divided by the signal measured for SARS-CoV-2S protein with S-framework sequence signal to normalize the signal for each S variant tested to the framework.

The S variants with the stability substitutions D614N, A892P and A942P showed higher ACE2-Fc binding compared to the soluble uncleaved S variant with the C-terminal folder domain (SEQ ID NO: 2) or the variant with additional PP (SEQ ID NO: 3) (FIG. 3).

The following were analyzed using analytical SEC: cell culture supernatants transfected with semi-stable uncleaved SARS-CoV-2 S+PP design and with the labile uncleaved SARS-CoV-2S protein, as well as cell culture supernatants with variants with single point mutations (D614N, A892P and A942P) as described above (FIG. 4). An ultra-high performance liquid chromatography system (Vanquish, siemens technologies (Thermo Scientific)) and a μDAWN TREOS instrument (Huai Yate) coupled to an Optilab μT-rEX refractive index detector (Huai Yate (Wyatt)) were combined with an online Nanostar DLS reader (Huai Yate) for analytical SEC experiments. The clarified crude cell culture supernatant was applied at 0.35mL/min to a SRT-10CSEC-500 15cm column (Sepax catalog number 235500-4615) with the corresponding guard column (Sedan technology Co., ltd.) equilibrated in flowing buffer (150 mM sodium phosphate, 50mM NaCl, pH 7.0). When analyzing supernatant samples, the μmals detector was offline and analytical SEC data was analyzed using the Chromeleon 7.2.8.0 software package. The signal of the supernatant of the untransfected cells was subtracted from the signal of the supernatant of the S-transfected cells. When the purified protein was analyzed using SEC-MALS, the mMALS detector was online and the data was analyzed using the Astra 7.3 software package. For the protein component, the dn/dc (mL/g) value of 0.1850 was used, and for the glycan component, the 0.1410 value was used. According to analytical SEC of culture supernatants, variants with additional stability substitutions D614N, A892P and in particular a942P showed higher trimer content compared to the semi-stable soluble uncleaved S variants with C-terminal folder domain + PP (fig. 4A-C). Similarly, according to analytical SEC of culture supernatants, variants with stability substitution D614N, A892P and in particular a942P showed higher trimer content compared to soluble uncleaved S variants with C-terminal foldback subunit domain. The a942P mutation has a stronger effect on trimer expression than the double proline mutation in the disclosed hinge loops, reflecting a higher stability during synthesis and transport (compare the dotted line of fig. 4B with the solid line of fig. 4E). SEC-MALS analysis was performed on the purified stabilized protein according to SEQ ID NO. 5 and showed that the peak at 5 minutes corresponds to the mass of trimeric S protein (FIG. 4G).

Example 3:analysis of stability Point mutations and disulfide bridges Using alpha LISA and analytical SEC

To stabilize the unstable pre-fusion conformation of the SARS-CoV-2S protein, a disulfide bridge is introduced between residues 880 and 888 or between residues 884 and 893, and point mutations are introduced at positions 532 and 572. Similar to example 2, plasmids encoding uncleaved SARS-CoV-2S protein with or without biproline in hinge loops were expressed in Expi293F cells and supernatants were tested for binding to ACE2-Fc using AlphaLISA as described in example 2 (fig. 5) 3 days after transfection.

Variants with a stable substitution T572I, N532P and introduction of a disulfide bridge between residues 880 and 888 showed higher ACE2-Fc binding compared to the soluble labile uncleaved S variant with a C-terminal folder (fig. 5, left panel).

In addition, variants with a stability substitution T572I, N532P, introduction of a disulfide bridge between residues 880 and 888 and introduction of a disulfide bridge between residues 884 and 893 showed higher ACE2-Fc binding compared to the soluble semi-stable uncleaved S variant with a C-terminal foldback domain and biproline (fig. 5, right panel).

As described in example 2, the following were analyzed using analytical SEC: cell culture supernatants transfected with the semi-stable uncleaved SARS-CoV-2 S+PP design and with the unstable uncleaved SARS-CoV-2S protein, as well as cell culture supernatants with either the introduced disulfide bridge or the variants of single point mutations (T572I, N532P, CYS880-CYS888 and CYS884-CYS 893) as described above (FIG. 6). According to analytical SEC of culture supernatants, variants with stability substitutions T572I, N532P and disulfide bridges 880C-888C and 884C-893C (fig. 6A-D) showed higher trimer content compared to the semi-stable soluble uncleaved S variants with C-terminal foldback domain + PP. Similarly, according to analytical SEC of culture supernatants, variants with stability substitution T572I, N532P and variants with disulfide bridge 880C-888C showed higher trimer content compared to soluble uncleaved S variants (fig. 6E-H).

Example 4:analysis of stability Point mutations Using Alphalisa and analytical SEC

To stabilize the unstable pre-fusion conformation of SARS-CoV-2S protein, point mutations were introduced at positions 941 and 944. Similar to example 2, plasmids encoding the unstable uncleaved SARS-CoV-2S protein were expressed in the Expi293F cells and supernatants were tested for binding to ACE2-Fc using the AlphaLISA as described in example 2 3 days post-transfection (FIG. 7A).

Variants with additional stability substitutions T941P and a944P showed higher ACE2-Fc binding compared to the soluble labile uncleaved S variant with C-terminal fold (fig. 7A).

As described in example 2, the following were analyzed using analytical SEC: cell culture supernatants transfected with the unstabilized uncleaved SARS-CoV-2S protein, as well as cell culture supernatants having variants with single point mutations (T941P and A944P) as described above (FIG. 7B). According to analytical SEC of culture supernatants, variants with stability substitutions T941P and a944P (fig. 7B) showed higher trimer content compared to the unstable soluble uncleaved S variant with C-terminal foldback domain.

Example 5: differential Scanning Calorimetry (DSC)

The melting temperature of the S trimer was determined using a MicroCal capillary DSC system. 400. Mu.L of 0.5mg/mL protein sample was used for each measurement. The measurement was performed at an initial temperature of 20℃and a final temperature of 110 ℃. Scanning speed is 100 ℃/h, and a feedback mode is adopted; low (=signal amplification). Data were analyzed using Origin J. Software (MicroCal VP-analysis tool). The purified uncleaved S trimer (SEQ ID NO: 2) shows a broad range of melting events, with the major melting event (Tm 2) at 64 ℃ (FIG. 8, upper panel). The introduction of the mutation K986P made the trimer very unstable, since most of the trimer melted at 48 ℃. The mutation V987P had relatively little effect because the main melting event (Tm 2) occurred at 64 ℃, and the biproline mutation (k986p+v987p) showed a slight increase in melting event at 47 ℃ (fig. 8, upper panel). The middle panel shows DSC thermostability data for an uncleaved semi-stable S trimer (furin KO+PP, SEQ ID NO:3, dashed line) with additional indicated point mutations in the S1 domain. N532P had no effect on Tm, and T572I and D614N showed strong stability effects (middle panel). The lower panel shows DSC thermostability data for an uncleaved semi-stable S trimer (SEQ ID NO:3, dashed line) with additional indicated mutations in the S2 domain. A942P had no effect on Tm, A892P and disulfide bridge F88C-G880C showed strong stability effect (bottom panel).

Example 6: s protein fusion

By transiently coexpression of GFP, ACE2, TMPRSS2 and full-length S variants in HEK293 cells, a cell-cell fusion assay was developed that mimics the entry pathway of SARS-CoV-2 on plasma membranes (see figure 9). After 18-24 hours, cell monolayers were observed using an EVOS microscope. All defined stability mutations were tested for furin KO and biproline mutations. Assays were performed at saturated concentrations of S protein to obtain yes or no answers regarding fusion ability. Although the furin KO, PP mutations and the two cystines completely abrogate the fusions, single point mutants still allowed fusion to occur. This means that these variants are folded and function correctly.

Example 7:analysis of combinations of stability mutations with analytical SEC and AlphaLISA

To stabilize the unstable pre-fusion conformation of SARS-CoV-2S protein, a combination of the indicated stability point mutations from the previous example was introduced in SEQ ID NO. 2 (FIG. 10). Similar to example 2, plasmids encoding the unstable uncleaved SARS-CoV-2S protein (dashed line) and stable variants were expressed in Expi293F cells and the supernatant was analyzed for trimer content by analytical SEC as described in example 2, 3 days post-transfection. As shown in FIG. 10A, the variants with four mutations A892P, A942P, D N and V987P showed the highest trimer content, much higher than the semi-stable S protein based on SEQ ID NO. 3 (furin KO+PP). Fig. 10B shows that trimers in cell culture supernatants also showed higher ACE2-Fc binding using AlphaLISA as described in example 2 (fig. 10B).

To obtain soluble natural S trimers, the C-terminal heterotrimeric domain is added as described by Pallesen et al (Pallesen, PNAS [ Proc. Natl. Acad. Sci. USA ], 2017). To investigate whether point mutations could sufficiently stabilize proteins to trimerize without addition of trimerization domains, variants with four stability mutations without a foldback (DF) were prepared (A892P-A942P-D614N-V987P-DF). As shown in fig. 10C and table 2, a large trimer peak was detected in analytical SEC at about 5.3 minutes. A slight shift to longer retention times was observed due to the lower molecular weight. Molecular weights were confirmed by MALS detection (table 2). Subsequently, the variants with the combination of stability mutations were tested for thermostability by DSC (D). The uncleaved S variant shown in panel D was compared with the semi-stable uncleaved variant with biproline at positions 986 and 987 (SEQ ID NO:3, dashed line). Variants with 4 or 5 mutations with or without a folder show a single Tm at 66 ℃.

Example 8: freeze-thaw stability of SARS-2S variants

Purified SARS-2S trimer (fig. 11, table 3) with indicated mutations was tested for freeze-thaw (F/T) stability using analytical SEC to measure the remaining amount of trimer after each F/T step. Proteins were diluted to 0.32mg/ml in Tris buffer (20mM Tris,150mM NaCl,pH7.4) without cryoprotectant and snap frozen with liquid nitrogen and thawed one (1 x) or more times (3 x, 5 x). Semi-stable variants with K986p+v987p were almost completely lost after 3F/T, but stable variants survived better under these conditions (fig. 11, table 3).

Example 9: stabilization of antigenicity of SARS-2S variants

RBD exposure is characterized by ACE2, by the neutralizing antibody SAD-S35 competing with ACE2 and the non-neutralizing antibody CR3022 (Yuan et al, (2020)). ACE2 and SAD-S35 can only bind RBDs in the up configuration, and CR3022 can only bind when 2 RBDs are in the up configuration (fig. 12A). The variant with K986P showed higher binding to SAD-S35, ACE2 and CR3022 compared to furin KO+PP (SEQ ID NO: 3), consistent with the results obtained with SEC and AlphaLISA in the supernatant, indicating that it causes more RBD exposure and thus more trimer opening. D614N and T572I showed very low binding to SAD-S35 and ACE2 compared to S-2P, and little CR3022 binding, indicating a more closed trimer with some 1 up configuration, but little any 2 up or 3 up RBD up configuration. A892P improved trimer closure to a lesser extent than the control, while a942 appeared to increase its patency. These mutants may exhibit a mixture of closed, 1 up and 2 up structures (fig. 12B). When the stability mutations were combined, ACE2-Fc and antibody showed lower binding compared to furin KO+PP (SEQ ID NO: 3), indicating that the trimer was predominantly in the closed conformation (FIG. 12C). Next, the storage stability of furin KO+PP at 4 degrees was tested using a biofilm interferometry technique at endpoint equilibrium compared to the stable variant (A892P-A942P-D614N-V987P-DF). ACE2-Fc binding and CR3022 binding were comparable to freshly purified material, indicating that the stabilized protein was still in a closed conformation. The strongly neutralizing monoclonal antibody S309 binding to RBD in the closed conformation was shown to bind to both proteins (Pinto et al 2020) (FIG. 12D). The difference in CR3022 and S309 in combination with the stable, fold-free S trimer suggests a well-folded RBD in the down (down) configuration.

Example 10: a942P increases S expression of FL S

Different Full Length (FL) S proteins were produced in Expi292F cells. COR200660 (660) has a furin knockout mutation, i.e. an amino acid at position 682 is mutated to S and/or an amino acid at position 685 is mutated to G. COR200662 (662) is identical to 660 but has an additional a942P substitution. COR200007 (007) has the furin knockout mutation and the additional mutations K986P and V987P. COR200664 is identical to 007 but has an additional a942P substitution.

The higher amount of spike detected in the supernatant when a942P was present (as shown in fig. 13) compared to when a942P was not present, indicating that a942P increased the expression of Full Length (FL) S protein, whereas the amount of β actin of the cell itself was not altered by a942P substitution. SDS-PAGE was applied to whole cell lysates, followed by Western blotting, showing that A942P increased expression of full-length S protein by about 1.4 fold (data not shown).

Example 11: antigenicity of stably substituted membrane bound S

The antigenicity of the seven membrane-bound S proteins encoded by the different DNA constructs was evaluated in a cell-based ELISA (CBE) as described below. Binding to three neutralizing ligands, angiotensin converting enzyme 2 (ACE 2-Fc) (Liu et al (2017)), and two monoclonal antibodies (mabs) COVA1-22 and COVA2-15 (Brouwer et al 2020) and two non-neutralizing mabs, CR3015 (van den Brink et al (2005)) and CR3046 (Bos et al (2020)) were evaluated. Substitution of D614N (e.g., D614G), a892P, and T572I reduced binding of CR3015 and CR3046 in the membrane compared to wild-type spikes, indicating that these substitutions stabilized the spikes. A892P and T572I reduced the binding of CR3015 and CR3046 with or without D614G (fig. 14).

Cell-based ELISA method

HEK293 cells were grown at 2X 10 ⁵ Each cell/mL was inoculated in an appropriate medium in a flat bottom 96-well microtiter plate (Corning). Plates were incubated overnight at 37 ℃ in 10% CO 2. After 24 hours, cell transfection was performed with 300ng DNA/well and the plates were incubated in 5% CO2 for 48 hours at 37 ℃. Two days after transfection, cells were washed with 100. Mu.l/well of blocking buffer containing 1% (w/v) BSA (Sigma), 1mM MgCl2,1.8mM CaCl2 and 5mM Tris (pH 8.0) in 1 XPBS (Ji Buke company (GIBCO)). After washing, non-specific binding was blocked at 4℃using 100. Mu.l/well of blocking solution20 minutes. Subsequently, the cells were incubated in 50. Mu.l/well blocking buffer containing 1. Mu.g/ml primary antibodies ACE2-Fc, COVA1-22, COVA2-15, CR3015 and CR3046 for 1 hour at 4 ℃. Plates were washed three times with 100. Mu.l/well of blocking buffer, three times with 100. Mu.l/well of washing buffer containing 1mM MgCl2,1.8mM CaCl2 in 1 XPBS, and then incubated with 100. Mu.l/well of blocking buffer for 5 minutes at 4 ℃. After blocking, cells were incubated with 50 μl/well of secondary anti-HRP conjugated mouse anti-human IgG (Jackson, 1:2500) or HRP conjugated goat anti-mouse IgG (Jackson, 1:2500) followed by 40 minutes at 4 ℃. Plates were washed three times with 100. Mu.l/well blocking buffer and three times with 100. Mu.l/well washing buffer. 30 μl/well of BM chemiluminescent ELISA substrate (Roche, 1:50) was added to the plate and the luminosity was measured immediately using an Ensight plate reader.

Example 12: three soluble proteins were tested for in vitro antigenicity in mice for immunogenicity.

The antigenicity of COR200617, COR200619 and COR201225 is assessed with a set of neutralizing antibodies (4 A8 (Chi et al, (2020))) against the N-terminal domain (NTD) and several antibodies binding to both up and down RBD or only up-configured RBD. See Henderson et al, (2020) for definitions of "up" and "down". The following antibodies were directed against the closed conformation of the spike and recognized the up and down states: COVA1-22 (Brouwers et al, (2020)) for RBD in the "up" or "down" position (S2M 11 (tortortorice et al, (2020)), C144 (Barnes et al, (2020)), 2-43 (Liu et al, (2020)) and COVA2-15 Brouwers et al, (2020)) the following antibodies directed against RBD in the open conformation of the spike and recognized an upward state (ACE 2-Fc and S35 (AcroBiosystems, inc.) as described below, as shown in fig. 14, non-neutralizing antibodies CR3022 (Yuan et al, (2020)), CR3015 and CR3046 do not bind to the pre-fusion conformation CR3022 only when 2 RBDs are in the up configuration, but do not bind to COR200619 and COR201225, indicating that COR200627 loses (part of) its pre-fusion conformation, whereas COR200619 and COR201225 bind to RBD in the "up" position "more closely to COR 24 than to RBD 35 and COR 8232 in the more than to COR 82352, thereby indicating that the more binding to the lower position of COR 82352 than to COR 82324.

Antibodies were immobilized on anti-hIgG (AHC) sensors (Ford biosystems, catalog No. 18-5060) in 1 Xkinetic buffer (Ford biosystems, catalog No. 18-1092) in 96-well black flat bottom polypropylene microwell plates (Fort Bio, catalog No. 3694). Experiments were performed on an Octet RED384 instrument (Pall-forte Bio) at 30℃with an oscillation speed of 1000 rpm. Activation for 600S, antibody immobilization for 900S, followed by washing for 600S, and then binding to protein S300S. Data analysis was performed using ford biosome data analysis 12.0 software (ford biosome).

Example 13: COR200619 and COR201225 are immunogenic in mice

In this example, the in vivo immunogenicity of the recombinant SARS-CoV-2 spike protein of the invention was evaluated. Production of SARS-CoV-2S protein stabilized in a predominantly closed conformation: COR201225 and COR200619. Immunogenicity was compared to a stable SARS-CoV-2 spike protein (COR 200627) with an open conformation.

Thus spike protein variants appear from the most closed (top) to the most open variant (bottom). All constructs had furin knockout mutations (R682S R685G). In addition, the constructs contained the stability mutations shown in the following table:

Groups of 7 female BALB/c mice (8-10 weeks of age at study initiation) were immunized intramuscularly with 5 or 0.5 μ g S protein containing 100 μg of aluminum hydroxide adjuvant on day 0 and day 28. Mice were bled on day 27 and day 41 to analyze neutralizing antibody responses against SARS-CoV-2b.1 (Wuhan-Hu-1+d 614 g) spike protein by pseudovirion neutralization assay (psVNA) or to SARS-CoV-2L-0008 isolate (lineage b.1) by wild-type VNA (wtVNA). For details of the assays used see Solforosi et al.

Results

The neutralizing antibody titer against SARS-CoV-2 isolate L-0008 (lineage b.1) was undetectable or near LLOD four weeks after immunization with a dose of COR200627 (open conformation) (day 27), as measured by wild-type virus neutralization assay (wtVNA) (fig. 16). In contrast, COR201225 and COR200619 (predominantly closed) elicit neutralizing antibodies against SARS-CoV-2 isolate L-0008 in a dose-dependent manner four weeks after immunization. This difference between COR200627 (open conformation) and COR201225 and COR200619 (predominantly closed) was also observed in pseudo-virion neutralization (psVNA) assays against SARS-CoV-2b.1 spike protein. In this assay, COR200627 (open conformation) did not induce neutralizing antibody titers against SARS-CoV-2b.1 spike protein, as measured by psVNA, whereas COR201225 and COR200619 (predominantly closed conformation) promoted neutralizing antibody titers in a dose-dependent manner (fig. 16). Two weeks after week 6 (day 41) the second dose, detectable neutralization titers against SARS-CoV-2b.1 spike protein were observed by psVNA for all constructs. However, higher neutralization titers were observed in mice immunized with COR201225 and COR200619 (predominantly closed conformation) compared to COR200627 (open conformation).

Conclusion(s)

According to the present invention, COR201225 and COR200619 (mainly closed and containing a stability mutation according to the present invention) have therefore been shown to be immunogenic in mice and induce higher neutralizing antibody levels against SARS-CoV-2 4 and 6 weeks after immunization compared to COR200627 (open conformation).

Example 14: stabilization without K986P and V987P

As shown in the previous examples, substitution of A942P, A944P, A892P and F880C-G888C significantly increased the trimer yield of soluble S protein without K986P and V987P (FIGS. 4 and 6 bottom panels). A892P, T572I and D614N significantly reduced binding of non-neutralizing antibodies to membrane-bound S without any further modification, indicating that S trimers are more stable (fig. 14). As shown by analytical SEC on cell culture supernatants, the trimer yields obtained by combining the substitutions N532P, T572I, D614G, F880C-G888C and A944P (in COR 201291) in addition to K986P or V987P were much higher compared to the trimer yields measured for the S-2P protein alone (COR 200017) (FIG. 17). S-2P has a fold for trimerization, whereas COR201291 does not and therefore also produces some monomers. These examples demonstrate that the mutations of the invention that increase trimer expression or stabilize trimers are independent of and superior to the previously described substitutions of K986P and V987P.

Example 15: frozen electron microscope structure of Wuhan-Hu-01 quadruple mutant d614n+a892p+a942p+v987p with folder

Four stability mutations D614N, A892P, A942P and V987P were introduced in the Wuhan-Hu-01 sequence. The quadruple mutant was then imaged by cryoelectron microscopy. The 2-step 3D classification indicated that of 833,000 classified particles, about 80% were closed, all RBDs were in the down state, 38% were classified as well-defined closed categories, and about 20% showed 1 RBD up (fig. 18). Further processing of 320,000 closed conformation particles allows us to obtain a closed conformationElectron potential and 1 RBD up (one up) conformation +.>Electron potential diagram (fig. 19). Is constructed as->Atomic models of electron potential patterns confirm that S maintains the pre-fusion spike conformation. The definition of NTD and RBD densities is smaller than the rest of the graph, indicating flexibility in these areas. The closed configuration is very similar to that previously addressed by Walls et al (Walls, park et al 2020). All atoms RMSD of these two structures differ +.>And there is no significant difference in the relative positions of the domains or in the domain conformation. Compared to the closed trimer described by Walls et al, RBDThe definition is relatively more definite and the NTD is less definite. The stability mutation does not significantly affect the backbone conformation of the closed trimer. Viral spikes are mostly closed and are very similar in structure to other known closed S-2P spike conformations (Walls, park et al 2020, xiong, qu et al 2020), particularly the closed wild type structure of Xiong et al (Xiong, qu et al 2020), where all atoms RMSD are- >

Preparation of grid of frozen electron microscope and data collection

SARS-CoV-2S protein samples were prepared in 20mM Tris,150mM NaCl,pH7 buffer at a concentration of 0.15mg/mL and applied to a glow discharge Quantifoil R2/2 mesh grid, then double sided blotted in Vitrobot Mark IV (Siemens technologies Co. (Thermo Fisher Scientific)) for 3 seconds and placed in chilled liquid ethane for freezing. The grids were loaded into a Titan Krios electron microscope (Siemens Feishier technologies Co.) operated at 300kV, equipped with a Gatan K3 BioQuantum direct electron detector. In two microscopes at the netherlands electron nano-microscopy center (The Netherlands Centre for Electron Nanoscopy, neCEN), 9,760 pictures (movie) were collected in total. The detailed data acquisition parameters are summarized in table 1.

Image processing for frozen electron microscope

The collected pictures were imported into RELION-3.1-beta (Zivanov, nakane et al 2018) and beam induced drift correction (beaminduced drift correction) was performed using MotionCor2 (Zheng, palovcak et al 2017) and CTF estimation was performed by CTFFIND-4.1.18 (Rohou and Grigoriieff 2015). The detailed steps of the image processing workflow are shown in fig. 17. The final reconstructed image is sharpened and locally filtered in RELION post-processing.

Model construction and optimization

SARS-CoV-2S PDBID 6VX and 6VSB structures (Walls, park et al 2020, wrapp, wang et al 2020) were used as starting models. PHENIX-1.18.261 (Liebschner, afonine et al 2019), coot (Emsley, lohkamp et al 2010), and Namdiator Web Server (Kidmose, juhl et al 2019) iterate to build an atomic model. The geometry and statistics are given in tables 4 and 5. The final map was displayed using UCSF ChimeraX (Goddard, huang et al 2018).

TABLE 1 Standard amino acids, abbreviations and Properties

Amino acids	Three letters	Single letter	Polarity of side chain	Side chain charge (pH 7.4)
					Alanine (Ala)	Ala	A	Nonpolar material	Neutral
Arginine (Arg)	Arg	R	Polarity of	Positive direction
					Asparagine derivatives	Asn	N	Polarity of	Neutral
Aspartic acid	Asp	D	Polarity of	Negative pole
					Cysteine (S)	Cys	C	Nonpolar material	Neutral
Glutamic acid	Glu	E	Polarity of	Negative pole
					Glutamine	Gln	Q	Polarity of	Neutral
Glycine (Gly)	Gly	G	Nonpolar material	Neutral
					Histidine	His	H	Polarity of	Positive (10%) neutral (90%)
Isoleucine (Ile)	Ile	I	Nonpolar material	Neutral
					Leucine (leucine)	Leu	L	Nonpolar material	Neutral
Lysine	Lys	K	Polarity of	Positive direction
					Methionine	Met	M	Nonpolar material	Neutral
Phenylalanine (Phe)	Phe	F	Nonpolar material	Neutral
					Proline (proline)	Pro	P	Nonpolar material	Neutral
Serine (serine)	Ser	S	Polarity of	Neutral
					Threonine (Thr)	Thr	T	Polarity of	Neutral
Tryptophan	Trp	W	Nonpolar material	Neutral
					Tyrosine	Tyr	Y	Polarity of	Neutral
Valine (valine)	Val	V	Nonpolar material	Neutral

TABLE 2 SEC-MALS analysis

TABLE 3S trimer content (%)

TABLE 4 data collection for cryoelectron microscopy

TABLE 5 model optimization and validation statistics

Reference to the literature

Belouzard et al.(2009),Proc Natl Acad Sci U S A 106:5871-6.

Bosch et al.(2008),J Virol 82:8887-90.

Follis et al.(2006)Virology 350:358-69.

Madu et al.(2009),J Virol 83:7411-21.

Walls et al.(2016),Nature 531:114-7.

Wrapp et.al.(2020)Science 367(6482):1260-1263.

Hoffmann et al.(2020)BioRxiv:doi:https://doi.org/10.1101/2020.01.31.929042

Bestle et al(2020)BioRxiv doi:https://doi.org/10.1101/2020.04.15.042085

Hastie et al.(2017),Science 356,923-928.

Krarup et al(2015),Nat Commun 6,8143.

Pallesen et al.(2017),Proc Natl Acad Sci USA 114,E7348-E7357.

Rutten et al.(2020),Cell Rep.30(13):4540-4550.

Letarov et al.(1993),Biochemistry Moscow 64:817-823.

S-Guthe et al.(2004),J.Mol.Biol.337:905-915

Pinto et al.(2020),Nature,May 18 ^th

Liu et al.,(2017),10.1016/j.kint.2018.01.029

Brouwers et al.,(2020),10.1126/science.abc5902

van den Brink et al.,(2005)10.1128/JVI.79.3.1635-1644.2005

Bos et al.,(2020)10.1038/s41541-020-00243-x

Chi(2020),10.1126/science.abc6952

Tortorice(2020),10.1126/science.abe3354

Barnes(2020)10.1038/s41586-020-2852-1

Liu et al.,(2020)10.1038/s41586-020-2571-7

Yuan et al,(2020)10.1126/science.abb7269

Solforosi et al.,J Exp Med.2021Jul 5；218(7):e20202756.doi:10.1084/jem.20202756.

Henderson et al.,doi:10.1038/s41594-020-0479-4.

Emsley et al.,(2010)Acta Crystallogr D Biol Crystallogr 66(Pt 4):486-501.

Goddard et al.,Protein Sci 27(1):14-25.

Kidmose et al.,(2019)IUCrJ 6(Pt 4):526-531.

Liebschner et al.,(2019)Acta Crystallogr D Struct Biol 75(Pt 10):861-877.

Rohou,A.and N.Grigorieff(2015)J Struct Biol 192(2):216-221.

Walls et al.,(2020)Cell 181(2):281-292 e286.

Wrapp et al.,(2020)Science 367(6483):1260-1263.

Xiong et al.,(2020)"Athermostable,closed SARS-CoV-2 spike protein trimer."Nat Struct Mol Biol.

Zheng et al.,(2017)Nat Methods 14(4):331-332.

Zivanov et al.,(2018)New tools for automated high-resolution cryo-EM structure determination in RELION-3 Elife 7.

Sequence(s)

SEQ ID NO 1: full-length S protein (underlined Signal peptide, double underlined TM and cytoplasmic domain deleted in soluble form)

SEQ ID NO 2: soluble S protein with furin KO, underlined signal peptide, double underlined linker, folder, tag, etc.

SEQ ID NO 3: a soluble S protein having furin KO and having biproline in the hinge loop. (underlined Signal peptide) double underlined linker, fold, tag, etc

SEQ ID NO 4: folding son

GYIPEAPRDGQAYVRKDGEWVLLSTFL

SEQ ID NO 5:SEQ ID NO 2+A942P

SEQ ID NO 6：SEQ ID NO 2+A892P

SEQ ID NO 7：SEQ ID NO 2+D614N

SEQ ID NO 8：SEQ ID NO 2+T572I

SEQ ID NO 9：SEQ ID NO 2+N532P

SEQ ID NO 10：SEQ ID NO 2+G880C+F888C

SEQ ID NO 11：SEQ ID NO 2+S884C+A893C

SEQ ID NO 12：SEQ ID NO 3+A942P

SEQ ID NO 13：SEQ ID NO 3+A892P

SEQ ID NO 14：SEQ ID NO 3+D614N

SEQ ID NO 15：SEQ ID NO 3+T572I

SEQ ID NO 16：SEQ ID NO 3+N532P

SEQ ID NO 17：SEQ ID NO 3+G880C+F888C

SEQ ID NO 18：SEQ ID NO 3+S884C+A893C

SEQ ID NO 19：SEQ ID NO 2+A942P+A892P

SEQ ID NO 20：SEQ ID NO 2+A942P+D614N

SEQ ID NO 21：SEQ ID NO 2+A942P+T572I

SEQ ID NO 22：SEQ ID NO 2+A942P+N532P

SEQ ID NO 23：SEQ ID NO 2+A942P+G880C+F888C

SEQ ID NO 24：SEQ ID NO 2+A942P+S884C+A893C

SEQ ID NO 25：SEQ ID NO 2+A892P+D614N

SEQ ID NO 26：SEQ ID NO 2+A892P+T572I

SEQ ID NO 27：SEQ ID NO 2+A892P+N532P

SEQ ID NO 28：SEQ ID NO 2+A892P+G880C+F888C

SEQ ID NO 29：SEQ ID NO 2+A892P+S884C+A893C

SEQ ID NO 30：SEQ ID NO 2+D614N+T572I

SEQ ID NO 31：SEQ ID NO 2+D614N+N532P

SEQ ID NO 32：SEQ ID NO 2+D614N+G880C+F888C

SEQ ID NO 33：SEQ ID NO 2+D614N+S884C+A893C

SEQ ID NO 34：SEQ ID NO 2+T572I+N532P

SEQ ID NO 35：SEQ ID NO 2+T572I+G880C+F888C

SEQ ID NO 36：SEQ ID NO 2+T572I+S884C+A893C

SEQ ID NO 37：SEQ ID NO 2+N532P+G880C+F888C

SEQ ID NO 38：SEQ ID NO 2+N532P+S884C+A893C

SEQ ID NO 39：SEQ ID NO 2+A942P+A892P+D614N

SEQ ID NO 40：SEQ ID NO 2+A942P+A892P+T572I

SEQ ID NO 41：SEQ ID NO 2+A942P+A892P+N532P

SEQ ID NO 42：SEQ ID NO 2+A942P+A892P+G880C+F888C

SEQ ID NO 43：SEQ ID NO 2+A942P+A892P+S884C+A893C

SEQ ID NO 44：SEQ ID NO 2+A942P+D614N+T572I

SEQ ID NO 45：SEQ ID NO 2+A942P+D614N+N532P

SEQ ID NO 46：SEQ ID NO 2+A942P+D614N+G880C+F888C

SEQ ID NO 47：SEQ ID NO 2+A942P+D614N+S884C+A893C

SEQ ID NO 48：SEQ ID NO 2+A942P+T572I+N532P

SEQ ID NO 49：SEQ ID NO 2+A942P+T572I+G880C+F888C

SEQ ID NO 50：SEQ ID NO 2+A942P+T572I+S884C+A893C

SEQ ID NO 51：SEQ ID NO 2+A942P+N532P+G880C+F888C

SEQ ID NO 52：SEQ ID NO 2+A942P+N532P+S884C+A893C

SEQ ID NO 53：SEQ ID NO 2+A892P+D614N+T572I

SEQ ID NO 54：SEQ ID NO 2+A892P+D614N+N532P

SEQ ID NO 55：SEQ ID NO 2+A892P+D614N+G880C+F888C

SEQ ID NO 56：SEQ ID NO 2+A892P+D614N+S884C+A893C

SEQ ID NO 57：SEQ ID NO 2+A892P+T572I+N532P

SEQ ID NO 58：SEQ ID NO 2+A892P+T572I+G880C+F888C

SEQ ID NO 59：SEQ ID NO 2+A892P+T572I+S884C+A893C

SEQ ID NO 60：SEQ ID NO 2+A892P+N532P+G880C+F888C

SEQ ID NO 61：SEQ ID NO 2+A892P+N532P+S884C+A893C

SEQ ID NO 62：SEQ ID NO 2+D614N+T572I+N532P

SEQ ID NO 63：SEQ ID NO 2+D614N+T572I+G880C+F888C

SEQ ID NO 64：SEQ ID NO 2+D614N+T572I+S884C+A893C

SEQ ID NO 65：SEQ ID NO 2+D614N+N532P+G880C+F888C

SEQ ID NO 66：SEQ ID NO 2+D614N+N532P+S884C+A893C

SEQ ID NO 67：SEQ ID NO 2+T572I+N532P+G880C+F888C

SEQ ID NO 68：SEQ ID NO 2+T572I+N532P+S884C+A893C

SEQ ID NO 69：SEQ ID NO 2+A942P+A892P+D614N+T572I

SEQ ID NO 70：SEQ ID NO 2+A942P+A892P+D614N+N532P

SEQ ID NO 71：SEQ ID NO 2+A942P+A892P+D614N+G880C+F888C

SEQ ID NO 72：SEQ ID NO 2+A942P+A892P+D614N+S884C+A893C

SEQ ID NO 73：SEQ ID NO 2+A942P+A892P+T572I+N532P

SEQ ID NO 74：SEQ ID NO 2+A942P+A892P+T572I+G880C+F888C

SEQ ID NO 75：SEQ ID NO 2+A942P+A892P+T572I+S884C+A893C

SEQ ID NO 76：SEQ ID NO 2+A942P+A892P+N532P+G880C+F888C

SEQ ID NO 77：SEQ ID NO 2+A942P+A892P+N532P+S884C+A893C

SEQ ID NO78：SEQ ID NO 2+A942P+D614N+T572I+N532P

SEQ ID NO 79：SEQ ID NO 2+A942P+D614N+T572I+G880C+F888C

SEQ ID NO 80：SEQ ID NO 2+A942P+D614N+T572I+S884C+A893C

SEQ ID NO 81：SEQ ID NO 2+A942P+D614N+N532P+G880C+F888C

SEQ ID NO 82：SEQ ID NO 2+A942P+D614N+N532P+S884C+A893C

SEQ ID NO 83：SEQ ID NO 2+A942P+T572I+N532P+G880C+F888C

SEQ ID NO 84：SEQ ID NO 2+A942P+T572I+N532P+S884C+A893C

SEQ ID NO 85：SEQ ID NO 2+A892P+D614N+T572I+N532P

SEQ ID NO 86：SEQ ID NO 2+A892P+D614N+T572I+G880C+F888C

SEQ ID NO 87：SEQ ID NO 2+A892P+D614N+T572I+S884C+A893C

SEQ ID NO 88：SEQ ID NO 2+A892P+D614N+N532P+G880C+F888C

SEQ ID NO 89：SEQ ID NO 2+A892P+D614N+N532P+S884C+A893C

SEQ ID NO 90：SEQ ID NO 2+A892P+T572I+N532P+G880C+F888C

SEQ ID NO 91：SEQ ID NO 2+A892P+T572I+N532P+S884C+A893C

SEQ ID NO 92：SEQ ID NO 2+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 93：SEQ ID NO 2+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 94：SEQ ID NO 2+A942P+A892P+D614N+T572I+N532P

SEQ ID NO 95：SEQ ID NO 2+A942P+A892P+D614N+T572I+G880C+F888C

SEQ ID NO 96：SEQ ID NO 2+A942P+A892P+D614N+T572I+S884C+A893C

SEQ ID NO 97：SEQ ID NO 2+A942P+A892P+D614N+N532P+G880C+F888C

SEQ ID NO 98：SEQ ID NO 2+A942P+A892P+D614N+N532P+S884C+A893C

SEQ ID NO 99：SEQ ID NO 2+A942P+A892P+T572I+N532P+G880C+F888C

SEQ ID NO 100：SEQ ID NO 2+A942P+A892P+T572I+N532P+S884C+A893C

SEQ ID NO 101：SEQ ID NO 2+A942P+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 102：SEQ ID NO 2+A942P+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 103：SEQ ID NO 2+A892P+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 104：SEQ ID NO 2+A892P+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 105：SEQ ID NO 2+A942P+A892P+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 106：SEQ ID NO 2+A942P+A892P+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 107：SEQ ID NO 3+A942P+A892P

SEQ ID NO 108：SEQ ID NO 3+A942P+D614N

SEQ ID NO 109：SEQ ID NO3+A942P+T572I

SEQ ID NO 110：SEQ ID NO 3+A942P+N532P

SEQ ID NO111：SEQ ID NO 3+A942P+G880C+F888C

SEQ ID NO 112：SEQ ID NO 3+A942P+S884C+A893C

SEQ ID NO 113：SEQ ID NO 3+A892P+D614N

SEQ ID NO 114：SEQ ID NO 3+A892P+T572I

SEQ ID NO 115：SEQ ID NO 3+A892P+N532P

SEQ ID NO 116：SEQ ID NO 3+A892P+G880C+F888C

SEQ ID NO 117：SEQ ID NO 3+A892P+S884C+A893C

SEQ ID NO 118：SEQ ID NO 3+D614N+T572I

SEQ ID NO 119：SEQ ID NO 3+D614N+N532P

SEQ ID NO 120：SEQ ID NO 3+D614N+G880C+F888C

SEQ ID NO121：SEQ ID NO 3+D614N+S884C+A893C

SEQ ID NO 122：SEQ ID NO 3+T572I+N532P

SEQ ID NO 123：SEQ ID NO 3+T572I+G880C+F888C

SEQ ID NO 124：SEQ ID NO 3+T572I+S884C+A893C

SEQ ID NO 125：SEQ ID NO 3+N532P+G880C+F888C

SEQ ID NO 126：SEQ ID NO 3+N532P+S884C+A893C

SEQ ID NO 127：SEQ ID NO 3+A942P+A892P+D614N

SEQ ID NO 128：SEQ ID NO 3+A942P+A892P+T572I

SEQ ID NO 129：SEQ ID NO 3+A942P+A892P+N532P

SEQ ID NO 130：SEQ ID NO 3+A942P+A892P+G880C+F888C

SEQ ID NO 131：SEQ ID NO 3+A942P+A892P+S884C+A893C

SEQ ID NO 132：SEQ ID NO 3+A942P+D614N+T572I

SEQ ID NO 133：SEQ ID NO 3+A942P+D614N+N532P

SEQ ID NO 134：SEQ ID NO 3+A942P+D614N+G880C+F888C

SEQ ID NO 135：SEQ ID NO 3+A942P+D614N+S884C+A893C

SEQ ID NO 136：SEQ ID NO 3+A942P+T572I+N532P

SEQ ID NO 137：SEQ ID NO 3+A942P+T572I+G880C+F888C

SEQ ID NO 138：SEQ ID NO 3+A942P+T572I+S884C+A893C

SEQ ID NO 139：SEQ ID NO 3+A942P+N532P+G880C+F888C

SEQ ID NO 140：SEQ ID NO 3+A942P+N532P+S884C+A893C

SEQ ID NO 141：SEQ ID NO 3+A892P+D614N+T572I

SEQ ID NO 142：SEQ ID NO 3+A892P+D614N+N532P

SEQ ID NO 143：SEQ ID NO 3+A892P+D614N+G880C+F888C

SEQ ID NO144：SEQ ID NO 3+A892P+D614N+S884C+A893C

SEQ ID NO 145：SEQ ID NO 3+A892P+T572I+N532P

SEQ ID NO 146：SEQ ID NO 3+A892P+T572I+G880C+F888C

SEQ ID NO 147：SEQ ID NO 3+A892P+T572I+S884C+A893C

SEQ ID NO 148：SEQ ID NO 3+A892P+N532P+G880C+F888C

SEQ ID NO 149：SEQ ID NO 3+A892P+N532P+S884C+A893C

SEQ ID NO 150：SEQ ID NO 3+D614N+T572I+N532P

SEQ ID NO 151：SEQ ID NO 3+D614N+T572I+G880C+F888C

SEQ ID NO 152：SEQ ID NO 3+D614N+T572I+S884C+A893C

SEQ ID NO 153：SEQ ID NO 3+D614N+N532P+G880C+F888C

SEQ ID NO 154：SEQ ID NO 3+D614N+N532P+S884C+A893C

SEQ ID NO 155：SEQ ID NO 3+T572I+N532P+G880C+F888C

SEQ ID NO 156：SEQ ID NO 3+T572I+N532P+S884C+A893C

SEQ ID NO 157：SEQ ID NO 3+A942P+A892P+D614N+T572I

SEQ ID NO 158：SEQ ID NO 3+A942P+A892P+D614N+N532P

SEQ ID NO 159：SEQ ID NO 3+A942P+A892P+D614N+G880C+F888C

SEQ ID NO 160：SEQ ID NO 3+A942P+A892P+D614N+S884C+A893C

SEQ ID NO 161：SEQ ID NO 3+A942P+A892P+T572I+N532P

SEQ ID NO 162：SEQ ID NO 3+A942P+A892P+T572I+G880C+F888C

SEQ ID NO 163：SEQ ID NO 3+A942P+A892P+T572I+S884C+A893C

SEQ ID NO 164：SEQ ID NO 3+A942P+A892P+N532P+G880C+F888C

SEQ ID NO 165：SEQ ID NO 3+A942P+A892P+N532P+S884C+A893C

SEQ ID NO 166：SEQ ID NO 3+A942P+D614N+T572I+N532P

SEQ ID NO 167：SEQ ID NO 3+A942P+D614N+T572I+G880C+F888C

SEQ ID NO 168：SEQ ID NO 3+A942P+D614N+T572I+S884C+A893C

SEQ ID NO 169：SEQ ID NO 3+A942P+D614N+N532P+G880C+F888C

SEQ ID NO 170：SEQ ID NO 3+A942P+D614N+N532P+S884C+A893C

SEQ ID NO 171：SEQ ID NO 3+A942P+T572I+N532P+G880C+F888C

SEQ ID NO 172：SEQ ID NO 3+A942P+T572I+N532P+S884C+A893C

SEQ ID NO 173：SEQ ID NO 3+A892P+D614N+T572I+N532P

SEQ ID NO 174：SEQ ID NO 3+A892P+D614N+T572I+G880C+F888C

SEQ ID NO 175：SEQ ID NO 3+A892P+D614N+T572I+S884C+A893C

SEQ ID NO 176：SEQ ID NO 3+A892P+D614N+N532P+G880C+F888C

SEQ ID NO177：SEQ ID NO 3+A892P+D614N+N532P+S884C+A893C

SEQ ID NO 178：SEQ ID NO 3+A892P+T572I+N532P+G880C+F888C

SEQ ID NO 179：SEQ ID NO 3+A892P+T572I+N532P+S884C+A893C

SEQ ID NO 180：SEQ ID NO 3+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 181：SEQ ID NO 3+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 182：SEQ ID NO 3+A942P+A892P+D614N+T572I+N532P

SEQ ID NO 183：SEQ ID NO 3+A942P+A892P+D614N+T572I+G880C+F888C

SEQ ID NO 184：SEQ ID NO 3+A942P+A892P+D614N+T572I+S884C+A893C

SEQ ID NO 185：SEQ ID NO 3+A942P+A892P+D614N+N532P+G880C+F888C

SEQ ID NO 186：SEQ ID NO 3+A942P+A892P+D614N+N532P+S884C+A893C

SEQ ID NO 187：SEQ ID NO 3+A942P+A892P+T572I+N532P+G880C+F888C

SEQ ID NO188：SEQ ID NO 3+A942P+A892P+T572I+N532P+S884C+A893C

SEQ ID NO 189：SEQ ID NO 3+A942P+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 190：SEQ ID NO 3+A942P+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 191：SEQ ID NO 3+A892P+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 192：SEQ ID NO 3+A892P+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 193：SEQ ID NO 3+A942P+A892P+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 194：SEQ ID NO 3+A942P+A892P+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 195: full-length S protein with biproline in hinge loops

SEQ ID NO 196: full-length S protein with furin KO

SEQ ID NO 197：SEQ ID NO 196+D614N+A892P+A942P

SEQ ID NO 198：SEQ ID NO 196+D614N+A892P+A942P+T941P

SEQ ID NO 199：SEQ ID NO 196+D614N+A892P+A942P+A944P

SEQ ID NO 200：SEQ ID NO 196+D614N+A892P+T941P

SEQ ID NO 201：SEQ ID NO 196+D614N+A892P+A944P

/>

SEQ ID NO 202：SEQ ID NO 196+D614N+A892P+A942P+V987P

SEQ ID NO 203：SEQ ID NO 196+D614N+A892P+A942P+T941P+V987P

SEQ ID NO 204：SEQ ID NO 196+D614N+A892P+A942P+A944P+V987P

SEQ ID NO 205：SEQ ID NO 196+D614N+A892P+T941P+V987P

SEQ ID NO 206：SEQ ID NO 196+D614N+A892P+A944P+V987P

SEQ ID NO 207：SEQ ID NO 196+D614N+A892P+A942P+K986P+V987P

SEQ ID NO 208：SEQ ID NO 196+D614N+A892P+A942P+T941P+K986P+V987P

SEQ ID NO 209：SEQ ID NO 196+D614N+A892P+A942P+A944P+K986P+V987P

SEQ ID NO 210：SEQ ID NO 196+D614N+A892P+T941P+K986P+V987P

SEQ ID NO 211：SEQ ID NO196+D614N+A892P+A944P+K986P+V987P

SEQ ID NO 212：SEQ ID NO 1+D614N+A892P+A942P

SEQ ID NO 213：SEQ ID NO 1+D614N+A892P+A942P+T941P

SEQ ID NO 214：SEQ ID NO 1+D614N+A892P+A942P+A944P

SEQ ID NO 215：SEQ ID NO 1+D614N+A892P+T941P

SEQ ID NO 216：SEQ ID NO 1+D614N+A892P+A944P

SEQ ID NO 217：SEQ ID NO 1+D614N+A892P+A942P+V987P

SEQ ID NO 218：SEQ ID NO1+ D614N+A892P+A942P+T941P+V987P

SEQ ID NO 219：SEQ ID NO 1+D614N+A892P+A942P+A944P+V987P

SEQ ID NO 220：SEQ ID NO 1+D614N+A892P+T941P+V987P

SEQ ID NO 221：SEQ ID NO 1+D614N+A892P+A944P+V987P

SEQ ID NO 222：SEQ ID NO 1+D614N+A892P+A942P+K986P+V987P

SEQ ID NO 223：SEQ ID NO 1+D614N+A892P+A942P+T941P+K986P+V987P

SEQ ID NO 224：SEQ ID NO 1+D614N+A892P+A942P+A944P+K986P+V987P

SEQ ID NO 225：SEQ ID NO 1+D614N+A892P+T941P+K986P+V987P

SEQ ID NO 226：SEQ ID NO 1+D614N+A892P+A944P+K986P+V987P

SEQ ID NO 227：SEQ ID NO 1+A942P

SEQ ID NO 228：SEQ ID NO 1+A892P

SEQ ID NO 229：SEQ ID NO 1+D614N

SEQ ID NO 230：SEQ ID NO1+T572I

SEQ ID NO 231：SEQ ID NO 1+N532P

SEQ ID NO 232：SEQ ID NO 1+G880C+F888C

SEQ ID NO 233：SEQ ID NO 1+S884C+A893C

SEQ ID NO 234：SEQ ID NO 1+A942P+A892P

SEQ ID NO 235：SEQ ID NO 1+A942P+D614N

SEQ ID NO 236：SEQ ID NO 1+A942P+T572I

SEQ ID NO 237：SEQ ID NO 1+A942P+N532P

SEQ ID NO 238：SEQ ID NO 1+A942P+G880C+F888C

SEQ ID NO 239：SEQ ID NO 1+A942P+S884C+A893C

SEQ ID NO 240：SEQ ID NO 1+A892P+D614N

SEQ ID NO 241：SEQ ID NO 1+A892P+T572I

SEQ ID NO 242：SEQ ID NO 1+A892P+N532P

SEQ ID NO 243：SEQ ID NO 1+A892P+G880C+F888C

SEQ ID NO 244：SEQ ID NO 1+A892P+S884C+A893C

SEQ ID NO 245：SEQ ID NO 1+D614N+T572I

SEQ ID NO 246：SEQ ID NO 1+D614N+N532P

SEQ ID NO 247：SEQ ID NO 1+D614N+G880C+F888C

SEQ ID NO 248：SEQ ID NO 1+D614N+S884C+A893C

SEQ ID NO 249：SEQ ID NO 1+T572I+N532P

SEQ ID NO 250：SEQ ID NO 1+T572I+G880C+F888C

SEQ ID NO 251：SEQ ID NO 1+T572I+S884C+A893C

SEQ ID NO 252：SEQ ID NO 1+N532P+G880C+F888C

SEQ ID NO 253：SEQ ID NO 1+N532P+S884C+A893C

SEQ ID NO 254：SEQ ID NO 1+A942P+A892P+D614N

SEQ ID NO 255：SEQ ID NO 1+A942P+892P+T572I

SEQ ID NO 256：SEQ ID NO 1+A942P+A892P+N532P

SEQ ID NO 257：SEQ ID NO 1+A942P+A892P+G880C+F888C

SEQ ID NO 258：SEQ ID NO 1+A942P+A892P+S884C+A893C

SEQ ID NO 259：SEQ ID NO 1+A942P+D614N+T572I

SEQ ID NO 260：SEQ ID NO 1+A942P+D614N+N532P

SEQ ID NO 261：SEQ ID NO 1+A942P+D614N+G880C+F888C

SEQ ID NO 262：SEQ ID NO 1+A942P+D614N+S884C+A893C

SEQ ID NO 263：SEQ ID NO 1+A942P+T572I+N532P

SEQ ID NO 264：SEQ ID NO 1+A942P+T572I+G880C+F888C

SEQ ID NO 265：SEQ ID NO 1+A942P+T572I+S884C+A893C

SEQ ID NO 266：SEQ ID NO 1+A942P+N532P+G880C+F888C

SEQ ID NO 267：SEQ ID NO 1+A942P+N532P+S884C+A893C

SEQ ID NO 268：SEQ ID NO 1+A892P+D614N+T572I

SEQ ID NO 269：SEQ ID NO 1+A892P+D614N+N532P

SEQ ID NO 270：SEQ ID NO 1+A892P+D614N+G880C+F888C

SEQ ID NO 271：SEQ ID NO 1+A892P+D614N+S884C+A893C

SEQ ID NO 272：SEQ ID NO 1+A892P+T572I+N532P

SEQ ID NO 273：SEQ ID NO 1+A892P+T572I+G880C+F888C

SEQ ID NO 274：SEQ ID NO 1+A892P+T572I+S884C+A893C

SEQ ID NO 275：SEQ ID NO 1+A892P+N532P+G880C+F888C

SEQ ID NO 276：SEQ ID NO 1+A892P+N532P+S884C+A893C

SEQ ID NO 277：SEQ ID NO 1+D614N+T572I+N532P

SEQ ID NO 278：SEQ ID NO 1+D614N+T572I+G880C+F888C

SEQ ID NO 279：SEQ ID NO 1+D614N+T572I+S884C+A893C

SEQ ID NO 280：SEQ ID NO 1+D614N+N532P+G880C+F888C

SEQ ID NO 281：SEQ ID NO 1+D614N+N532P+S884C+A893C

SEQ ID NO 282：SEQ ID NO 1+T572I+N532P+G880C+F888C

SEQ ID NO 283：SEQ ID NO 1+T572I+N532P+S884C+A893C

SEQ ID NO 284：SEQ ID NO 1+A942P+A892P+D614N+T572I

SEQ ID NO 285：SEQ ID NO 1+A942P+A892P+D614N+N532P

SEQ ID NO 286：SEQ ID NO 1+A942P+A892P+D614N+G880C+F888C

SEQ ID NO 287：SEQ ID NO 1+A942P+A892P+D614N+S884C+A893C

SEQ ID NO 288：SEQ ID NO 1+A942P+A892P+T572I+N532P

SEQ ID NO 289：SEQ ID NO 1+A942P+A892P+T572I+G880C+F888C

SEQ ID NO 290：SEQ ID NO 1+A942P+A892P+T572I+S884C+A893C

SEQ ID NO 291：SEQ ID NO 1+A942P+A892P+N532P+G880C+F888C

SEQ ID NO 292：SEQ ID NO 1+A942P+A892P+N532P+S884C+A893C

SEQ ID NO 293：SEQ ID NO 1+A942P+D614N+T572I+N532P

SEQ ID NO 294：SEQ ID NO 1+A942P+D614N+T572I+G880C+F888C

SEQ ID NO 295：SEQ ID NO 1+A942P+D614N+T572I+S884C+A893C

SEQ ID NO 296：SEQ ID NO 1+A942P+D614N+N532P+G880C+F888C

SEQ ID NO 297：SEQ ID NO 1+A942P+D614N+N532P+S884C+A893C

SEQ ID NO 298：SEQ ID NO 1+A942P+T572I+N532P+G880C+F888C

SEQ ID NO 299：SEQ ID NO 1+A942P+T572I+N532P+S884C+A893C

SEQ ID NO 300：SEQ ID NO 1+A892P+D614N+T572I+N532P

SEQ ID NO 301：SEQ ID NO 1+A892P+D614N+T572I+G880C+F888C

SEQ ID NO 302：SEQ ID NO 1+A892P+D614N+T572I+S884C+A893C

SEQ ID NO 303：SEQ ID NO 1+A892P+D614N+N532P+G880C+F888C

SEQ ID NO 304：SEQ ID NO 1+A892P+D614N+N532P+S884C+A893C

SEQ ID NO 305：SEQ ID NO 1+A892P+T572I+N532P+G880C+F888C

SEQ ID NO 306：SEQ ID NO 1+A892P+T572I+N532P+S884C+A893C

SEQ ID NO 307：SEQ ID NO 1+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 308：SEQ ID NO 1+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 309：SEQ ID NO 1+A942P+A892P+D614N+T572I+N532P

SEQ ID NO 310：SEQ ID NO 1+A942P+A892P+D614N+T572I+G880C+F888C

SEQ ID NO 311：SEQ ID NO 1+A942P+A892P+D614N+T572I+S884C+A893C

SEQ ID NO 312：SEQ ID NO 1+A942P+A892P+D614N+N532P+G880C+F888C

SEQ ID NO 313：SEQ ID NO 1+A942P+A892P+D614N+N532P+S884C+A893C

SEQ ID NO 314：SEQ ID NO 1+A942P+A892P+T572I+N532P+G880C+F888C

SEQ ID NO 315：SEQ ID NO 1+A942P+A892P+T572I+N532P+S884C+A893C

SEQ ID NO 316：SEQ ID NO1+A942P+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 317：SEQ ID NO 1+A942P+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 318：SEQ ID NO 1+A892P+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 319：SEQ ID NO 1+A892P+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 320：SEQ ID NO 1+A942P+A892P+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 321：SEQ ID NO 1+A942P+A892P+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 322：SEQ ID NO 195+A942P

SEQ ID NO 323：SEQ ID NO 195+A892P

SEQ ID NO 324：SEQ ID NO 195+D614N

SEQ ID NO 325：SEQ ID NO 195+T572I

SEQ ID NO 326：SEQ ID NO 195+N532P

SEQ ID NO 327：SEQ ID NO 195+G880C+F888C

SEQ ID NO 328：SEQ ID NO 195+S884C+A893C

SEQ ID NO 329：SEQ ID NO 195+A942P+A892P

SEQ ID NO 330：SEQ ID NO 195+A942P+D614N

SEQ ID NO 331：SEQ ID NO 195+A942P+T572I

SEQ ID NO 332：SEQ ID NO 195+A942P+N532P

SEQ ID NO 333：SEQ ID NO 195+A942P+G880C+F888C

SEQ ID NO 334：SEQ ID NO 195+A942P+S884C+A893C

SEQ ID NO 335：SEQ ID NO 195+A892P+D614N

SEQ ID NO 336：SEQ ID NO 195+A892P+T572I

SEQ ID NO 337：SEQ ID NO 195+A892P+N532P

SEQ ID NO 338：SEQ ID NO 195++A892P+G880C+F888C

SEQ ID NO 339：SEQ ID NO 195+A892P+S884C+A893C

SEQ ID NO 340：SEQ ID NO 195+D614N+T572I

SEQ ID NO 341：SEQ ID NO 195+D614N+N532P

SEQ ID NO 342：SEQ ID NO 195+D614N+G880C+F888C

SEQ ID NO 343：SEQ ID NO 195+D614N+S884C+A893C

SEQ ID NO 344：SEQ ID NO195+T572I+N532P

SEQ ID NO 345：SEQ ID NO 195+T572I+G880C+F888C

SEQ ID NO346：SEQ ID NO 195+T572I+S884C+A893C

SEQ ID NO 347：SEQ ID NO 195+N532P+G880C+F888C

SEQ ID NO 348：SEQ ID NO 195+N532P+S884C+A893C

SEQ ID NO 349：SEQ ID NO 195+A942P+A892P+D614N

SEQ ID NO 350：SEQ ID NO 195+A942P+A892P+T572I

SEQ ID NO351：SEQ ID NO 195+A942P+A892P+N532P

SEQ ID NO 352：SEQ ID NO 195+A942P+A892P+G880C+F888C

SEQ ID NO 353：SEQ ID NO 195+A942P+A892P+S884C+A893C

SEQ ID NO 354：SEQ ID NO 195+A942P+D614N+T572I

SEQ ID NO 355：SEQ ID NO 195+A942P+D614N+N532P

SEQ ID NO 356：SEQ ID NO 195+A942P+D614N+G880C+F888C

SEQ ID NO 357：SEQ ID NO 195+A942P+D614N+S884C+A893C

SEQ ID NO 358：SEQ ID NO 195+A942P+T572I+N532P

SEQ ID NO 359：SEQ ID NO 195+A942P+T572I+G880C+F888C

SEQ ID NO 360：SEQ ID NO 195+A942P+T572I+S884C+A893C

SEQ ID NO 361：SEQ ID NO 195+A942P+N532P+G880C+F888C

SEQ ID NO 362：SEQ ID NO 195+A942P+N532P+S884C+A893C

SEQ ID NO 363：SEQ ID NO1 95+A892P+D614N+T572I

SEQ ID NO 364：SEQ ID NO 195+A892P+D614N+N532P

SEQ ID NO 365：SEQ ID NO 195+A892P+D614N+G880C+F888C

SEQ ID NO 366：SEQ ID NO 195+A892P+D614N+S884C+A893C

SEQ ID NO 367：SEQ ID NO 195+A892P+T572I+N532P

SEQ ID NO368：SEQ ID NO 195+A892P+T572I+G880C+F888C

SEQ ID NO 369：SEQ ID NO 195+A892P+T572I+S884C+A893C

SEQ ID NO 370：SEQ ID NO 195+A892P+N532P+G880C+F888C

SEQ ID NO 371：SEQ ID NO 195+A892P+N532P+S884C+A893C

SEQ ID NO 372：SEQ ID NO 195+D614N+T572I+N532P

SEQ ID NO 373：SEQ ID NO 195+D614N+T572I+G880C+F888C

SEQ ID NO 374：SEQ ID NO 195+D614N+T572I+S884C+A893C

SEQ ID NO 375：SEQ ID NO 195+D614N+N532P+G880C+F888C

SEQ ID NO 376：SEQ ID NO 195+D614N+N532P+S884C+A893C

SEQ ID NO 377：SEQ ID NO 195+T572I+N532P+G880C+F888C

SEQ ID NO 378：SEQ ID NO 195+T572I+N532P+S884C+A893C

SEQ ID NO 379：SEQ ID NO 195+A942P+A892P+D614N+T572I

SEQ ID NO 380：SEQ ID NO 195+A942P+A892P+D614N+N532P

SEQ ID NO 381：SEQ ID NO 195+A942P+A892P+D614N+G880C+F888C

SEQ ID NO 382：SEQ ID NO 195+A942P+A892P+D614N+S884C+A893C

SEQ ID NO 383：SEQ ID NO 195+A942P+A892P+T572I+N532P

SEQ ID NO 384：SEQ ID NO 195+A942P+A892P+T572I+G880C+F888C

SEQ ID NO 385：SEQ ID NO 195+A942P+A892P+T572I+S884C+A893C

SEQ ID NO 386：SEQ ID NO 195+A942P+A892P+N532P+G880C+F888C

SEQ ID NO 387：SEQ ID NO 195+A942P+A892P+N532P+S884C+A893C

SEQ ID NO 388：SEQ ID NO 195+A942P+D614N+T572I+N532P

SEQ ID NO 389：SEQ ID NO 195+A942P+D614N+T572I+G880C+F888C

SEQ ID NO 390：SEQ ID NO 195+A942P+D614N+T572I+S884C+A893C

SEQ ID NO 391：SEQ ID NO 195+A942P+D614N+N532P+G880C+F888C

SEQ ID NO 392：SEQ ID NO 195+A942P+D614N+N532P+S884C+A893C

SEQ ID NO 393：SEQ ID NO 195+A942P+T572I+N532P+G880C+F888C

SEQ ID NO 394：SEQ ID NO 195+A942P+T572I+N532P+S884C+A893C

SEQ ID NO 395：SEQ ID NO 195+A892P+D614N+T572I+N532P

SEQ ID NO 396：SEQ ID NO 195+A892P+D614N+T572I+G880C+F888C

SEQ ID NO 397：SEQ ID NO 195+A892P+D614N+T572I+S884C+A893C

SEQ ID NO 398：SEQ ID NO 195+A892P+D614N+N532P+G880C+F888C

SEQ ID NO 399：SEQ ID NO 195+A892P+D614N+N532P+S884C+A893C

SEQ ID NO 400：SEQ ID NO 195+A892P+T572I+N532P+G880C+F888C

SEQ ID NO 401：SEQ ID NO 195+A892P+T572I+N532P+S884C+A893C

SEQ ID NO 402：SEQ ID NO 195+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 403：SEQ ID NO 195+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 404：SEQ ID NO 195+A942P+A892P+D614N+T572I+N532P

SEQ ID NO 405：SEQ ID NO 195+A942P+A892P+D614N+T572I+G880C+F888C

SEQ ID NO 406：SEQ ID NO 195+A942P+A892P+D614N+T572I+S884C+A893C

SEQ ID NO 407：SEQ ID NO 195+A942P+A892P+D614N+N532P+G880C+F888C

SEQ ID NO 408：SEQ ID NO 195+A942P+A892P+D614N+N532P+S884C+A893C

SEQ ID NO 409：SEQ ID NO 195+A942P+A892P+T572I+N532P+G880C+F888C

SEQ ID NO 410：SEQ ID NO 195+A942P+A892P+T572I+N532P+S884C+A893C

SEQ ID NO 411：SEQ ID NO 195+A942P+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 412：SEQ ID NO 195+A942P+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 413：SEQ ID NO 195+A892P+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 414：SEQ ID NO 195+A892P+D614N+T572I+N532P+S884C+A893C

SEQ ID NO 415：SEQ ID NO 195+A942P+A892P+D614N+T572I+N532P+G880C+F888C

SEQ ID NO 416：SEQ ID NO 195+A942P+A892P+D614N+T572I+N532P+S884C+A893C

SEQ ID NO. 417 (COR 201225) -S protein-R682S R685G N532P T572ID614GG880C F888C A944P V987P non-folding child

Signal peptides

SEQ ID NO. 418 (COR 200619) -S protein-R682S R685G A35892P A942P D NV987P non-folding subunit

Signal peptides

SEQ ID NO:419 (COR 200627) -soluble stable SARS-CoV-S S protein (furin ko+pp+ fold) has a sortase a-C tag and an a942P mutation

Signal peptides

SEQ ID NO：420(COR201291)

SEQ ID NO：421(COR200017)

/>

Claims (32)

1. A recombinant pre-fusion SARS-CoV-2S protein or fragment thereof comprising S1 and S2 domains and comprising at least one mutation selected from the group consisting of: mutations corresponding to at least one amino acid in the loop region of amino acid residues 941-945 to P, mutation of amino acid 892, mutation of amino acid 614, mutation of 572, mutation of 532, disulfide bridge between residues 880 and 888, and disulfide bridge between residues 884 and 893, wherein the numbering of these amino acid positions is according to SEQ ID NO:1, and the numbering of these amino acid positions in 1.

2. The protein or fragment thereof according to claim 1, comprising an amino acid sequence, wherein the amino acid at position 892 is not alanine (a), the amino acid at position 614 is not aspartic acid (D), the amino acid at position 532 is not asparagine (N) and/or the amino acid at position 572 is not threonine (T).

3. The protein or fragment thereof according to claim 1 or 2, comprising a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 to P, and a mutation selected from the group consisting of: mutation of the 892 oxy acid, mutation of the 614 amino acid, mutation of the 572 position, mutation of the 532 position, disulfide bridge between residues 880 and 888 and disulfide bridge between residues 884 and 893.

4. A protein or fragment thereof according to claim 3, comprising a mutation of at least one amino acid in the loop region corresponding to amino acid residues 941-945 to P, and a mutation selected from the group consisting of: mutations at amino acid 892, at amino acid 614, at 572, at 532, between residues 880 and 888 and between residues 884 and 893, provided that these proteins do not contain both a disulfide bridge between residues 880 and 888 and a disulfide bridge between residues 884 and 893.

5. The protein or fragment thereof according to any one of the preceding claims, wherein the disulfide bridge is a disulfide bridge between residues 880 and 888.

6. The protein or fragment thereof according to any one of the preceding claims, wherein the at least one mutation in the loop region corresponding to amino acid residues 941-945 is an amino acid mutation at position 942 to P.

7. The protein or fragment thereof according to any one of the preceding claims, wherein the at least one mutation in the loop region corresponding to amino acid residues 941-945 is an amino acid mutation at position 941 to P.

8. The protein or fragment thereof according to any one of the preceding claims, wherein the at least one mutation in the loop region corresponding to amino acid residues 941-945 is an amino acid mutation at position 944 to P.

9. The protein or fragment thereof according to any one of the preceding claims, wherein the mutation at position 892 is a mutation to P.

10. The protein or fragment thereof according to any one of the preceding claims, wherein the mutation at position 614 is a mutation to N or G.

11. The protein or fragment thereof according to any one of the preceding claims, wherein the mutation at position 532 is a mutation to P.

12. The protein or fragment thereof according to any one of the preceding claims, wherein the mutation at position 572 is a mutation to I.

13. The protein or fragment thereof according to any one of the preceding claims, comprising an amino acid sequence, wherein the amino acid at position 892 is proline (P), the amino acid at position 614 is glycine (G), the amino acid at position 942 is proline (P) and/or the amino acid at position 944 is proline (P).

14. The protein or fragment thereof of any one of the preceding claims, further comprising a deletion of the furin cleavage site.

15. The protein or fragment thereof of claim 14, wherein the deletion of the furin cleavage site comprises a mutation of the amino acid at position 682 to S and/or a mutation of the oxy acid at position 685 to G.

16. The protein or fragment thereof of any one of the preceding claims, further comprising an amino acid mutation at position 986 and/or 987 to P.

17. The protein of any one of the preceding claims, comprising a sequence selected from the group consisting of SEQ ID NOs: 5-194 and SEQ ID NO:197-418, SEQ ID NO:420 and SEQ ID NO:421, or a fragment thereof.

18. The protein or fragment thereof according to any one of the preceding claims, wherein the protein or fragment thereof does not comprise a signal peptide or tag sequence.

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

20. The protein or fragment thereof of claim 18, wherein the transmembrane and cytoplasmic domains have been removed.

21. The protein or fragment thereof according to claim 18 or 19, wherein a heterotrimeric domain is linked to the truncated S2 domain.

22. The protein or fragment thereof according to claim 21, wherein the heterotrimeric domain is a polypeptide comprising SEQ ID NO:4, and a folding subdomain of the amino acid sequence of seq id no.

23. A nucleic acid molecule encoding the protein or fragment thereof according to any one of the preceding claims 1-22.

24. The nucleic acid according to claim 23, wherein the nucleic acid molecule is DNA or RNA.

25. A vector comprising the nucleic acid of claim 23 or 24.

26. The vector of claim 25, wherein the vector is a human recombinant adenovirus vector.

27. A composition comprising the protein of any one of claims 1-22, the nucleic acid of claim 23 or 243, and/or the vector of claim 25 or 26.

28. A covd-19 vaccine comprising the protein or fragment thereof according to any one of claims 1-22, the nucleic acid according to claim 23 or 24 and/or the vector according to claim 25 or 26.

29. A method for vaccinating a subject with a covd-19 vaccine, the method comprising administering to the subject a vaccine according to claim 28.

30. A method for reducing SARS-CoV-2 infection and/or replication in a subject, the method comprising administering to the subject a composition according to claim 27 or a vaccine according to claim 28.

31. An isolated host cell comprising the nucleic acid of claim 23.

32. An isolated host cell comprising a recombinant human adenovirus of serotype 26, the recombinant human adenovirus of serotype 26 comprising the nucleic acid of claim 23.