SARS-cov-2 vaccine

Abstract

Provided herein are recombinant SARS-CoV-2 Spike proteins and fragments thereof comprising the receptor binding domain (RBD), which have utility, for example, for elicitation of an immune response to SARS-CoV-2 in a subject. Also provided are nucleic acid molecules and vectors encoding these proteins, as well as methods of their use and production. In several implementations, the disclosed recombinant SARS-CoV-2 Spike proteins and fragments thereof comprising the RBD, can be used to generate an immune response to SARS-CoV-2 in a subject, for example to treat or prevent or reduce the severity of SARS-CoV-2 infection.

Description

4239-112474-02SARS-CoV-2 VACCINE CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 678,464, filed August 1, 2024, which is incorporated by reference in its entirety. FIELD

[0002] This disclosure relates to recombinant SARS-CoV-2 spike (S) proteins and their use to elicit an immune response to SARS-CoV-2. BACKGROUND

[0003] Coronaviruses are enveloped, positive-sense single-stranded RNA viruses. They have the largest genomes (26–32 kb) among known RNA viruses, and are phylogenetically divided into four genera (α, β, γ, δ), with betacoronaviruses further subdivided into four lineages (A, B, C, D). Coronaviruses infect a wide range of avian and mammalian species, including humans.

[0004] In 2019, a novel coronavirus (later designated SARS-CoV-2 by the World Health Organization) was identified as the causative agent of an outbreak of pneumonia that was later termed COVID-19. As of 2024, SARS-CoV-2 had infected more than 700 million people worldwide, leading to more than 7 million deaths.

[0005] Current COVID-19 vaccines provide safe and effective protection against severe disease and hospitalization caused by SARS-CoV-2 viruses characterized by sequences that closely match those of the vaccine. However, they generally exhibit reduced neutralizing antibody responses and protection against mismatched viruses. Therefore, COVID-19 vaccines must be updated frequently. Thus, although multiple SARS-CoV-2 vaccines have been fully approved or granted emergency use authorization, a need exists for additional SARS-CoV-2 vaccines that elicit a superior and longer-lasting immune response, for example that targets SARS-CoV-2 variants, SARS-CoV-1 as well as related viruses. SUMMARY

[0006] Provided herein are implementations of a recombinant SARS-CoV-2 Spike protein or fragment thereof comprising the receptor binding domain (RBD) of the recombinant SARS- CoV-2 Spike protein, which have utility, for example, for elicitation of an immune response4239-112474-02to SARS-CoV-2 in a subject. The recombinant SARS-CoV-2 Spike protein or fragment thereof contains amino acid substitutions that are shown to provide superior antigenicity and immunogenicity for elicitation of a neutralizing immune response to SARS-CoV-2. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof optionally further comprises one, two or three of phenylalanine, leucine, and arginine amino acids at positions, 455, 456, and 475, respectively. The amino acid numbering is according to the reference SARS-CoV-2 Spike sequence set forth as SEQ ID NO: 131.

[0007] In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof can be linked to a trimerization domain (such as T4 Fibritin trimerization domain). In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof can be membrane anchored, for example, by linkage to a transmembrane domain.

[0008] In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof is fused to a heterologous protein, such as a protein tag (e.g., a SpyTag moiety) or a self-assembling nanoparticle subunit. In some implementations, the recombinant SARS- CoV-2 Spike protein or fragment thereof is linked to a carrier molecule.

[0009] Nanoparticles including a disclosed recombinant SARS-CoV-2 Spike protein or fragment thereof are also provided. In some implementations, the nanoparticles are self- assembling encapsulin nanoparticles linked to the recombinant SARS-CoV-2 Spike protein or fragment thereof using an isopeptide bond conjugation system, such as the SpyTag:SpyCatcher isopeptide bond conjugation system.

[0010] Immunogenic compositions including the recombinant SARS-CoV-2 Spike or fragment thereof that are suitable for administration to a subject are also provided, and may also be contained in a unit dosage form. The compositions can further include an adjuvant. The recombinant SARS-CoV-2 Spike or fragment thereof may also be conjugated to a carrier to facilitate presentation to the immune system.

[0011] Methods of inducing an immune response in a subject are disclosed, as are methods of treating, inhibiting or preventing a SARS-CoV-2 infection in a subject, by administering to the subject an effective amount of a disclosed recombinant SARS-CoV-2 Spike or fragment thereof, or nucleic acid molecule or vector encoding the recombinant SARS-CoV-2 Spike or4239-112474-02fragment thereof, or nanoparticle comprising the recombinant SARS-CoV-2 Spike or fragment thereof.

[0012] The foregoing and other features and advantages of this disclosure will become more apparent from the following detailed description of several examples which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG.1. Endemic CoV S prime with SARS-CoV-2 S boost did not induce higher neutralizing antibodies against SARS-CoV-2. Mice were primed at weeks 0 and 4 with 10ug of the indicated CoV S DNA vaccines and boosted at week 8 with10ug of SARS-CoV-2 S DNA by intramuscular injection plus electroporation (IM+EP). Serum samples were assessed for neutralizing antibodies at week 10 using a SARS-CoV-2 S pseudotyped lentivirus neutralization assay on 293-TMPRSS2-ACE2 cells. Neutralization ID50 / ID80 titers are shown.

[0014] FIGs.2A-2B. SARS1 and SARS2 S prime / boost did not induce higher neutralizing antibodies against either. Mice were primed at weeks 0 and 4 and boosted at week 8 with 10ug of indicated SARS-CoV-2 or SARS-CoV-1 S DNA by intramuscular injection plus electroporation (IM+EP). Serum samples were assessed for neutralizing antibodies at week 10 using a SARS-CoV-2 and SARS-CoV-1 S pseudotyped lentivirus neutralization assay on 293-TMPRSS2-ACE2 cells. Neutralization ID50 / ID80 titers are shown.

[0015] FIG.3. SARS2-SARS1 or MERS S1-S1 protein induced neutralizing antibodies against both SARS-CoV-2 and SARS-CoV or MERS-CoV. Mice were immunized at weeks 0 and 4 with 10ug of SARS2 S1, SARS1 S1 and SARS2-SARS1 S1-S1 protein (top) or SARS2 S1, MERS S1 and SARS2-MERS S1-S1 protein (bottom) plus ribi as adjuvant by intramuscular injection (IM). Serum samples were assessed for neutralizing antibodies at week 8 using a pseudotyped lentivirus neutralization assay against SARS-CoV-2 and SARS- CoV on 293-TMPRSS2-ACE2 cells or against MERS-CoV on Huh7.5 cells. Neutralization ID80 titers are shown.

[0016] FIG.4. SARS2 S1R 8mut4-SARS1 S1 protein induced neutralizing antibodies against SARS-CoV-2 variants and SARS-CoV. Mice were immunized at weeks 0 and 4 with 10ug of SARS2 S1, SARS1 S1 and SARS2 S1R 8mut4-SARS1 S1 protein plus ribi as adjuvant by intramuscular injection (IM). Serum samples were assessed for neutralizing antibodies at4239-112474-02week 8 using a pseudotyped lentivirus neutralization assay against indicated SARS-CoV-2 variants and SARS-CoV on 293-TMPRSS2-ACE2 cells. Neutralization ID50 titers are shown.

[0017] FIG.5. SARS2 S1R 8mut4-SARS1 S1 protein induced neutralizing antibodies against SARS-CoV-2 variants and SARS-CoV. Mice were immunized at weeks 0 and 4 with 10ug of SARS2 S1R, SARS1 S1, SARS2 S1R-8mut4 and SARS2 S1R 8mut4-SARS1 S1 protein plus ribi as adjuvant by intramuscular injection (IM). Serum samples were assessed for neutralizing antibodies at week 6 using a pseudotyped lentivirus neutralization assay against indicated SARS-CoV-2 variants and SARS-CoV on 293-TMPRSS2-ACE2 cells. Neutralization ID50 titers are shown.

[0018] FIG.6. SARS2 RBD 8mut4 nanoparticle protein induced neutralizing antibodies against SARS-CoV-2 variants and SARS-CoV. Mice were immunized at weeks 0 and 4 with 10, 2 or 0.4ug of SARS2 RBD-8mut4 nanoparticle (EnDS, stabilized Encapsulin) or 10ug SARS2 RBD-8mut4 protein plus ribi as adjuvant by intramuscular injection (IM). Serum samples were assessed for neutralizing antibodies at week 6 using a pseudotyped lentivirus neutralization assay against indicated SARS-CoV-2 variants and SARS-CoV on 293- TMPRSS2-ACE2 cells. Neutralization ID50 titers are shown.

[0019] FIGs.7A-7H. Purification of SARS-CoV-2 S1, S1R proteins. SARS-CoV-2 S1 and S1R proteins were purified using HiLoad 16600 Sephedex 200pg column. SEC peaks are shown. FIG.7A. SARS2 S1R. FIG.7B. SARS2 S1R 8mut4. FIG.7C. SARS2 S1R 9mut. FIG.7D. SARS2 S1R 11mut. FIG.7E. SARS2 S1. FIG.7F. SARS2 RBD 8mut4. FIG.7G. SARS2 S19mut. FIG.7H. SARS2 S111mut.

[0020] FIGs.8A-8F. Purification of SARS-CoV-2 S1, S1R-SARS1 S1 fusion proteins. SARS-CoV-2 S1 and S1R-SARS1 S1 fusion proteins were purified using HiLoad 16600 Sephedex 200pg or HiLoad 16600 Superose 6pg column. SEC peaks are shown. FIG.8A. SARS2 S1R-SARS1 S1. FIG.8B. SARS2 S1R 8mut4-SARS1 S1. FIG.8 C. SARS2 S1R 9mut-SARS1 S1. FIG.8D. SARS2 S1R 11mut-SARS1 S1. FIG.8E. SARS2 S19mut- SARS1 S1. FIG.8F. SARS2 S111mut-SARS1 S1.

[0021] FIG.9. SDS-PAGE of SEC-purified SARS-CoV-2 and SARS-CoV proteins. A. SDS-PAGE in reducing condition. B. SDS-PAGE in non-reducing condition.

[0022] FIGs.10A-10C. Antigenicity of purified SARS-CoV-2 and SARS-CoV proteins. Purified proteins (left column) were evaluated to bind to a panel of SARS-CoV-2 RBD-4239-112474-02directed antibodies (class I, II, III, IV, V) and SARS-CoV mAbs using ELISA. OD450nm readings with mAbs at 0.08ug / ml are shown. SARS2 S1R 8mut4 and SARS2 RBD 8mut4- EnDS bind class IV, V, III mAbs that neutralize SARS-CoV-2 variants, SARS-CoV and other sarbecoviruses.8mut4 designs do not bind mAbs that only neutralize early variants, but not recent Omicron variants.

[0023] FIG.11. Schematic diagram illustrating nanoparticle production.

[0024] FIGs.12A-12E. Production and antigenicity of SARS-CoV-2 S1R-8mut4-EnDS. FIG.12A. SEC of SARS-CoV-2 S1R-8mut4-EnDS purification. FIG.12B. Reducing SDS- PAGE of different proteins. FIG.12C. Negative stained EM of SARS-CoV-2 S1R-8mut4- EnDS. FIG.12D.2D average image of SARS-CoV-2 S1R-8mut4-EnDS. FIG.12E. Antibody binding data with fitted curve measured with Octet. SARS-CoV-2 S1R-8mut4 -EnDS bind A18-448.1 (broadly neutralizing antibody targeting class IV epitope) and S652-118 (cross- reactive NTD antibody), but not LY-CoV555 (only neutralize WA-1 and alpha variant).

[0025] FIGs.13A-13D. Production and antigenicity of SARS-CoV-2 RBD 8mut4-EnDS. FIG.13A. SEC of SARS-CoV-2 RBD 8mut4-EnDS purification. FIG.13B. Reducing SDS- PAGE of different proteins. FIG.13C. Negative stained EM of SARS-CoV-2 RBD 8mut4- EnDS. FIG.13D. Antibody binding data with fitted curve measured with Octet. SARS-CoV- 2 RBD 8mut4 and SARS-CoV-2 RBD 8mut4-EnDS bind A18-448.1 (broadly neutralizing antibody targeting class IV epitope), but not S652-16 (SARS-CoV RBD-specific) and LY- CoV555 (only neutralize WA-1 and alpha variant).

[0026] FIGs.14A-14F. Production and antigenicity of SARS-CoV S1-EnDS. FIG.14A. SEC of SARS-CoV S1-EnDS purification. FIG.14B. Reducing SDS-PAGE of different proteins. FIG.14C. Negative stained EM of SARS-CoV S1-EnDS. D. Protein yield of different proteins. FIGs.14D-14E. Antibody binding data with fitted curve measured with Octet. SARS-CoV S1-EnDS bind A18-448.1 (broadly neutralizing antibody targeting class IV epitope), S652-16 (SARS-CoV RBD-specific) and S652-118 (cross-reactive NTD antibody), but not LY-CoV555 (SARS-CoV-2 RBD-specific).

[0027] FIGs.15A-15E. Production and antigenicity of SARS-CoV-2 S1R-8mut4-SARS1 S1- EnDS. FIG.15A. SEC of SARS-CoV-2 S1R-8mut4-SARS1 S1-EnDS purification. FIG.15B. Reducing SDS-PAGE of different proteins. FIG.15C. Negative stained EM of SARS-CoV-2 S1R-8mut4-SARS1 S1-EnDS. FIGs.15D-15E. Antibody binding data with fitted curve measured with Octet. SARS-CoV-2 S1R-8mut4-SARS1 S1-EnDS bind S652-16 (SARS-CoV4239-112474-02RBD-specific) and S652-118 (cross-reactive NTD antibody) and A18-448.1 (broadly neutralizing antibody targeting class IV epitope), but not LY-CoV555 (SARS-CoV-2 RBD- specific).

[0028] FIGs.16A-16E. Production and antigenicity of SARS2 S1R-8mut4-15ln-EnDS (Longer linker). FIG.16A. SEC of SARS2 S1R 8mut4-15ln-EnDS (Longer linker) purification. FIG.16B. Reducing SDS-PAGE of different proteins. FIG.16C. Negative stained EM of SARS2 S1R 8mut4-15ln-EnDS (Longer linker). FIGs.16D-16E. Protein yield of different proteins. Antibody binding data with fitted curve measured with Octet. SARS2 S1R-8mut4-15ln-EnDS bind A18-448.1 (broadly neutralizing antibody targeting class IV epitope) and S652-118 (cross-reactive NTD antibody), but not LY-CoV555 (only neutralize WA-1 and alpha variant) and S652-16 (SARS-CoV RBD-specific).

[0029] FIGs.17A-17E. Production and antigenicity of NP-SARS2 S1R 8mut4-Nter-EnDS (N-terminal SpyT). FIG.17A. SEC of NP-SARS2 S1R -8mut4-Nter-EnDS (N-ter SpyT) purification. FIG.17B. Reducing SDS-PAGE of different proteins. FIG.17C. Negative stained EM of NP-SARS2 S1R 8mut4-Nter-EnDS (N-ter SpyT). FIGs.17D-17E. Antibody binding data with fitted curve measured with Octet. NP-SARS2 S1R 8mut4-Nter-EnD bind A18-448.1 (broadly neutralizing antibody targeting class IV epitope) and S652-118 (cross- reactive NTD antibody), but not LY-CoV555 (only neutralize WA-1 and alpha variant) and S652-16 (SARS-CoV RBD-specific).

[0030]

[0031] FIGs.18A-18C. Monoclonal antibody insights guide rational vaccine designs. FIG. 18A. Rationale of designs. The footprints of the representative mAbs (in bold) from each class (class I, II, III, I / IV or IV, and V) are shown on the RBD. The ACE2 binding interface is outlined with a black line. Examples of class I, II and III mAbs have lost neutralization activity against emerging variants (column 5), with specific residue changes in the variants (column 6) identified as key contributors to this reduced efficacy. In contrast, class IV and V mAbs target more conserved region of the RBD and most of them retain broad reactivity across variants. FIGs.18B-18C. RBD residue substitutions on designed vaccines. The location of substituted residues on the RBD used in the designed vaccines is illustrated. Combinations of amino acid substitutions were used to generate sequential vaccine variants (3mut, 5mut, 8mut, 9mut, 10 mut and 11mut).4239-112474-02

[0032] FIGs.19A-19B. SARS2 S1R 8mut, 10mut and 11mut proteins induced potent neutralizing antibodies against SARS-CoV-2 variants and SARS-CoV. Mice were immunized at weeks 0 and 4 with 10ug of SARS2 S1R-8mut, S1R-9mut, S1R-10mut or S1R-11mut proteins plus ribi as adjuvant by intramuscular injection (IM). Serum samples were assessed at week 6 for binding using Octet and for neutralization using a pseudotyped lentivirus neutralization assay against SARS-CoV-2 variants and SARS-CoV on 293-TMPRSS2-ACE2 cells. FIG.19A. Binding to indicated RBD proteins. Shown are binding responses (nm) at serum dilution at 1:100. FIG.19B. Neutralization against indicated SARS-CoV-2 variants and SARS-CoV. Neutralization ID50 titers are shown.

[0033] FIG.20. S1R 8mut-np, not WA1-np induced potent neutralization antibodies against SARS2 variants. Mice were immunized at weeks 0 and 4 with 2ug of indicated S1R proteins plus ribi as adjuvant by intramuscular injection (IM). Serum samples were assessed at week 6 for neutralization using a pseudotyped lentivirus neutralization assay against SARS-CoV-2 variants on 293-TMPRSS2-ACE2 cells. Neutralization ID50 titers are shown.

[0034] FIGs.21A-22C. N-terminal np-S1R 8mut (N-EnDS-NP-S1R 8mut) induced more consistent, broad and potent neutralization antibodies against SARS2 variants and SARS1. Mice were immunized at weeks 0 and 4 with 2ug of indicated S1R proteins plus ribi as adjuvant by intramuscular injection (IM). Serum samples were assessed at week 6 for binding to indicated RBD proteins (FIG.21A) by Octet, and neutralization (FIG.21B) using a pseudotyped lentivirus neutralization assay against SARS-CoV-2 variants and SARS-CoV on 293-TMPRSS2-ACE2 cells. FIG.21A. Binding to indicated RBD proteins. Shown are binding responses (nm) at serum dilution at 1:100. FIG.21B. Neutralization against SARS- CoV-2 variants and SARS-CoV. Neutralization ID50 titers are shown. FIG.21C. Diagram of S1R (NTD-RBD) positions on EnDS-NP of three nanoparticles. RBD is exposed in the N- EnDS-NP-S1R 8mut. DETAILED DESCRIPTION I. Summary of Terms

[0035] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.4239-112474-02

[0036] As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes single or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0037] Adjuvant: A component of an immunogenic composition used to enhance antigenicity. In some aspects, an adjuvant can include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion, for example, in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and / or causes influx of macrophages). In some aspects, the adjuvant used in a disclosed immunogenic composition is a combination of lecithin and carbomer homopolymer (such as the ADJUPLEX™ adjuvant available from Advanced BioAdjuvants, LLC; see also Wegmann, Clin Vaccine Immunol 22(9): 1004-1012, 2015). Additional adjuvants for use in the disclosed immunogenic compositions include the QS21 purified plant extract, Matrix M, AS01, MF59, and ALFQ adjuvants. Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants. Adjuvants include biological molecules (a “biological adjuvant”), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL and toll-like receptor (TLR) agonists, such as TLR-9 agonists. The person of ordinary skill in the art is familiar with adjuvants (see, e.g., Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley- Interscience, 2007).

[0038] Administration: The introduction of an agent, such as a disclosed immunogen, into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intranasal, the agent (such as an immunogen comprising a recombinant4239-112474-02SARS-CoV-2 Spike protein or fragment thereof) is administered by introducing the composition into the nasal passages of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.

[0039] Amino acid substitution: The replacement of one amino acid in a polypeptide with a different amino acid.

[0040] Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen) such as a SARS-CoV-2 S protein. The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols.1-2, 2ndEd., Springer Press, 2010). Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs” (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to an epitope of an antigen.

[0041] Carrier: An immunogenic molecule to which an antigen can be linked. When linked to a carrier, the antigen may become more immunogenic. Carriers are chosen to increase the immunogenicity of the antigen and / or to elicit antibodies against the carrier which are diagnostically, analytically, and / or therapeutically beneficial. Useful carriers include polymeric carriers, which can be natural (for example, proteins from bacteria or viruses),4239-112474-02semi-synthetic or synthetic materials containing one or more functional groups to which a reactant moiety can be attached.

[0042] Conservative variants: “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to induce an immune response when administered to a subject. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Furthermore, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some examples less than 1%) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.

[0043] The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0044] Non-conservative substitutions are those that reduce an activity or function of the recombinant SARS-CoV-2 Spike or fragment thereof, such as the ability to induce an immune response when administered to a subject. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. Thus, a conservative substitution does not alter the basic function of a protein of interest.

[0045] Control: A reference standard. In some implementations, the control is a negative control sample obtained from a healthy patient. In other implementations, the control is a positive control sample obtained from a patient diagnosed with a SARS-CoV-2 infection. In still other implementations, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients infected with a SARS-CoV-2 with known prognosis or outcome, or group of samples that represent baseline or normal values).4239-112474-02

[0046] A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some implementations, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%.

[0047] Coronavirus: A large family of positive-sense, single-stranded RNA viruses that can infect humans and non-human animals. Coronaviruses get their name from the crown-like spikes on their surface. The viral envelope is comprised of a lipid bilayer containing the viral membrane (M), envelope (E) and spike (S) proteins. Most coronaviruses cause mild to moderate upper respiratory tract illness, such as the common cold. However, three coronaviruses have emerged that can cause more serious illness and death: severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1), SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Other coronaviruses that infect humans include human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63- CoV), human coronavirus OC43 (OC43-CoV), and human coronavirus HKU1 (HKU1-CoV).

[0048] Coronavirus Disease 2019 (COVID-19): A disease caused by SARS-CoV-2 infection. Common symptoms include fever, cough, fatigue, shortness of breath or breathing difficulties, and loss of smell and taste. The incubation period may range from one to fourteen days. While most patients have mild symptoms, some develop severe COVID-19 disease, often characterized by acute respiratory distress syndrome (ARDS) that is precipitated by cytokine storm, multi-organ failure, septic shock, and blood clots, and often requiring hospitalization and possible ventilation-based breathing assistance.

[0049] A host of underlying medical conditions are known to lead to increased risk of COVID-19, and severe COVID-19, following infection with SARS-CoV-2. Non-limiting examples include heart disease, cancer, chronic obstructive pulmonary disease, type 2 diabetes, type 1 diabetes, obesity, chronic kidney disease, sickle cell disease, asthma, liver disease, chronic lung disease, high blood pressure, or a suppressed immune system due to medical treatment, infection with a pathogen other than SARS-CoV-2, or an autoimmune disorder.4239-112474-02

[0050] The World Health Organization (WHO) has published testing guidelines for COVID- 19 diagnosis (see, e.g., Laboratory Guidelines for the Detection and Diagnosis of COVID-19 virus infection, July 2020). The standard method of testing for SARS-CoV-2 infection is real-time reverse transcription polymerase chain reaction (rRT-PCR) on respiratory samples obtained by a nasopharyngeal swab. Standard diagnostic methods of the detection of symptoms of COVID-19 are also utilized (e.g., lung inflammation, shortness of breath, low oxygen saturation, etc).

[0051] Covalent bond: An interatomic bond between two atoms, characterized by the sharing of one or more pairs of electrons by the atoms. The terms “covalently bound” or “covalently linked” refer to making two separate molecules into one contiguous molecule. The terms include reference to joining an antigen (such as a SARS-CoV-2 S protein or fragment thereof) either directly or indirectly to a carrier molecule, for example indirectly with an intervening linker molecule, such as a peptide or non-peptide linker.

[0052] Degenerate variant: In the context of the present disclosure, a “degenerate variant” refers to a polynucleotide encoding a polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0053] Effective amount: An amount of agent, such as an immunogen, that is sufficient to elicit a desired response, such as an immune response in a subject. It is understood that to obtain a protective immune response against an antigen of interest can require multiple administrations of a disclosed immunogen, and / or administration of a disclosed immunogen as the “prime” in a prime boost protocol wherein the boost immunogen can be different from the prime immunogen. Accordingly, an effective amount of a disclosed immunogen can be the amount of the immunogen sufficient to elicit a priming immune response in a subject that can be subsequently boosted with the same or a different immunogen to elicit a protective immune response.

[0054] In one implementation, a desired response is to elicit an immune response that inhibits or prevents SARS-CoV-2 infection. SARS-CoV-2 infection does not need to be completely eliminated or prevented for the composition to be effective. For example, administration of an effective amount of the immunogen can induce an immune response that inhibits the4239-112474-02SARS-CoV-2 infection (for example, as measured by infection of cells, by number or percentage of subjects infected by the SARS-CoV-2, or by severity of COVID-19) by a desired amount, for example by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SARS-CoV-2 infection or COVID19 symptoms), as compared to a suitable control.

[0055] Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response, for example, an epitope is the region of an antigen to which B and / or T cells respond. An antibody can bind to a particular antigenic epitope, such as an epitope on SARS-CoV-2 Spike. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.

[0056] Expression: Transcription or translation of a nucleic acid sequence. For example, a gene is expressed when its DNA is transcribed into an RNA or RNA fragment, which in some examples is processed to become mRNA. A gene may also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or a protein fragment. In a particular example, a heterologous gene is expressed when it is transcribed into an RNA. In another implementation, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. The term “expression” is used herein to denote either transcription or translation. Regulation of expression can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.

[0057] Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader4239-112474-02sequences and fusion partner sequences. Expression control sequences can include a promoter.

[0058] A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. In one implementation, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences.

[0059] Expression vector: A vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0060] GCN4 trimerization domain: A trimerization domain from the GCN4 protein that comprises a leucine zipper amino acid sequence that naturally forms a trimeric structure. Examples of the GCN4 trimerization domain is described, for example, Harbury et al. (1993 Science 262:1401-1407). In some implementations, a GCN4 trimerization domain can be included in the amino acid sequence of a disclosed recombinant protein so that the recombinant protein will trimerize. A non-limiting example of a GCN4 trimerization domain sequence for use with the disclosed examples is provided as MKQIEDKIEEILSKIYHIENEIARIKKLIGER (SEQ ID NO: 132).

[0061] Heterologous: A heterologous polypeptide or polynucleotide refers to a polypeptide or polynucleotide derived from a different source or species.4239-112474-02

[0062] Host cells: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used.

[0063] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one implementation, the response is specific for a particular antigen (an “antigen-specific response”). In one implementation, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another implementation, the response is a B cell response, and results in the production of specific antibodies.

[0064] “Priming an immune response” refers to treatment of a subject with a “prime” immunogen to induce an immune response that is subsequently “boosted” with a boost immunogen. Together, the prime and boost immunizations produce the desired immune response in the subject. “Enhancing an immune response” refers to co-administration of an adjuvant and an immunogenic agent, wherein the adjuvant increases the desired immune response to the immunogenic agent compared to administration of the immunogenic agent to the subject in the absence of the adjuvant.

[0065] Immunogenic conjugate: A composition composed of at least two heterologous molecules (such as a recombinant SARS-CoV-2 Spike protein or fragment thereof and a carrier, such as a protein carrier) linked together that stimulates or elicits an immune response to a molecule in the conjugate in a vertebrate. In some examples where the conjugate includes a viral antigen, the immune response is protective in that it enables the vertebrate animal to better resist infection from the virus from which the antigen is derived.

[0066] Immunogen: A protein or a portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen.

[0067] Immunogenic composition: A composition comprising a disclosed immunogen, or a nucleic acid molecule or vector encoding a disclosed immunogen, that elicits a measurable CTL response against the immunogen, or elicits a measurable B cell response (such as production of antibodies) against the immunogen, when administered to a subject. It further refers to isolated nucleic acids encoding an immunogen, such as a nucleic acid that can be used to express the immunogen (and thus be used to elicit an immune response against this4239-112474-02immunogen). For in vivo use, the immunogenic composition will typically include the protein or nucleic acid molecule in a pharmaceutically acceptable carrier and may also include other agents, such as an adjuvant.

[0068] Inhibiting or treating a disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as a SARS-CoV-2 infection or COVID19 disease. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment.

[0069] Inhibiting a disease can include preventing or reducing the risk of the disease, such as preventing or reducing the risk of viral infection. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well-being of the subject, or by other parameters that are specific to the particular disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

[0070] Isolated: An “isolated” biological component has been substantially separated or purified away from other biological components, such as other biological components in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides, nucleic acids, and viruses that have been “isolated” include those purified by standard purification methods. Isolated does not require absolute purity, and can include protein, peptide, nucleic acid, or virus molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

[0071] Linked: The term “linked” means joined together, either directly or indirectly. For example, a first moiety may be covalently or noncovalently (e.g., electrostatically) linked to a second moiety. This includes, but is not limited to, covalently bonding one molecule to another molecule, noncovalently bonding one molecule to another (e.g. electrostatically bonding), non-covalently bonding one molecule to another molecule by hydrogen bonding, non-covalently bonding one molecule to another molecule by van der Waals forces, and any and all combinations of such couplings. Indirect attachment is possible, such as by using a4239-112474-02“linker”. In several implementations, linked components are associated in a chemical or physical manner so that the components are not freely dispersible from one another, at least until contacting a cell, such as an immune cell.

[0072] Linker: One or more molecules or groups of atoms positioned between two moieties. Typically, linkers are bifunctional, i.e., the linker includes a functional group at each end, wherein the functional groups are used to couple the linker to the two moieties. The two functional groups may be the same, i.e., a homobifunctional linker, or different, i.e., a heterobifunctional linker. In several implementations, a peptide linker can be used to link the C-terminus of a first protein to the N-terminus of a second protein. Non-limiting examples of peptide linkers include glycine-serine peptide linkers, which are typically not more than 10 amino acids in length. Typically, such linkage is accomplished using molecular biology techniques to genetically manipulate DNA encoding the first polypeptide linked to the second polypeptide by the peptide linker.

[0073] Native protein or sequence: A polypeptide or sequence that has not been modified, for example, by selective mutation. Native protein or native sequence are also referred to as wild-type protein or wild-type sequence.

[0074] Nanoparticle: A nanoscale moiety of from 1-1000 nm in diameter. In some implementations, a protein nanoparticle is provided that comprises a multi-subunit, self- assembling, protein-based polyhedron shaped structure optionally linked to a recombinant SARS-CoV-2 Spike or fragment thereof as described herein. The subunits are each composed of proteins, for example a glycosylated polypeptide. In some implementations, recombinant SARS-CoV-2 Spike protein or fragment thereof can be fused to the subunits of the protein nanoparticles to provide multiple copies of the recombinant SARS-CoV-2 Spike protein or fragment thereof on each protein nanoparticle. Non-limiting examples of protein nanoparticles include ferritin nanoparticles (see, e.g., Zhang, Y. Int. J. Mol. Sci., 12:5406- 5421, 2011), encapsulin nanoparticles (see, e.g., Sutter et al., Nature Struct. and Mol. Biol., 15:939-947, 2008), Sulfur Oxygenase Reductase (SOR) nanoparticles (see, e.g., Urich et al., Science, 311:996-1000, 2006), lumazine synthase nanoparticles (see, e.g., Zhang et al., J. Mol. Biol., 306: 1099-1114, 2001), and pyruvate dehydrogenase nanoparticles (see, e.g., Izard et al., PNAS 96: 1240-1245, 1999). Ferritin, encapsulin, SOR, lumazine synthase, and pyruvate dehydrogenase are monomeric proteins that self-assemble into a globular protein complexes that in some cases consists of 24, 60, 24, 60, and 60 protein subunits, respectively. Additional protein nanoparticle structures are described by Heinze et al., J Phys Chem B.,4239-112474-02120(26):5945-52, 2016; Hsia et al., Nature, 535(7610):136-9, 2016; and King et al., Nature, 510(7503):103-8, 2014.

[0075] Nucleic acid molecule: A polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, mRNA, circular RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. “cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form. “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, an mRNA, or a circular RNA to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.

[0076] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0077] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, 22nded., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents.

[0078] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional4239-112474-02non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as non-natural preservatives), and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection. In some aspects, the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as a pill or in a selected quantity in a vial. Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed).

[0079] Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). “Polypeptide” applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymer as well as in which one or more amino acid residue is a non-natural amino acid, for example, an artificial chemical mimetic of a corresponding naturally occurring amino acid. A “residue” refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide” is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues.

[0080] Prime-boost immunization: An immunotherapy including administration of multiple immunogens over a period of time to elicit the desired immune response.

[0081] Recombinant: A recombinant biological molecule (e.g., nucleic acid, protein, vector, or virus) is one that comprises a sequence that is not naturally occurring. For example, the sequence is altered from nature by the artificial manipulation of isolated segments of nucleic acids or proteins, for example, using genetic engineering techniques

[0082] SARS-CoV-2: A positive-sense, single stranded RNA virus of the genus betacoronavirus that has emerged as a highly fatal cause of severe acute respiratory infection. The viral genome is capped, polyadenylated, and covered with nucleocapsid proteins. The SARS-CoV-2 virion includes a viral envelope with large spike glycoproteins. The SARS- CoV-2 genome, like most coronaviruses, has a common genome organization with the4239-112474-02replicase gene included in the 5'-two thirds of the genome, and structural genes included in the 3'-third of the genome. The SARS-CoV-2 genome encodes the canonical set of structural protein genes in the order 5' - spike (S) - envelope (E) - membrane (M) and nucleocapsid (N) - 3'. Symptoms of SARS-CoV-2 infection include fever and respiratory illness, such as dry cough and shortness of breath. Cases of severe infection can progress to severe pneumonia, multi-organ failure, and death. The time from exposure to onset of symptoms is approximately 2 to 14 days.

[0083] Standard methods for detecting viral infection may be used to detect SARS-CoV-2 infection, including but not limited to, assessment of patient symptoms and background and genetic tests such as reverse transcription-polymerase chain reaction (rRT-PCR). The test can be done on patient samples such as respiratory or blood samples.

[0084] SARS-CoV-2 Spike (S): A class I fusion glycoprotein initially synthesized as a precursor protein of approximately 1273 amino acids in size. Individual precursor S polypeptides form a homotrimer and undergo glycosylation within the Golgi apparatus as well as processing to remove the signal peptide. The S polypeptide includes S1 and S2 proteins separated by a protease cleavage site between approximately position 685 / 686. Cleavage at this site generates separate S1 and S2 polypeptide chains, which remain associated as S1 / S2 protomers within the homotrimer. It is believed that the beta coronaviruses are generally not cleaved prior to the low pH cleavage that occurs in the late endosome-early lysosome by the TMPRSS2 protease, at the start of the fusion peptide. The S1 subunit is distal to the virus membrane and contains the receptor-binding domain (RBD) that is believed to mediate virus attachment to its host receptor. The S2 subunit is believed to contain the fusion protein machinery, such as the fusion peptide, two heptad-repeat sequences (HR1 and HR2) and a central helix typical of fusion glycoproteins, a transmembrane domain, and the cytosolic tail domain.

[0085] Unless context indicates otherwise, the numbering used in the disclosed SARS-CoV-2 S proteins and fragments thereof is relative to the S protein of SARS-CoV-2, the sequence of which is provided as SEQ ID NO: 131, and deposited as NCBI Ref. No. YP_009724390.1, which is incorporated by reference herein in its entirety.

[0086] Sequence identity: The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity; the higher the4239-112474-02percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.

[0087] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res.16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol.215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0088] Homologs and variants of a polypeptide (such as a SARS-CoV-2 S ectodomain) are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet.

[0089] As used herein, reference to “at least 90% identity” or similar language refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.

[0090] Signal Peptide: A short amino acid sequence (e.g., approximately 10-35 amino acids in length) that directs newly synthesized secretory or membrane proteins to and through membranes (for example, the endoplasmic reticulum membrane). Signal peptides are typically located at the N-terminus of a polypeptide and are removed by signal peptidases. Signal peptide sequences typically contain three common structural features: an N-terminal polar basic region (n-region), a hydrophobic core, and a hydrophilic c-region).4239-112474-02

[0091] Specifically bind: When referring to the formation of an antibody:antigen protein complex, or a protein:protein complex, refers to a binding reaction which determines the presence of a target protein, peptide, or polysaccharide (for example, a glycoprotein), in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated conditions, a particular antibody or protein binds preferentially to a particular target protein, peptide or polysaccharide (such as an antigen present on the surface of a pathogen, for example, gp120) and does not bind in a significant amount to other proteins or polysaccharides present in the sample or subject. Specific binding can be determined by standard methods. A first protein or antibody specifically binds to a target protein when the interaction has a KD of less than 10-7Molar, such as less than 10-8Molar, less than 10-9, or even less than 10-10Molar.

[0092] Subject: Living multicellular vertebrate organisms, a category that includes human and non-human mammals. In some aspects, the subject is a human. In some implementations, a subject who is in need of inhibiting or preventing a SARS-CoV-2 infection is selected. For example, the subject can be uninfected and at risk of SARS-CoV-2 infection.

[0093] T4 Fibritin trimerization domain: Also referred to as a “foldon” domain, the T4 Fibritin trimerization domain comprises an amino acid sequence that naturally forms a trimeric structure. In some implementations, a T4 Fibritin trimerization domain can be linked to the C-terminus of a disclosed recombinant SARS-CoV-2 S protein ectodomain. In one implementation, a T4 Fibritin trimerization domain comprises the amino acid sequence set forth as (GYIPEAPRDGQAYVRKDGEWVLLSTF, SEQ ID NO: 133).

[0094] Transmembrane domain: An amino acid sequence that inserts into a lipid bilayer, such as the lipid bilayer of a cell or virus or virus-like particle. A transmembrane domain can be used to anchor an antigen to a membrane. In some examples a transmembrane domain is a SARS-CoV-2 S transmembrane domain.

[0095] Under conditions sufficient for: A phrase that is used to describe any environment that permits a desired activity.

[0096] Vaccine: A pharmaceutical composition that induces a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine induces an antigen-specific immune response to an antigen of a pathogen, for example a viral pathogen, or to a cellular constituent correlated with a4239-112474-02pathological condition. A vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus, a cell or one or more cellular constituents. In a non-limiting example, a vaccine induces an immune response that reduces the severity of the symptoms associated with a SARS-CoV-2 infection and / or decreases the viral load compared to a control. In another non-limiting example, a vaccine induces an immune response that reduces and / or prevents a SARS-CoV-2 infection compared to a control.

[0097] Vector: An entity containing a DNA or RNA molecule bearing a promoter(s) that is operationally linked to the coding sequence of an antigen(s) of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and / or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication-incompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses.

[0098] Virus-like particle (VLP): A non-replicating, viral shell, derived from any of several viruses. VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques, such as by electron microscopy, biophysical characterization, and the like. Further, VLPs can be isolated by known techniques, e.g., density gradient centrifugation and identified by characteristic density banding. See, for example, Baker et al. (1991) Biophys. J.60:1445-1456; and Hagensee et al. (1994) J. Virol.68:4503-4505; Vincente, J Invertebr Pathol., 2011; Schneider-Ohrum and Ross, Curr. Top. Microbiol. Immunol., 354: 53073, 2012). II. Recombinant SARS-CoV-2 Spike and fragments thereof

[0099] Disclosed herein are recombinant SARS-CoV-2 S proteins and fragments thereof comprising one or more amino acid substitutions in variable regions of the RBD, while4239-112474-02preserving conserved epitopes recognized by potently neutralizing antibodies. Further provided are examples of these modified SARS-CoV-2 S proteins and fragments thereof fused to heterologous proteins (such as a SARS-CoV-1 S protein) and presented on a nanoparticle. The disclosed recombinant SARS-CoV-2 S proteins and fragments thereof elicit a superior immune response compared to prior SARS-CoV-2 immunogens.

[0100] An exemplary sequence of native SARS-CoV-2 S protein (including the ectodomain and TM and CT domains) is provided as SEQ ID NO: 131 (NCBI Ref. No. YP_009724390.1, incorporated by reference herein): MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNV TWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNAT NVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNF KNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSY LTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEK GIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSA SFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVI AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYG FQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKK FLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEV PVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPR RARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICG DSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQIL PDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDE MIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSA IGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQ IDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQ SAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIIT TDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVN IQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSC CSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT (SEQ ID NO: 131)

[0101] The amino acid numbering used herein for residues of the SARS-CoV-2 S protein is with reference to the SARS-CoV-2 S sequence provided as SEQ ID NO: 131. With reference to the SARS-CoV-2 S protein sequence provided as SEQ ID NO: 131, the ectodomain of the SARS-CoV-2 S protein includes about residues 14-1208. Residues 1-13 are the signal peptide, which is removed during cellular processing. The S1 / S2 cleavage site is located at position 685 / 686. The HR1 is located at about residues 915-983. The central helix is located at about residues 988-1029. The HR2 is located at about 1162-1194. The C-terminal end of the S2 ectodomain is located at about residue 1208. In some aspects, the fragment of the recombinant SARS-CoV-2 S protein can have a C-terminal residue of the C-terminal residue of the HR2 (e.g., position 1194), or the ectodomain (e.g., position 1208), or from one of4239-112474-02positions 1194-1208. The position numbering of the S protein may vary between SARS- CoV-2 stains, but the sequences can be aligned to determine relevant structural domains and cleavage sites. It will be appreciated that a few residues (such as up to 10) on the N- and / or C-terminal ends of the ectodomain can be removed or modified in the disclosed immunogens without decreasing the utility of the S ectodomain as an immunogen.

[0102] The recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain) comprises amino acid substitutions at one or more (such as 1, 2, 3, 4, 5, 6, 7, or 8) of the following SARS-CoV-2 S positions: 346, 417, 445, 446, 452, 460, 484, and 486. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain) comprises amino acid substitutions at the following SARS-CoV-2 S positions: 346, 417, 445, 446, 452, 460, 484, and 486. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, one or more (such as all) of which may be present as a result of an amino acid substitution. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids, or a conservative variant thereof, at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, one or more (such as all) of which may be present as a result of an amino acid substitution.

[0103] In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain) further comprises amino acid substitutions at one, two or three of the following SARS-CoV-2 S positions: 455, 456, and 475. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain) further comprises one, two or three of phenylalanine, leucine, and arginine amino acids at positions, 455, 456, and 475, respectively, one or more (such as all) of which may be present as a result of an amino acid substitution. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), further comprises one, two or three of phenylalanine, leucine, and arginine amino acids at positions, 455, 456, and 475, respectively, one or more (such as all) of which may be present as a result of an amino acid substitution.4239-112474-02

[0104] In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises phenylalanine at position 455. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids, or a conservative variant thereof, at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises phenylalanine, or a conservative variant thereof, at position 455.

[0105] In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises leucine at position 456. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids, or a conservative variant thereof, at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises leucine, or a conservative variant thereof, at position 456.

[0106] In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises arginine at position 475. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids, or a conservative variant thereof, at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises arginine, or a conservative variant thereof, at position 475.

[0107] In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises phenylalanine and4239-112474-02leucine at positions 455 and 456, respectively. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids, or a conservative variant thereof, at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises phenylalanine and leucine, or a conservative variant thereof, at positions 455 and 456, respectively.

[0108] In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises phenylalanine and arginine at positions 455 and 475, respectively. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids, or a conservative variant thereof, at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises phenylalanine and arginine, or a conservative variant thereof, at positions 455 and 475, respectively.

[0109] In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises leucine and arginine at positions 456 and 475, respectively. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids, or a conservative variant thereof, at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises leucine and arginine, or a conservative variant thereof, at positions 456 and 475, respectively.

[0110] In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises phenylalanine, leucine, and arginine at positions 455, 456 and 475, respectively. In some implementations, the recombinant SARS-CoV-2 Spike protein or fragment thereof (e.g., comprising the RBD, such as an RBD or ectodomain), comprises threonine, asparagine, alanine, serine, arginine,4239-112474-02lysine, alanine, and proline amino acids, or a conservative variant thereof, at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, and further comprises phenylalanine, leucine, and arginine, or a conservative variant thereof, at positions 455, 456 and 475, respectively.

[0111] In some implementations, the amino acid substitutions described above for the threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, are due to one or more of R346T, K417N, V445A, G446S, L452R, N460K, E484A, and F486P amino acid substitutions; and the one, two, or three of arginine, phenylalanine, and leucine amino acids at positions 475, 455, and 456, respectively, are due to one or more of A475R, L455F, and F456L amino acid substitutions.

[0112] In some implementations, the fragment of the recombinant SARS-CoV-2 Spike protein comprises or consists of the RBD of the SARS-CoV-2 Spike protein, for example comprising about SARS-CoV-2 Spike residues 319-537. In some such implementations, the fragment of the recombinant SARS-CoV-2 Spike protein comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to residues 319-537 of any one of SEQ ID NOs: 127-130, and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids. In some such implementations, the fragment of the recombinant SARS-CoV-2 Spike protein comprises the amino acid sequence set forth as residues 319-537 of any one of SEQ ID NOs: 127-130.

[0113] In some implementations, the fragment of the recombinant SARS-CoV-2 Spike protein comprises or consists of the S1 subunit of the SARS-CoV-2 Spike protein, for example comprising about SARS-CoV-2 Spike residues 14-537 or 14-680. In some such implementations, the fragment of the recombinant SARS-CoV-2 Spike protein comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to residues 14-537 or 14-680 of any one of SEQ ID NOs: 127-130, and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids. In some such implementations, the fragment of the recombinant SARS-CoV-2 Spike protein comprises the amino acid sequence set forth as residues 14-537 or 14-680 of any one of SEQ ID NOs: 127-130.4239-112474-02

[0114] In some implementations, the fragment of the recombinant SARS-CoV-2 Spike protein comprises or consists of the ectodomain of the SARS-CoV-2 Spike protein, for example comprising about SARS-CoV-2 Spike residues 14-1208. In some such implementations, the fragment of the recombinant SARS-CoV-2 Spike protein comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to residues 14-1208 of any one of SEQ ID NOs: 127-130, and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids. In some such implementations, the fragment of the recombinant SARS-CoV-2 Spike protein comprises the amino acid sequence set forth as residues 14-1208 of any one of SEQ ID NOs: 127-130.

[0115] In some implementations, the recombinant SARS-CoV-2 Spike protein is provided. In some such implementations, the recombinant SARS-CoV-2 Spike protein comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 127-130, and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids. In some such implementations, the recombinant SARS-CoV-2 Spike comprises the amino acid sequence set forth as residues 14-1208 of any one of SEQ ID NOs: 127-130.

[0116] The recombinant SARS-CoV-2 Spike or fragment thereof can be presented in any format of the spike protein (e.g., RBD only, S1 subunit, soluble ectodomain trimer, or full- length transmembrane protein). Additionally, recombinant SARS-CoV-2 Spike or fragment thereof can further comprise additional mutations as disclosed herein, such as the 2P substitution (K986P and V987P substitutions) and / or modification to remove the S1 / S2 protease cleavage site (e.g., RRAR682-685GSAS substitution).

[0117] In some aspects, the recombinant SARS-CoV-2 S protein or fragment thereof disclosed herein further comprises one or more (such as two, for example two consecutive) proline substitutions between the HR1 domain and the central helix domain to stabilize the SRAS-CoV-2 ectodomain in a prefusion conformation, such as 986P and 987P substitutions (referred to as the “2P substitutions), for example, K986P and V987P substitutions (with reference to SEQ ID NO: 131).4239-112474-02

[0118] In some aspects that include an S ectodomain (such as full-length S), a S1 / S2 protease cleavage site of the S ectodomain is mutated to inhibit protease cleavage. For example, the S1 and S2 components of the SARS-CoV-2 S ectodomain are joined by a linker, such as a peptide linker. Examples of peptide linkers that can be used include glycine, serine, and glycine-serine linkers. In a non-limiting example, a RRAR682-685GSAS substitution is used to remove the S1 / S2 protease cleavage site. In any of the examples including a native S1 / S2 cleavage site, a peptide linker (such as a GGGSGGGG linker, SEQ ID NO: 134) can be inserted immediately following the cleavage site. In some implementations, a peptide linker (such as GGGSGGGG linker, SEQ ID NO: 134) is inserted following the PSKR site located at residues 812-815 of SEQ ID NO: 131.

[0119] In some implementations, the C-terminal residue of an ectodomain of a disclosed recombinant SARS-CoV-2 S protein can be linked to a trimerization domain to promote trimerization of the ectodomain, and to stabilize the membrane proximal aspect of the ectodomain in a trimeric configuration. Non-limiting examples of heterologous multimerization domains that promote stable trimers of soluble recombinant proteins include: the GCN4 leucine zipper (Harbury et al.1993 Science 262:1401-1407), the trimerization motif from the lung surfactant protein (Hoppe et al.1994 FEBS Lett 344:191-195), collagen (McAlinden et al.2003 J Biol Chem 278:42200-42207), and the phage T4 fibritin (Miroshnikov et al.1998 Protein Eng 11:329-414), any of which can be linked to a recombinant SARS-CoV-2 ectodomain (e.g., by linkage to the C-terminus of ectodomain) to promote trimerization.

[0120] In some implementations, the C-terminal residue of the S ectodomain can be linked to a T4 fibritin domain. In specific examples, the T4 fibritin domain can include the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTF (SEQ ID NO: 131), which adopts a β- propeller conformation, and can fold and trimerize in an autonomous way (Tao et al.1997 Structure 5:789-798).

[0121] Optionally, the heterologous trimerization is connected to the recombinant SARS- CoV-2 S ectodomain via a peptide linker, such as an amino acid linker. Non-limiting examples of peptide linkers that can be used include glycine, serine, and glycine-serine linkers.

[0122] In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof can be membrane anchored, for examples where the recombinant SARS-CoV-2 S4239-112474-02protein or fragment thereof is expressed on an attenuated viral vaccine, or a virus like particle, or by recombinant nucleic acid. In such implementations, the recombinant SARS- CoV-2 S protein or fragment thereof typically comprises a C-terminal linkage to a transmembrane domain, such as the transmembrane domain (and optionally the cytosolic tail) of SARS-CoV-2 S protein. In some implementations, one or more peptide linkers (such as a gly-ser linker, for example, a 10 amino acid glycine-serine peptide linker can be used to link the recombinant SARS-CoV-2 S protein or fragment thereof to the transmembrane domain.

[0123] In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof is linked to a heterologous moiety, such as a heterologous protein. In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof are fused C- terminally to a linkage tag or a purification tag, such as a SpyTag moiety. For example, the recombinant SARS-CoV-2 S protein or fragment thereof is fused C-terminally to a SpyTag moiety that allows subsequent linkage to a heterologous moiety (such as a self-assembling protein nanoparticle) containing a corresponding SpyCatcher moiety. Optionally, a linker (such as a glycine, serine, or glycine-serine linker) can be included between the recombinant SARS-CoV-2 S protein or fragment thereof (or recombinant SARS-CoV-2 S protein or fragment thereof fused to an additional heterologous protein) and the linkage tag or purification tag. In one example, the SpyTag moiety (e.g., AHIVMVDAYKPTK, SEQ ID NO: 136) is genetically fused to the recombinant SARS-CoV-2 S protein or fragment thereof.

[0124] In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof is fused directly or indirectly via a peptide linker to a heterologous protein. In some such implementations, the heterologous protein is a Spike protein or a fragment thereof (such as RBD, S1, or ectodomain) of a heterologous coronavirus. For example, the spike protein or a fragment thereof (such as RBD, S1, or ectodomain) of SARS-CoV-1 , MERS-CoV, 229E- CoV, NL63-CoV, OC43-CoV, and HKU1-CoV.

[0125] In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof is fused directly or indirectly via a peptide linker to a Spike protein or a fragment thereof (such as RBD, S1, or ectodomain) of SARS-CoV-1. In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof is fused directly or indirectly via a peptide linker to a S1 subunit of SARS-CoV-1 Spike. In some such implementations, the recombinant SARS-CoV-2 S protein or fragment thereof fused directly or indirectly via a peptide linker to the S1 subunit of SARS-CoV-1 comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or4239-112474-02at least 99%) identical to any one of SEQ ID NOs: 6-9, 19-22, 54-57, 63-66, 72-75, or 94- 105, and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids. In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof fused directly or indirectly via a peptide linker to the S1 subunit of SARS-CoV-1 comprises the amino acid sequence set forth as any one of SEQ ID NOs: 6-9, 19-22, 54-57, 63-66, 72-75, or 94-105.

[0126] In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof is fused directly or indirectly via a peptide linker to a Spike protein or a fragment thereof (such as RBD, S1, or ectodomain) of MERS-CoV. In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof is fused directly or indirectly via a peptide linker to a S1 subunit of MERS-CoV Spike. In some such implementations, the recombinant SARS-CoV-2 S protein or fragment thereof fused directly or indirectly via a peptide linker to the S1 subunit of MERS-CoV comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 10-13, 23-26, 58-61, 67-70, 76-79, or 106- 117, and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids. In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof fused directly or indirectly via a peptide linker to the S1 subunit of MERS-CoV Spike comprises the amino acid sequence set forth as any one of SEQ ID NOs: 10-13, 23-26, 58-61, 67-70, 76-79, or 106-117.

[0127] In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof is fused directly or indirectly via a peptide linker to a SpyTag moiety. In some such implementations, the recombinant SARS-CoV-2 S protein or fragment thereof fused directly or indirectly via a peptide linker to the SpyTag moiety comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to any one of SEQ ID NOs: 38-61, 63-70, 72-83, 85-88, or 90-125, and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids. In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof fused directly or indirectly via a peptide linker to the SpyTag moiety comprises the amino acid sequence set forth as any one of SEQ ID NOs: 38-61, 63-70, 72-83, 85-88, or 90-125.4239-112474-02

[0128] The recombinant SARS-CoV-2 S protein or fragment thereof and variants thereof can be produced using recombinant techniques, or chemically or enzymatically synthesized.

[0129] Analogs and variants of the recombinant SARS-CoV-2 S protein or fragment thereof may be used in the methods and systems of the present disclosure. Through the use of recombinant DNA technology, variants of the recombinant SARS-CoV-2 S protein or fragment thereof may be prepared by altering the underlying DNA. All such variations or alterations in the structure of the recombinant SARS-CoV-2 S protein or fragment thereof resulting in variants are included within the scope of this disclosure. Such variants include insertions, substitutions, or deletions of one or more amino acid residues, glycosylation variants, unglycosylated recombinant SARS-CoV-2 S protein or fragment thereof, organic and inorganic salts, covalently modified derivatives of the recombinant SARS-CoV-2 S protein or fragment thereof, or a precursor thereof. Such variants may maintain one or more of the functional, biological activities of the recombinant SARS-CoV-2 S protein or fragment thereof, such as binding to cell surface receptor. The recombinant SARS-CoV-2 S protein or fragment thereof can be modified, for example, by PEGylation, to increase the half-life of the protein in the recipient, and / or to make the protein more stable for delivery to a subject.

[0130] In some implementations, a recombinant SARS-CoV-2 S protein or fragment thereof useful within the disclosure is modified by replacement of one or more naturally occurring side chains of the 20 genetically encoded amino acids (or D-amino acids) with other side chains, for example with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclics. For example, proline analogs can be made in which the ring size of the proline residue is changed from a 5-membered ring to a 4-, 6-, or 7-membered ring. Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups can contain one or more nitrogen, oxygen, and / or sulphur heteroatoms. Nanoparticles containing recombinant SARS-CoV-2 S protein or fragment thereof

[0131] In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof is provided on a nanoparticle. In some implementations, a nanoparticle (such as a self-assembling protein nanoparticle) is provided that includes a recombinant SARS-CoV-2 S4239-112474-02protein or fragment thereof displayed on its surface. Any suitable nanoparticle format can be used.

[0132] In some implementations, a nanoparticle (such as a self-assembling protein nanoparticle) is provided that includes a recombinant SARS-CoV-2 S protein or fragment thereof displayed on its surface. Any suitable nanoparticle format can be used.

[0133] In some implementations, to construct protein nanoparticles, nucleic acid encoding a recombinant SARS-CoV-2 S protein or fragment thereof can be fused to nucleic acid encoding a subunit of the protein nanoparticle (such as a ferritin protein, an encapsulin protein, a SOR protein, or a lumazine synthase protein) and expressed in cells under appropriate conditions. The fusion protein self-assembles into a nanoparticle any can be purified.

[0134] In several implementations, to construct such protein nanoparticles, a purified recombinant SARS-CoV-2 S protein or fragment thereof can be linked (for example, via bioconjugation) to subunits of a purified self-assembling protein nanoparticle (such as a ferritin protein, an encapsulin protein, a SOR protein, or a lumazine synthase protein) and the resulting nanoparticle / S trimer purified.

[0135] In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof is included in a self-assembling protein nanocage that directs its own release from cells inside small vesicles in a manner that resembles viruses, for example, as described in Votteler et al., “Designed proteins induce the formation of nanocage-containing extracellular vesicles,” Nature 540, 292–29, 2016. This hybrid biomaterial can fuse its membranes with target cells and deliver its contents, thereby transferring cargoes from one cell to another.

[0136] In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof is linked to a self-assembling protein nanoparticle. Non-limiting example of self- assembling protein nanoparticles include ferritin nanoparticles, encapsulin nanoparticles, Sulfur Oxygenase Reductase (SOR) nanoparticles, and lumazine synthase nanoparticles, which are comprised of an assembly of monomeric subunits including ferritin proteins, encapsulin proteins, SOR proteins, and lumazine synthase, respectively. Additional protein nanoparticle structures are described by Heinze et al., J Phys Chem B., 120(26):5945-52, 2016; Hsia et al., Nature, 535(7610):136-9, 2016; and King et al., Nature, 510(7503):103-8, 2014.4239-112474-02

[0137] Linkage of the recombinant SARS-CoV-2 S protein or fragment thereof to the nanoparticle surface may be accomplished using any suitable means. In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof are C- terminally fused to subunits of the self-assembling nanoparticle, for example, as described in Kanekiyo et al. (Nature, 499:102-106, 2013) for ferritin-based nanoparticles and Sutter et al. (Nature Struct. and Mol. Biol., 15:939-947, 2008) for encapsulin-based nanoparticles.

[0138] In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof is linked to the protein nanoparticle using an isopeptide bond conjugation system, such as the SpyTag:SpyCatcher system (Brune, K. D. et al. Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization. Sci Rep 6, 19234, 2016) to display antigens on nanoparticle surface. The SpyTag:SpyCatcher system is highly specific and stable with an isopeptide bond and has been used for conjugation of antigens on nanoparticle surfaces (See, e.g., WO2011098772, US9547003, US10247727, and US10527609, Zakeri, B. et al. “Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.” Proc Natl Acad Sci U S A 109, E690-697, (2012); Brune, K. D. et al. Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization. Sci Rep 6, 19234, 2016). In such implementations, the recombinant SARS-CoV-2 S protein or fragment thereof and the self-assembling protein nanoparticles are separately produced with fusion to either the SpyTag or SpyCatcher moiety and then combined under conditions suitable for isopeptide bond formation between the SpyTag and SypCatcher moieties.

[0139] In one example, the SpyTag moiety (e.g., AHIVMVDAYKPTK, SEQ ID NO: 136) is genetically fused to the recombinant SARS-CoV-2 S protein or fragment thereof, and the nanoparticle with SpyCatcher is produced under standard conditions. The SpyCatcher moiety is genetically fused to the nanoparticle subunit, and the nanoparticle with SpyCatcher is produced under standard conditions. Non-limiting examples of SpyCatcher moieties are provided as: SEQ ID NO: 137 DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNE QGQVTVNGKATKGDAHI SEQ ID NO: 138 VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTF VETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDAHTGSSGS4239-112474-02

[0140] The nanoparticle / SpyCatcher and recombinant SARS-CoV-2 S protein or fragment thereof / SpyCatcher are subsequently mixed under conditions sufficient for the SpyCatcher / SpyTag to form an isopeptide bond and covalently link the nanoparticle and recombinant SARS-CoV-2 S protein or fragment thereof.

[0141] In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof is linked to the protein nanoparticle using the SpyTag:SpyCatcher system, wherein the recombinant SARS-CoV-2 S protein or fragment thereof are C-terminally fused to SpyTag (as in SEQ ID NO: 136) and the protein nanoparticle is a self-assembled encapsulin nanoparticle with subunits fused to the SpyCatcher moiety. A non-limiting example of an encapsulin subunit sequence fused to SpyCatcher for use in the examples provided herein is set forth as residues 57-394 of SEQ ID NO: 126: PYGWEYAAHPLCEVEVLSDENEVVKWGLRKSLPLIELRATFTLLWELDNLECGKPNVDLSSLEETVRKVAEFEDE VIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEK RVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTMLLKFGSGSGSVTTLS GLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAA PDGYEVATAITFTVNEQGQVTVNGEATKGDAHTGSSGS

[0142] In the above sequence, residues 1-214 are the encapsulin subunit sequence, which is C-terminally fused via a glycine-serine linker (residues 215-220) to SpyCatcher (residues 221-328). Additionally, the encapsulin sequence contains cysteine residues introduced by amino acid substitutions at positions 68 and 108 (relative to SEQ ID NO: 126), which form a non-natural disulfide bond that increases stabilization of the nanoparticle.

[0143] In some implementations, the recombinant SARS-CoV-2 S protein or fragment thereof is linked to the protein nanoparticle using the SpyTag:SpyCatcher system, wherein the recombinant SARS-CoV-2 S protein or fragment thereof are C-terminally fused to SpyTag (as in SEQ ID NO: 136) and the protein nanoparticle is a self-assembled encapsulin nanoparticle with subunits fused to the SpyCatcher moiety. A non-limiting example of an encapsulin subunit sequence fused to SpyCatcher for use in the examples provided herein is SEQ ID NO: 126: MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGGGGGHHHHHHGGGGGPYGWEYAAHPLCEVEVLSD ENEVVKWGLRKSLPLIELRATFTLLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEE RKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIED ALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTMLLKFGSGSGSVTTLSGLSGEQGPSGDMTTEEDSA THIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQ VTVNGEATKGDAHTGSSGS

[0144] In the above sequence, residues 1-270 are the encapsulin subunit sequence, which is C-terminally fused via a glycine-serine linker (residues 271-276) to SpyCatcher (residues 277-394). Additionally, the encapsulin sequence contains cysteine residues introduced by4239-112474-02amino acid substitutions at positions 68 and 108 (relative to SEQ ID NO: 126), which form a non-natural disulfide bond that increases stabilization of the nanoparticle. Additionally, residues 41-56 are inserted in the encapsulin loop region for yield improvement purposes.

[0145] For production purposes, in some aspects, the recombinant SARS-CoV-2 S protein or fragment thereof linked to the nanoparticle subunit can include an N-terminal signal peptide that is cleaved during cellular processing. For example, the recombinant SARS-CoV-2 S protein or fragment thereof linked to the protein nanoparticle subunit can include a signal peptide at its N-terminus including, for example, a native recombinant SARS-CoV-2 S signal peptide.

[0146] The protein nanoparticles can be expressed in appropriate cells (e.g., HEK 293 Freestyle cells) and fusion proteins are secreted from the cells self-assembled into nanoparticles. The nanoparticles can be purified using known techniques, for example by a few different chromatography procedures, e.g. Mono Q (anion exchange) followed by size exclusion (SUPEROSE® 6) chromatography. Virus-Like Particles containing recombinant SARS-CoV-2 S protein or fragment thereof

[0147] In some implementations, a virus-like particle (VLP) is provided that includes a disclosed recombinant SARS-CoV-2 Spike protein or fragment thereof. Typically, such VLPs include a recombinant SARS-CoV-2 Spike protein or fragment thereof that is membrane anchored by a C-terminal transmembrane domain, for example the recombinant SARS-CoV-2 Spike protein or fragment thereof is fused to or includes a transmembrane domain and cytosolic tail from SARS-CoV-2. VLPs lack the viral components that are required for virus replication and thus represent a highly attenuated, replication-incompetent form of a virus. However, the VLP can display a polypeptide (e.g., a recombinant SARS- CoV-2 Spike protein or fragment thereof) that is analogous to that expressed on infectious virus particles and can eliciting an immune response to SARS-CoV-2 Spike when administered to a subject. Virus-like particles and methods of their production are known, and viral proteins from several viruses are known to form VLPs, including human papillomavirus, HIV (Kang et al., Biol. Chem.380: 353-64 (1999)), Semliki-Forest virus (Notka et al., Biol. Chem.380: 341-52 (1999)), human polyomavirus (Goldmann et al., J. Virol.73: 4465-9 (1999)), rotavirus (Jiang et al., Vaccine 17: 1005-13 (1999)), parvovirus (Casal, Biotechnology and Applied Biochemistry, Vol 29, Part 2, pp 141-150 (1999)), canine4239-112474-02parvovirus (Hurtado et al., J. Virol.70: 5422-9 (1996)), hepatitis E virus (Li et al., J. Virol. 71: 7207-13 (1997)), and Newcastle disease virus. The formation of such VLPs can be detected by any suitable technique. Examples of suitable techniques known in the art for detection of VLPs in a medium include, e.g., electron microscopy techniques, dynamic light scattering (DLS), selective chromatographic separation (e.g., ion exchange, hydrophobic interaction, and / or size exclusion chromatographic separation of the VLPs) and density gradient centrifugation. III. Polynucleotides and Expression

[0148] Polynucleotides encoding a disclosed recombinant SARS-CoV-2 Spike protein or fragment thereof are also provided. These polynucleotides include DNA, cDNA and RNA sequences which encode the protomer, as well as vectors including the DNA, cDNA and RNA sequences, such as a DNA or RNA vector used for immunization. The genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same protein sequence, or encode a conjugate or fusion protein including the nucleic acid sequence.

[0149] The encoding sequences of native SARS-CoV-2 Spike proteins are well known and readily available. An exemplary nucleic acid sequence encoding SARS-CoV-2 S protein of SEQ ID NO: 131 is provided as SEQ ID NO: 135 (Spike sequence from GenBank Accession No. NC_045512.2, incorporated by reference herein): atgtttgtttttcttgttttattgccactagtctctagtcagtgtgttaatcttacaaccagaactcaattaccc cctgcatacactaattctttcacacgtggtgtttattaccctgacaaagttttcagatcctcagttttacattca actcaggacttgttcttacctttcttttccaatgttacttggttccatgctatacatgtctctgggaccaatggt actaagaggtttgataaccctgtcctaccatttaatgatggtgtttattttgcttccactgagaagtctaacata ataagaggctggatttttggtactactttagattcgaagacccagtccctacttattgttaataacgctactaat gttgttattaaagtctgtgaatttcaattttgtaatgatccatttttgggtgtttattaccacaaaaacaacaaa agttggatggaaagtgagttcagagtttattctagtgcgaataattgcacttttgaatatgtctctcagcctttt cttatggaccttgaaggaaaacagggtaatttcaaaaatcttagggaatttgtgtttaagaatattgatggttat tttaaaatatattctaagcacacgcctattaatttagtgcgtgatctccctcagggtttttcggctttagaacca ttggtagatttgccaataggtattaacatcactaggtttcaaactttacttgctttacatagaagttatttgact cctggtgattcttcttcaggttggacagctggtgctgcagcttattatgtgggttatcttcaacctaggactttt ctattaaaatataatgaaaatggaaccattacagatgctgtagactgtgcacttgaccctctctcagaaacaaag tgtacgttgaaatccttcactgtagaaaaaggaatctatcaaacttctaactttagagtccaaccaacagaatct attgttagatttcctaatattacaaacttgtgcccttttggtgaagtttttaacgccaccagatttgcatctgtt tatgcttggaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataattccgcatcattttcc acttttaagtgttatggagtgtctcctactaaattaaatgatctctgctttactaatgtctatgcagattcattt gtaattagaggtgatgaagtcagacaaatcgctccagggcaaactggaaagattgctgattataattataaatta ccagatgattttacaggctgcgttatagcttggaattctaacaatcttgattctaaggttggtggtaattataat tacctgtatagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggcc ggtagcacaccttgtaatggtgttgaaggttttaattgttactttcctttacaatcatatggtttccaacccact aatggtgttggttaccaaccatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgt ggacctaaaaagtctactaatttggttaaaaacaaatgtgtcaatttcaacttcaatggtttaacaggcacaggt4239-112474-02gttcttactgagtctaacaaaaagtttctgcctttccaacaatttggcagagacattgctgacactactgatgct gtccgtgatccacagacacttgagattcttgacattacaccatgttcttttggtggtgtcagtgttataacacca ggaacaaatacttctaaccaggttgctgttctttatcaggatgttaactgcacagaagtccctgttgctattcat gcagatcaacttactcctacttggcgtgtttattctacaggttctaatgtttttcaaacacgtgcaggctgttta ataggggctgaacatgtcaacaactcatatgagtgtgacatacccattggtgcaggtatatgcgctagttatcag actcagactaattctcctcggcgggcacgtagtgtagctagtcaatccatcattgcctacactatgtcacttggt gcagaaaattcagttgcttactctaataactctattgccatacccacaaattttactattagtgttaccacagaa attctaccagtgtctatgaccaagacatcagtagattgtacaatgtacatttgtggtgattcaactgaatgcagc aatcttttgttgcaatatggcagtttttgtacacaattaaaccgtgctttaactggaatagctgttgaacaagac aaaaacacccaagaagtttttgcacaagtcaaacaaatttacaaaacaccaccaattaaagattttggtggtttt aatttttcacaaatattaccagatccatcaaaaccaagcaagaggtcatttattgaagatctacttttcaacaaa gtgacacttgcagatgctggcttcatcaaacaatatggtgattgccttggtgatattgctgctagagacctcatt tgtgcacaaaagtttaacggccttactgttttgccacctttgctcacagatgaaatgattgctcaatacacttct gcactgttagcgggtacaatcacttctggttggacctttggtgcaggtgctgcattacaaataccatttgctatg caaatggcttataggtttaatggtattggagttacacagaatgttctctatgagaaccaaaaattgattgccaac caatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaagat gtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttggtgcaatttcaagt gttttaaatgatatcctttcacgtcttgacaaagttgaggctgaagtgcaaattgataggttgatcacaggcaga cttcaaagtttgcagacatatgtgactcaacaattaattagagctgcagaaatcagagcttctgctaatcttgct gctactaaaatgtcagagtgtgtacttggacaatcaaaaagagttgatttttgtggaaagggctatcatcttatg tccttccctcagtcagcacctcatggtgtagtcttcttgcatgtgacttatgtccctgcacaagaaaagaacttc acaactgctcctgccatttgtcatgatggaaaagcacactttcctcgtgaaggtgtctttgtttcaaatggcaca cactggtttgtaacacaaaggaatttttatgaaccacaaatcattactacagacaacacatttgtgtctggtaac tgtgatgttgtaataggaattgtcaacaacacagtttatgatcctttgcaacctgaattagactcattcaaggag gagttagataaatattttaagaatcatacatcaccagatgttgatttaggtgacatctctggcattaatgcttca gttgtaaacattcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctc caagaacttggaaagtatgagcagtatataaaatggccatggtacatttggctaggttttatagctggcttgatt gccatagtaatggtgacaattatgctttgctgtatgaccagttgctgtagttgtctcaagggctgttgttcttgt ggatcctgctgcaaatttgatgaagacgactctgagccagtgctcaaaggagtcaaattacattacacataa

[0150] The encoding sequences of native SARS-CoV-2 Spike proteins can be modified to introduce the amino acid substitutions and deletions disclosed herein to prepare the recombinant SARS-CoV-2 Spike proteins and fragments thereof disclosed herein.

[0151] In several implementations, the nucleic acid molecule encodes a precursor of a disclosed SARS-CoV-2 S protein is provided, that, when expressed in an appropriate cell, is processed into SARS-CoV-2 S trimer. For example, the nucleic acid molecule can encode a recombinant SARS-CoV-2 S protein including a N-terminal signal sequence for entry into the cellular secretory system that is proteolytically cleaved in the during processing of the recombinant SARS-CoV-2 S protein in the cell.

[0152] In several implementations, the nucleic acid molecule encodes a precursor SARS- CoV-2 S polypeptide that, when expressed in an appropriate cell, is processed into a disclosed recombinant SARS-CoV-2 S ectodomain protomer including S1 and S2 polypeptides, wherein the recombinant SARS-CoV-2 S ectodomain protomer includes any of the appropriate stabilizing modifications described herein, and optionally can be linked to a trimerization domain, such as a T4 Fibritin trimerization domain.4239-112474-02

[0153] Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are known (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4thed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013).

[0154] Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription- based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

[0155] The polynucleotides encoding a disclosed recombinant SARS-CoV-2 Spike or fragment thereof can include a recombinant DNA which is incorporated into a vector (such as an expression vector) into an autonomously replicating plasmid or virus or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (such as a cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double forms of DNA.

[0156] Polynucleotide sequences encoding a disclosed recombinant SARS-CoV-2 Spike or fragment thereof can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.

[0157] DNA sequences encoding the disclosed recombinant SARS-CoV-2 Spike or fragment thereof can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.4239-112474-02

[0158] Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archea, insect, fungi (for example, yeast), plant, and animal cells (for example, mammalian cells, such as human). Exemplary cells of use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines. Techniques for the propagation of mammalian cells in culture are well-known (see, e.g., Helgason and Miller (Eds.), 2012, Basic Cell Culture Protocols (Methods in Molecular Biology), 4thEd., Humana Press). Examples of commonly used mammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although cell lines may be used, such as cells designed to provide higher expression, desirable glycosylation patterns, or other features. In some implementations, the host cells include HEK293 cells or derivatives thereof, such as GnTI- / -cells (ATCC® No. CRL-3022), or HEK-293F cells.

[0159] Transformation of a host cell with recombinant DNA can be carried out by conventional techniques. Where the host is prokaryotic, such as, but not limited to, E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl2method using standard procedures. Alternatively, MgCl2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.

[0160] When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or viral vectors can be used. Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding a disclosed antigen, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Viral Expression Vectors, Springer press, Muzyczka ed., 2011). Appropriate expression systems such as plasmids and vectors of use in producing proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.4239-112474-02

[0161] In one non-limiting example, a disclosed immunogen is expressed using the pVRC8400 vector (described in Barouch et al., J. Virol., 79 ,8828-8834, 2005, which is incorporated by reference herein).

[0162] Modifications can be made to a nucleic acid encoding a disclosed recombinant SARS- CoV-2 S ectodomain protomer without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, termination codons, a methionine added at the amino terminus to provide an initiation, site, additional amino acids placed on either terminus to create conveniently located restriction sites, or additional amino acids (such as poly His) to aid in purification steps.

[0163] In some implementations, the disclosed recombinant SARS-CoV-2 S ectodomain protomer can be expressed in cells under conditions where the recombinant SARS-CoV-2 S ectodomain protomer can self-assemble into trimers which are secreted from the cells into the cell media. In such implementations, each recombinant SARS-CoV-2 S ectodomain protomer contains a leader sequence (signal peptide) that causes the protein to enter the secretory system, where the signal peptide is cleaved and the protomers form a trimer, before being secreted in the cell media. The medium can be centrifuged and recombinant SARS- CoV-2 S ectodomain trimer purified from the supernatant. IV. Viral Vectors

[0164] A nucleic acid molecule encoding a disclosed recombinant SARS-CoV-2 Spike or fragment thereof can be included in a viral vector, for example, for expression of the immunogen in a host cell, or for immunization of a subject as disclosed herein. In some implementations, the viral vectors are administered to a subject as part of a prime-boost vaccination. In several implementations, the viral vectors are included in a vaccine, such as a primer vaccine or a booster vaccine for use in a prime-boost vaccination.

[0165] In several implementations, the viral vector can be replication-competent. For example, the viral vector can have a mutation in the viral genome that does not inhibit viral replication in host cells. The viral vector also can be conditionally replication-competent. In other examples, the viral vector is replication-deficient in host cells.4239-112474-02

[0166] A number of viral vectors have been constructed, that can be used to express the disclosed antigens, including polyoma, i.e., SV40 (Madzak et al., 1992, J. Gen. Virol., 73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407- 4412; Quantin et al., 1992, Proc. Natl. Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239; Stratford- Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256), vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol., 158:91-123; On et al., 1990, Gene, 89:279-282), herpes viruses including HSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J. Virol., 66:29522965; Fink et al., 1992, Hum. Gene Ther.3:11-19; Breakfield et al., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199), Sindbis viruses (H. Herweijer et al., 1995, Human Gene Therapy 6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol.11:18-22; I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377) and retroviruses of avian (Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J. Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol., 54:401-407), and human origin (Page et al., 1990, J. Virol., 64:5370- 5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739). Baculovirus (Autographa californica multinuclear polyhedrosis virus; AcMNPV) vectors are also known in the art, and may be obtained from commercial sources (such as PharMingen, San Diego, Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).

[0167] In several implementations, the viral vector can include an adenoviral vector that expresses a protomer of a disclosed recombinant SARS-CoV-2 Spike protein or fragment thereof. Adenovirus from various origins, subtypes, or mixture of subtypes can be used as the source of the viral genome for the adenoviral vector. Non-human adenovirus (e.g., simian, chimpanzee, gorilla, avian, canine, ovine, or bovine adenoviruses) can be used to generate the adenoviral vector. For example, a simian adenovirus can be used as the source of the viral genome of the adenoviral vector. A simian adenovirus can be of serotype 1, 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, 39, 48, 49, 50, or any other simian adenoviral serotype. A simian adenovirus can be referred to by using any suitable abbreviation known in the art, such as, for example, SV, SAdV, SAV or sAV. In some implementations, a simian adenoviral vector is a4239-112474-02simian adenoviral vector of serotype 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, or 39. In one implementation, a chimpanzee serotype C Ad3 vector is used (see, e.g., Peruzzi et al., Vaccine, 27:1293-1300, 2009). Human adenovirus can be used as the source of the viral genome for the adenoviral vector. Human adenovirus can be of various subgroups or serotypes. For instance, an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49 and 51), or any other adenoviral serotype. Examples of replication-deficient adenoviral vectors, including multiply replication-deficient adenoviral vectors, are disclosed in U.S. Patent Nos.5,837,511; 5,851,806; 5,994,106; 6,127,175; 6,482,616; and 7,195,896, and International Patent Application Nos. WO 94 / 28152, WO 95 / 02697, WO 95 / 16772, WO 95 / 34671, WO 96 / 22378, WO 97 / 12986, WO 97 / 21826, and WO 03 / 022311. V. Immunogenic Compositions

[0168] Immunogenic compositions comprising a disclosed immunogen (e.g., recombinant SARS-CoV-2 Spike or fragment thereof) and a pharmaceutically acceptable carrier are also provided. Such compositions can be administered to subjects by a variety of administration modes, for example, intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes. Methods for preparing administrable compositions are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 22nded., London, UK: Pharmaceutical Press, 2013.

[0169] Thus, an immunogen described herein can be formulated with pharmaceutically acceptable carriers to help retain biological activity while also promoting increased stability during storage within an acceptable temperature range. Potential carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffer saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents. The resulting aqueous solutions may be packaged for4239-112474-02use as is or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple dosing.

[0170] Formulated compositions, especially liquid formulations, may contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually ≦1% w / v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben. A bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component.

[0171] The immunogenic compositions of the disclosure can contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

[0172] The immunogenic composition may optionally include an adjuvant to enhance an immune response of the host. Suitable adjuvants are, for example, toll-like receptor agonists, alum, AlPO4, alhydrogel, Lipid-A and derivatives or variants thereof, oil-emulsions, saponins, neutral liposomes, liposomes containing the vaccine and cytokines, non-ionic block copolymers, and chemokines. Non-ionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL^ (3-O- deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA), may be used as an adjuvant. These adjuvants have the advantage in that they help to stimulate the immune system in a non-specific way, thus enhancing the immune response to a pharmaceutical product.

[0173] In some implementations, the composition can be provided as a sterile composition. The pharmaceutical composition typically contains an effective amount of a disclosed immunogen and can be prepared by conventional techniques. Typically, the amount of immunogen in each dose of the immunogenic composition is selected as an amount which elicits an immune response without significant, adverse side effects. In some implementations, the composition can be provided in unit dosage form for use to elicit an immune response in a subject, for example, to prevent or inhibit SARS-CoV-2 infection or COVID19 disease in the subject. A unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or4239-112474-02more preselected unit dosages, and / or a metering mechanism for administering the unit dose or multiples thereof. In other examples, the composition further includes an adjuvant. VI. Methods of Inducing an Immune Response

[0174] The disclosed immunogens (e.g., recombinant SARS-CoV-2 Spike or fragment thereof, a nucleic acid molecule (such as an RNA molecule) or vector encoding a recombinant SARS-CoV-2 Spike or fragment thereof, or a nanoparticle or virus like particle comprising a disclosed recombinant SARS-CoV-2 Spike or fragment thereof) can be administered to a subject to induce an immune response to SARS-CoV-2 S protein in the subject. In a particular example, the subject is a human. The immune response can be a protective immune response, for example a response that inhibits subsequent infection with SARS-CoV-2. Elicitation of the immune response can also be used to treat or inhibit SARS- CoV-2 infection and illnesses associated with the SARS-CoV-2 infection.

[0175] A subject can be selected for treatment that has or is at risk for developing SARS- CoV-2 infection, for example because of exposure or the possibility of exposure to the SARS-CoV-2. Following administration of a disclosed immunogen, the subject can be monitored for infection or symptoms associated with SARS-CoV-2 infection.

[0176] Typical subjects intended for immunization with the immunogens and methods of the present disclosure include humans, as well as non-human primates and other animals. To identify subjects for immunization according to the methods of the disclosure, accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease or condition, or to determine the status of an existing disease or condition in a subject. These screening methods include, for example, conventional work-ups to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods, such as various ELISA and other immunoassay methods to detect and / or characterize SARS- CoV-2 infection. These and other routine methods allow the clinician to select patients in need of immunization using the methods and pharmaceutical compositions of the disclosure.

[0177] The administration of a disclosed immunogen can be for prophylactic or therapeutic purpose. When provided prophylactically, the immunogen is provided in advance of any symptom, for example, in advance of infection. The prophylactic administration of the immunogen serves to prevent or ameliorate any subsequent infection. When provided4239-112474-02therapeutically, the immunogen is provided at or after the onset of a symptom of infection, for example, after development of a symptom of SARS-CoV-2 infection or after diagnosis with the SARS-CoV-2 infection. The immunogen can thus be provided prior to the anticipated exposure to the SARS-CoV-2 so as to attenuate the anticipated severity, duration or extent of an infection and / or associated disease symptoms, after exposure or suspected exposure to the SARS-CoV-2, or after the actual initiation of an infection.

[0178] The immunogens described herein, and immunogenic compositions thereof, are provided to a subject in an amount effective to induce or enhance an immune response against the SARS-CoV-2 S protein in the immunogen in the subject, preferably a human. The actual dosage of disclosed immunogen will vary according to factors such as the disease indication and particular status of the subject (for example, the subject’s age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the composition for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response.

[0179] An immunogenic composition including one or more of the disclosed immunogens can be used in coordinate (or prime-boost) vaccination protocols or combinatorial formulations. In certain examples, novel combinatorial immunogenic compositions and coordinate immunization protocols employ separate immunogens or formulations, each directed toward eliciting an anti-viral immune response, such as an immune response to SARS-CoV-2 S protein. Separate immunogenic compositions that elicit the anti-viral immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic compositions) in a coordinate (or prime-boost) immunization protocol.

[0180] There can be several boosts, and each boost can be a different disclosed immunogen. In some examples that the boost may be the same immunogen as another boost, or the prime. The prime and boost can be administered as a single dose or multiple doses, for example two doses, three doses, four doses, five doses, six doses or more can be administered to a subject over days, weeks or months. Multiple boosts can also be given, such one to five (e.g., 1, 2, 3, 4 or 5 boosts), or more. Different dosages can be used in a series of sequential4239-112474-02immunizations. For example a relatively large dose in a primary immunization and then a boost with relatively smaller doses.

[0181] In some implementations, the boost can be administered about two, about three to eight, or about four, weeks following the prime, or about several months after the prime. In some implementations, the boost can be administered about 5, about 6, about 7, about 8, about 10, about 12, about 18, about 24, months after the prime, or more or less time after the prime. Periodic additional boosts can also be used at appropriate time points to enhance the subject's “immune memory.” The adequacy of the vaccination parameters chosen, e.g., formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program. In addition, the clinical condition of the subject can be monitored for the desired effect, e.g., prevention of infection or improvement in disease state (e.g., reduction in viral load). If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of immunogenic composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response.

[0182] In some implementations, the prime-boost method can include DNA-primer and protein-boost vaccination protocol to a subject. The method can include two or more administrations of the nucleic acid molecule or the protein.

[0183] For protein therapeutics, typically, each human dose will comprise 1-1000 µg of protein, such as from about 1 µg to about 100 µg, for example, from about 1 µg to about 50 µg, such as about 1 µg, about 2 µg, about 5 µg, about 10 µg, about 15 µg, about 20 µg, about 25 µg, about 30 µg, about 40 µg, or about 50 µg.

[0184] The amount utilized in an immunogenic composition is selected based on the subject population (e.g., infant or elderly). An optimal amount for a particular composition can be ascertained by standard studies involving observation of antibody titers and other responses in subjects. It is understood that a therapeutically effective amount of a disclosed immunogen, such as a disclosed recombinant SARS-CoV-2 Spike or fragment thereof, viral vector, nanoparticle, or nucleic acid molecule, in an immunogenic composition, can include an amount that is ineffective at eliciting an immune response by administration of a single dose, but that is effective upon administration of multiple dosages, for example in a prime- boost administration protocol.4239-112474-02

[0185] Upon administration of a disclosed immunogen of this disclosure, the immune system of the subject typically responds to the immunogenic composition by producing antibodies specific for SARS-CoV-2 Spike . Such a response signifies that an immunologically effective dose was delivered to the subject.

[0186] In some implementations, the antibody response of a subject will be determined in the context of evaluating effective dosages / immunization protocols. In most instances it will be sufficient to assess the antibody titer in serum or plasma obtained from the subject. Decisions as to whether to administer booster inoculations and / or to change the amount of the therapeutic agent administered to the individual can be at least partially based on the antibody titer level. The antibody titer level can be based on, for example, an immunobinding assay which measures the concentration of antibodies in the serum which bind to an antigen including, for example, the recombinant SARS-CoV-2 Spike or fragment thereof included in the immunogen.

[0187] SARS-CoV-2 infection does not need to be completely eliminated or reduced or prevented for the methods to be effective. For example, elicitation of an immune response to SARS-CoV-2 with one or more of the disclosed immunogens can reduce or inhibit SARS- CoV-2 infection by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable infected cells), as compared to SARS-CoV-2 infection in the absence of the immunogen. In additional examples, SARS- CoV-2 replication can be reduced or inhibited by the disclosed methods. SARS-CoV-2 replication does not need to be completely eliminated for the method to be effective. For example, the immune response elicited using one or more of the disclosed immunogens can reduce SARS-CoV-2 replication by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SARS-CoV-2 replication, as compared to SARS-CoV-2 replication in the absence of the immune response.

[0188] In some implementations, the disclosed immunogen is administered to the subject simultaneously with the administration of the adjuvant. In other examples, the disclosed immunogen is administered to the subject after the administration of the adjuvant and within a sufficient amount of time to induce the immune response.4239-112474-02

[0189] One approach to administration of nucleic acids is direct immunization with plasmid DNA, such as with a mammalian expression plasmid. Immunization by nucleic acid constructs is well known in the art and taught, for example, in U.S. Patent No.5,643,578 (which describes methods of immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell-mediated or a humoral response), and U.S. Patent No.5,593,972 and U.S. Patent No.5,817,637 (which describe operably linking a nucleic acid sequence encoding an antigen to regulatory sequences enabling expression). U.S. Patent No.5,880,103 describes several methods of delivery of nucleic acids encoding immunogenic peptides or other antigens to an organism. The methods include liposomal delivery of the nucleic acids (or of the synthetic peptides themselves), and immune-stimulating constructs, or ISCOMSTM, negatively charged cage-like structures of 30-40 nm in size formed spontaneously on mixing cholesterol and Quil ATM(saponin). Protective immunity has been generated in a variety of experimental models of infection, including toxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMSTMas the delivery vehicle for antigens (Mowat and Donachie, Immunol. Today 12:383, 1991). Doses of antigen as low as 1 µg encapsulated in ISCOMSTMhave been found to produce Class I mediated CTL responses (Takahashi et al., Nature 344:873, 1990).

[0190] In some implementations, a plasmid DNA vaccine is used to express a disclosed immunogen in a subject. For example, a nucleic acid molecule encoding a disclosed immunogen can be administered to a subject to induce an immune response to the recombinant SARS-CoV-2 Spike or fragment thereof included in the immunogen. In some implementations, the nucleic acid molecule can be included on a plasmid vector for DNA immunization, such as the pVRC8400 vector (described in Barouch et al., J. Virol, 79, 8828- 8834, 2005).

[0191] In another approach to using nucleic acids for immunization, a disclosed recombinant SARS-CoV-2 Spike or fragment thereof can be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalo virus or other viral vectors can be used to express the peptide or protein, thereby eliciting a CTL response. For example, vaccinia vectors and methods useful in immunization protocols are described in U.S. Patent No.4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the peptides (see Stover, Nature 351:456-460, 1991).4239-112474-02

[0192] In one implementation, a nucleic acid encoding a disclosed recombinant SARS-CoV- 2 Spike or fragment thereof is introduced directly into cells. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOS^ Gene Gun. The nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter. Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 µg / kg to about 50 mg / kg, and typically are about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Patent No.5,589,466).

[0193] In another implementation, an mRNA-based immunization protocol can be used to deliver a nucleic acid encoding a recombinant SARS-CoV-2 Spike or fragment thereof to elicit an immune response to Sars-CoV-2 Spike. mRNA vaccines preclude safety concerns about DNA integration into the host genome and can be directly translated in the host cell cytoplasm. Moreover, cell-free, in vitro synthesis of RNA avoids the manufacturing complications associated with viral vectors.

[0194] In some implementations, mRNA vaccination is achieved using mRNA encoding a recombinant SARS-CoV-2 Spike or fragment thereof as described herein and formulated as a lipid nanoparticle according to known methods, such as those described in WO2021154763, US20210228707, WO2017070626 and US2019 / 0192646. See, also, Jackson et al., N Engl J Med., 383(20):1920-1921, 2020. For example the mRNA component is a modified mRNA with 1-methylpseudouridine in place of uridine and a 7mG(5’)ppp(5’)N1mpNp cap. The mRNA sequence includes a 5’ untranslated region (UTR), immunogen encoding sequence (recombinant SARS-CoV-2 Spike protein or fragment thereof), a 3’ UTR, and a polyA tail. In some implementations, the ORF sequence is codon optimized relative to native sequence for mRNA expression in a human and to increase stability. In several implementations, the mRNA is formulated in a lipid nanoparticle; for example, comprising a PEG-modified lipid, a non-cationic lipid, a sterol, an ionizable lipid, or any combination thereof. In some implementations, the lipid nanoparticle is composed of 50 mol% ionizable lipid ((2 hydroxyethyl)(6 oxo 6-(undecycloxy)hexyl)amino)octanoate, 10 mol% 1,2 distearoyl sn glycerol-3 phosphocholine (DSPC), 38.5 mol% cholesterol, and 1.5 mol% 1- monomethoxypolyethyleneglycol-2,3,dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG). The mRNA / lipid nanoparticle composition may be provided in any suitable carrier, such as a sterile liquid for injection at a concentration of4239-112474-020.5 mg / mL in 20 mM trometamol (Tris) buffer containing 87 mg / mL sucrose and 10.7 mM sodium acetate, at pH 7.5 and with appropriate diluent.

[0195] Additional exemplary forms of RNA-based vaccination that can be used to deliver a nucleic acid encoding a recombinant SARS-CoV-2 Spike or fragment thereof as described herein include conventional non-amplifying mRNA immunization (see, e.g., Petsch et al., “Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection,” Nature biotechnology, 30(12):1210–6, 2012) and self-amplifying mRNA immunization (see, e.g., Geall et al., “Nonviral delivery of self-amplifying RNA vaccines,” PNAS, 109(36): 14604-14609, 2012; Magini et al., “Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge,” PLoS One, 11(8):e0161193, 2016; and Brito et al., “Self-amplifying mRNA vaccines,” Adv Genet., 89:179-233, 2015). In another implementation, a circular RNA (circRNA)-based immunization protocol can be used to deliver a nucleic acid encoding the recombinant SARS-CoV-2 Spike or fragment thereof to elicit an immune response to the SARS-CoV-2 Spike. In contrast to linear RNA, circRNA is stable due to its covalently closed ring structure, which protects it from exonuclease- mediated degradation. Although circRNA lacks the essential elements for cap-dependent translation, it can be engineered to enable protein translation through internal ribosome entry site (IRES) or the m6A modification incorporated to its 5’ UTR region. (See, e.g., Wesselhoeft, P. S. Kowalski, D. G. Anderson, Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat Commun.9, 2629, 2018; Yang et al., Extensive translation of circular RNAs driven by N(6)-methyladenosine. Cell Res.27, 626-641, 2017; Kristensen et al. The biogenesis, biology, and characterization or circular RNAs. Nat. Rev. Genetics, 20, 675-691, 2029).

[0196] In some implementations, administration of a therapeutically effective amount of one or more of the disclosed immunogens to a subject induces a neutralizing antibody response in the subject. To assess neutralization activity, following immunization of a subject, serum can be collected from the subject at appropriate time points, frozen, and stored for neutralization testing. Methods to assay for neutralization activity include, but are not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry based assays, single-cycle infection assays.4239-112474-02EXAMPLES

[0197] The following examples are provided to illustrate certain particular features and / or aspects of the disclosed technology. These examples should not be construed to limit the disclosure to the particular features or aspects described. Example 1

[0198] Provided herein are examples of vaccines that induce broad and potent neutralizing antibodies against SARS-CoV-2 variants, including most recent variants KP.2 and KP.3, as well as against SARS-CoV or MERS-CoV when tested in a mouse model. Summary

[0199] Current COVID-19 vaccines provide safe and effective protection against severe disease and hospitalization caused by SARS-CoV-2 viruses characterized by sequences that closely match those of the vaccine. However, they generally exhibit reduced neutralizing antibody responses and protection against mismatched viruses. Therefore, COVID-19 vaccines must be updated frequently. The SARS-CoV-2 S glycoprotein is targeted by a diverse array of antibodies, with RBD-directed antibodies accounting for most of the serum neutralizing activity. However, most of these antibodies are variant-specific, with a limited capacity to neutralize a broad spectrum of evolving variants.

[0200] To develop a vaccine that induces a very broad and potent antibody response against SARS-CoV-2 variants as well as more distantly related coronavirus, we employed three strategies: 1) We designed SARS2 S1R (NTD+RBD) subunit vaccines with amino acid substitutions in variable regions of the RBD, while preserving conserved epitopes recognized by potently neutralizing antibodies. 2) We developed novel vaccine antigens that link the S1R of SARS2 with the S1 subunit of SARS1 as fusion protein (SARS2 S1R-SARS1 S1).3) We displayed designed SARS2 S1R or RBD, or SARS2 S1R-SARS1 S1 proteins on nanoparticles to enhance immunogenicity. Our designed vaccines induced broadly neutralizing antibodies against SARS-CoV-2 variants, ranging from the earliest to the most current variants, and also generated neutralizing antibodies against SARS-CoV. When displayed on nanoparticles, the designed vaccines induced potently neutralizing antibodies against both SARS-CoV-2 variants and SARS-CoV. A nanoparticle displaying S1R protein with the NTD linked to the nanoparticle and the RBD exposed elicited more potent neutralizing antibodies against SARS-CoV-2 variants and SARS-CoV. Although the results4239-112474-02generated with a protein subunit vaccine, our designs can be adapted for use with gene-based platforms, such as DNA, mRNA or viral vector. Additionally, these designs can also be displayed on other nanoparticles, such as H. Pylori ferritin, insect ferritin or lumazine synthase. These vaccines demonstrate potential utility against new SARS-CoV-2 variants that may emerge, and against coronaviruses with pandemic potential. As described herein:

[0201] SARS-CoV-2 S1R protein (SARS2 S1R 8mut4) induced potent neutralizing antibodies against SARS-CoV-2 variants, including D614G, BA.4 / 5, XBB.1.5, JN.1 and the newly emerged variants, KP.2 and KP.3. This vaccine also induced neutralizing antibodies against SARS-CoV. In contrast, the S1R from the original SARS-CoV-2 (SARS2 S1R) induced neutralizing antibodies mainly against the original SARS-CoV-2 isolate. The breadth of neutralizing antibody responses induced by the SARS-CoV-2 S1R (SARS2 S1R 8mut4) protein has not been reported by other vaccine platforms, including currently licensed vaccines. (see results in Fig.5).

[0202] SARS-CoV-2 S1R and SARS-CoV S1 fusion protein (SARS2 S1R 8mut4-SARS1 S1) induced potent neutralizing antibodies against SARS-CoV-2 variants, including D614G, BA.4 / 5, XBB.1.5, JN.1 and the most recent variants, KP.2 and KP.3. This vaccine also induced potent neutralizing antibodies against SARS-CoV. The breadth of neutralizing antibody responses induced by the SARS-CoV-2 S1R-SARS1 S1 fusion protein has not been reported by other vaccine platforms, including currently licensed vaccines. (see results in Figs.4-5).

[0203] SARS-CoV-2 RBD 8mut4 protein displayed on nanoparticles (SARS2 RBD 8mut4- np) induced potent neutralizing antibodies against SARS-CoV-2 variants, including D614G, BA.4 / 5, XBB.1.5, JN.1 and the most recent variants, KP.2 and KP.3. This vaccine also induced neutralizing antibodies against SARS-CoV. In contrast, the designed SARS2 RBD 8mut4 protein alone induced low titers of neutralizing antibodies against the variants tested. The breadth and potency of neutralizing antibody responses induced by the Designed SARS- CoV-2 RBD 8mut4-np protein has not been reported by other vaccine platforms, including currently licensed vaccines. (see results in Fig.6). Introduction

[0204] Multiple COVID-19 vaccines have been approved worldwide to generate antibody responses targeting the SARS-CoV-2 spike (S) protein. These vaccines provided safe and4239-112474-02effective protection against severe disease and hospitalization with the original SARS-CoV-2 isolate, which emerged in 2019 and quickly swept the globe, creating a pandemic associated with millions of deaths. Continued viral evolution led to the sequential emergence of multiple SARS-CoV-2 variants with distinct antigenic properties relative to the earlier isolates, allowing escape from neutralizing antibodies. As a result, breakthrough infections have become common, and the neutralizing activity of monoclonal antibody therapies has been compromised, resulting in the withdrawal of their regulatory authorization.

[0205] The SARS-CoV-2 S glycoprotein is targeted by a wide array of antibodies, with RBD- directed antibodies contributing the most to serum-neutralizing activity in convalescent and vaccinated individuals. In contrast, the N-terminal domain (NTD) is predominantly targeted by variant-specific neutralizing antibodies. The SARS-CoV-2 S2 subunit is considerably more conserved than the S1 subunit, which includes the NTD, RBD and two subdomains. However, S2-directed antibodies generally exhibit limited or no neutralization activity as compared to highly effective RBD-directed antibodies. Therefore, vaccines capable of inducing broad and potent neutralizing antibodies hold the promise in reducing the necessity for frequent vaccine updates.

[0206] Our aim was to develop a SARS-CoV-2 S1R (NTD+RBD) subunit vaccine that elicits potent neutralizing antibodies against both SARS-CoV-2 variants and SARS-CoV. We achieved this by introducing eight amino acid substitutions into the more variable antigenic sites on the RBD, while preserving conserved antigenic sites to focus vaccine-elicited responses on conserved epitopes recognized by antibodies characterized by broad neutralizing activity. Immunization of mice with these designed SARS-CoV-2 S1R vaccines resulted in the production of broadly neutralizing antibodies against SARS-CoV-2 variants, including the original virus, variants evolve sequentially during the pandemic, and currently circulating variants, KP.2 and KP.3. These vaccines also elicit antibodies that neutralize SARS-CoV, demonstrating potential utility against new SARS-CoV-2 variants that may emerge, and against coronaviruses with pandemic potential. Neutralizing antibody titers elicited by our designed S1R vaccines were comparable against the original virus and the immune evasive variants. Additionally, the immunization of mice with the designed SARS- CoV-2 RBD displayed on nanoparticles induced broad and potent neutralizing antibodies against SARS-CoV-2 variants, including the earliest to the most recent variants, KP.2 and KP.3, as well as against SARS-CoV. The breadth and potency of neutralization induced by4239-112474-02these designed vaccines, as described in this application has not been observed with other vaccines, including currently licensed vaccines. Methods for vaccine production and antigenicity assessment in vitro and Immunogenicity in vivo

[0207] Constructs. The sequences of protein subunit vaccines described within contain the murine IL-2 signal sequence, SARS-CoV-2 S1, S1R or RBD, followed by a GS liker, the HRV 3C (human rhinovirus protease 3C) cleavage site and 6 histidine tag. The sequences described within used for producing nanoparticles contain a ScFc-purification tag-HRV 3C followed by SARS-CoV-2 S1, S1R or RBD and a SpyTag. The sequences of DNA vaccines described within contain IL-2 signal sequence, SARS-CoV-2 S1, S1R or RBD. S2P contains amino acid 1-1208, 2P (K986P / V987P) and furin cleavage site replaced with GSAS. S2P-FL contains amino acid 1-1273, 2P (K986P / V987P) and native furin cleavage site.

[0208] Protein expression and purification. All proteins were expressed on Expi293 cells. His-tagged proteins were purified with NTA column followed by HiLoad 16 / 600 Superdex 200pg. SpyTag proteins containing ScFc on the N-terminal were purified using a Protein A column and Superose 6 SEC column. The EnDS-SpyCatcher protein was expressed in E. coli and purified by heat treatment, saturated ammonium sulfate precipitation and SEC purification.

[0209] Nanoparticle conjugation and purification. SpyTag and SpyCatcher proteins were mixed in PBS for conjugation at a molar ratio of 1.2:1. After conjugation, the mixture was purified using a Superose 6 SEC column to remove the excess SpyTag proteins. SDS-PAGE was used to determine the conjugation and protein components. Different nanoparticles were analyzed by negative stain EM as well.

[0210] Antigenic analysis with Octet / ELISA. Monomeric proteins and / or nanoparticles’ antigenic properties were measured by Octet / ELISA with mAbs targeting diverse epitopes.

[0211] Immunization of mice. Balb / c mice were immunized intramuscularly with proteins or nanoparticles at week 0 and 4. Serum were collected 2-4 weeks after the boost to measure binding and neutralizing antibodies.

[0212] Binding by ELISA. Serum binding to spike proteins from SARS-CoV-2 variants and SARS-CoV was performed by ELISA. Mapping to determine serum binding properties on S-4239-112474-022P or RBD was performed by ELISA-based or Octet / BLI-based competition assays using previously characterized mAbs specific for defined RBD epitopes.

[0213] Neutralization. Serum neutralizing antibody activity was measured using pseudotyped lentivirus particles bearing coronavirus spike protein in a quantitative infection- inhibition assay. Neutralization against SARS-CoV-2 variants,including D614G, BA.4 / 5, XBB.1.5, JN.1, and recent variants KP.2, KP.3, SARS-CoV were tested. Results

[0214] Endemic CoV S prime with SARS-CoV-2 S boost didn’t induce higher neutralizing antibodies against SARS-CoV-2 (FIG.1). Mice were primed at weeks 0 and 4 with 10µg of the indicated CoV S as a DNA vaccine and boosted at week 8 with10 µg of SARS-CoV-2 S DNA by intramuscular injection plus electroporation (IM+EP). Serum samples were assessed for neutralizing antibodies at week 10 using a SARS-CoV-2 S pseudotyped lentivirus neutralization assay on 293-TMPRSS2-ACE2 cells. SARS-1 (Group3), HKU1 (Group5), OC43 (Group6), NL63 (Group 7), 229E (Group 8) spike prime did not induce higher neutralization to SARS-2. The titers were similar to single SARS-2 S immunization. Further, SARS-1 (Group2) did not boost anti-SARS-2 neutralization titers, and a 3rddose administrated 4 weeks after the 2nddose did not boost anti-SARS-2 neutralization titers (Group1).

[0215] SARS1 and SARS2 S prime / boost didn’t induce higher neutralizing antibodies against either (FIGs.2A-2B). Mice were primed at weeks 0 and 4 and boosted at week 8 with 10µg of indicated SARS-CoV-2 or SARS-CoV-1 S DNA by IM+EP. Serum samples were assessed for neutralizing antibodies at week 10 using a SARS-CoV-2 and SARS-CoV-1 S pseudotyped lentivirus neutralization assay on 293-TMPRSS2-ACE2 cells. SARS-1 (Gr3) spike prime did not induce higher neutralization to SARS-2 (titers are similar to single SARS-2 S immunization). SARS-1 (Gr2) did not boost anti-SARS-2 neutralization titers. SARS-2 (Gr2) spike prime did not induce higher neutralization titers to SARS-1 (titters are similar to single SARS-2 immunization). SARS-2 (Gr3) did not boost anti-SARS-1 neutralization titers. A 3rddose administrated 4 weeks after the 2nddose did not boost anti- SARS-2 (Gr1) or anti-SARS-1 (Gr4) neutralization titers.

[0216] SARS2-SARS1 or MERS S1-S1 protein induced neutralizing antibodies against both SARS-CoV-2 and SARS-CoV or MERS-CoV (FIG.3). Mice were immunized at weeks 04239-112474-02and 4 with 10 µg of SARS2 S1, SARS1 S1 and SARS2-SARS1 S1-S1 protein (top) or SARS2 S1, MERS S1 and SARS2-MERS S1-S1 protein (bottom) plus ribi as adjuvant by intramuscular injection (IM). Serum samples were assessed for neutralizing antibodies at week 8 using a pseudotyped lentivirus neutralization assay against SARS-CoV-2 and SARS- CoV on 293-TMPRSS2-ACE2 cells or against MERS-CoV on Huh7.5 cells. SARS2 S1- SARS S1 immunized mice induced high titers of neutralizing antibodies against both SARS- CoV-2 and SARS-CoV. SARS2 S1-MERS S1 immunized mice induced high titers of neutralizing antibodies against both SARS-CoV-2 and MERS-CoV.

[0217] SARS2 S1R 8mut4-SARS1 S1 protein induced neutralizing antibodies against SARS- CoV-2 variants and SARS-CoV (FIG.4). Mice were immunized at weeks 0 and 4 with 10 µg of SARS2 S1R, SARS1 S1 and SARS2 S1R 8mut4-SARS1 S1 protein plus ribi as adjuvant by IM. Serum samples were assessed for neutralizing antibodies at week 8 using a pseudotyped lentivirus neutralization assay against SARS-CoV-2 variants and SARS-CoV on 293-TMPRSS2-ACE2 cells. SARS2 S1R induced neutralization titers mainly to D614G. SARS1 S1 induced neutralization titers to SARS-CoV. SARS2-CoV-2 S1R 8mut4-SARS1 S1 induced antibodies neutralization titers to D614G and omicron variants as well as neutralization titers to SARS-CoV.

[0218] SARS2 S1R 8mut4-SARS1 S1 protein induced neutralizing antibodies against SARS- CoV-2 variants and SARS-CoV (FIG.5). Mice were immunized at weeks 0 and 4 with 10 µg of SARS2 S1R, SARS1 S1, SARS2 S1R-8mut4 and SARS2 S1R 8mut4-SARS1 S1 protein plus ribi as adjuvant by IM. Serum samples were assessed for neutralizing antibodies at week 6 using a pseudotyped lentivirus neutralization assay against SARS-CoV-2 variants and SARS-CoV on 293-TMPRSS2-ACE2 cells. SARS2 S1R induced neutralization titers mainly to D614G. SARS2-CoV-2 S1R 8mut4 induced neutralization titers cross SARS-CoV-2 variants including most recent variants, KP.2, KP.3 and SARS-CoV. SARS1 S1 induced neutralization titers only to SARS-CoV. SARS2-CoV-2 S1R 8mut4-SARS1 S1 induced antibodies neutralization titers to D614G and omicron variants as well as neutralization titers to SARS-CoV.

[0219] SARS2 RBD 8mut4 nanoparticle protein induced neutralizing antibodies against SARS-CoV-2 variants and SARS-CoV (FIG.6). Mice were immunized at weeks 0 and 4 with 10, 2 or 0.4 µg of SARS2 RBD-8mut4 nanoparticle (EnDS, stabilized Encapsulin) or 10 µg SARS2 RBD-8mut4 protein plus adjuvant by IM. Serum samples were assessed for neutralizing antibodies at week 6 using a pseudotyped lentivirus neutralization assay against4239-112474-02SARS-CoV-2 variants and SARS-CoV on 293-TMPRSS2-ACE2 cells. SARS2-CoV-2 RBD 8mut4 induced weakly neutralization titers to D614G, BA.4 / 5 and XBB.1.5. SARS2-CoV-2 RBD 8mut4-np induced high titers of neutralizing antibodies against SARS-CoV-2 variants, including most recent variants, KP.2 and KP.3, and SARS-CoV.

[0220] Figures 7-17 provide additional details regarding the production and antigenicity of the immunogens described herein.

[0221] Figure 11 provides a schematic diagram illustrating nanoparticle production for some of the immunogens assessed herein. For additional detail on encapsuling-based nanoparticles linked to antigen using an iso-peptide bond system, see Wang et al., Extraordinary Titer and Broad Anti-SARS-CoV-2 Neutralization Induced by Stabilized RBD Nanoparticles from Strain BA.5, Vaccines, 12(1), 37.2024, which is incorporated by reference herein.

[0222] Figure 18A-18C describes rational vaccine designs guided by mAb insights. Figure 18A shows rational of designs. The footprints of the representative mAb (Column4 in bold) from each class (class I, II, III, I / IV or IV, and V) are shown on the RBD (column2). The ACE2 binding interface is outlined with a black line (column2). Examples of class I, II and III mAbs have lost neutralization activity against emerging variants (column 5), with specific residue changes in the variants (column 6) identified as key contributors to this reduced efficacy. In contrast, class IV and V mAbs target more conserved region of the RBD and most of them retain broad reactivity across variants. Figure 18B-18C shows RBD residue substitutions on designed vaccines. These panels illustrate the location of substituted residues on the RBD used in the designed vaccines. Combinations of amino acid substitutions were used to generate sequential vaccine variants (3mut, 5mut, 8mut4, 9mut, 10 mut and 11mut). Substituted residues are color-coated to indicate incremental additions.

[0223] Figure 19. SARS2 S1R 8mut, 10mut and 11mut proteins induced potent neutralizing antibodies against SARS-CoV-2 variants and SARS-CoV. Mice were immunized at weeks 0 and 4 with 10ug of SARS2 S1R-8mut, S1R-9mut, S1R-10mut or S1R-11mut proteins plus ribi as adjuvant by intramuscular injection (IM). Serum samples were assessed at week 6 for binding using Octet and for neutralization using a pseudotyped lentivirus neutralization assay against SARS-CoV-2 variants and SARS-CoV on 293-TMPRSS2-ACE2 cells. SARS2 S1R 8mut4, 10mut and 11mut proteins induced potent antibodies that bound to the RBD proteins from SARS-CoV-2 variants (D614G, XBB.1.5, JN.1, KP.2, KP.3) as well as SARS-CoV (Fig 19A). SARS2 S1R 8mut4, 10mut and 11mut proteins induced potent neutralizing antibodies4239-112474-02against SARS-CoV-2 variants (D614G, XBB.1.5, JN.1, KP.2, KP.3) and SARS-CoV (Fig 19B).

[0224] Figure 20. S1R 8mut-np, not WA1-np induced potent neutralization antibodies against SARS2 variants. Mice were immunized at weeks 0 and 4 with 2ug of indicated S1R proteins plus ribi as adjuvant by intramuscular injection (IM). Serum samples were assessed at week 6 for neutralization using a pseudotyped lentivirus neutralization assay against SARS-CoV-2 variants on 293-TMPRSS2-ACE2 cells. Neutralization ID50 titers are shown. SARS2 S1R induced potent neutralizing antibodies against D614G, but not against XBB.1.5. Displaying WA1 on np (SARS2 WA1 S1R-np) increased neutralization titers against D614G by 30-fold, but remained no neutralization activity against XBB.1.5. SARS2 S1R 8mut4 induced potent neutralizing antibodies against both D614G and XBB.1.5. Displaying S1R 8mut4 on np (SARS2 S1R 8mut-np) further increased neutralization titer against both D614G and XBB.1.5 by 3- to 40- folds.

[0225] Figure 21. N-terminal np-S1R 8mut induced more consistent, broad and potent neutralization antibodies against SARS2 variants and SARS1. Mice were immunized at weeks 0 and 4 with 2ug of indicated S1R proteins by intramuscular injection (IM). Serum samples were assessed at week 6 for binding to indicated RBD proteins (left panel) by Octet, and neutralization (right panel) using a pseudotyped lentivirus neutralization assay against SARS-CoV-2 variants and SARS-CoV on 293-TMPRSS2-ACE2 cells. SARS2 S1R-8mut4 nanoparticle vaccine (N-EnDS-NP-S1R 8mut4), when the NTD was linked to nanoparticle and the RBD was exposed, induced more potent neutralizing antibodies against SARS-CoV-2 variants and SARS-CoV than the version with the NTD and RBD in the opposite position. The SARS2 S1R-8mut4-15aa-np induced similar level of neutralizing antibodies as the SARS2 S1R-8mut-np with 7-aa linker. Example 2 Sequences

[0226] The following table and list provide the sequences and brief description of constructs disclosed herein. Protein list and description SEQ Name Description4239-112474-021 SARS2 S1R (14-537): SARS-CoV-2 spike amino acid sequence 14-537 including NTD and RBD (S1R) from the ancestor virus e e e e e e4239-112474-0227 SARS2 RBD-8mut4 SARS-CoV-2 RBD with 8 amino acid substitutions on the RBD 28 SARS2 S2P 8mut (1-1208): SARS-CoV-2 S2P (1-1208) with 8 amino acid substitutions ns ns ns4239-112474-0254 SARS2_S1R 8mut4- linker- SARS-CoV-2 S1R 8mut4 linked with SARS1 S1 with SARS1 S1-linker-SpyT SpyTag at the C-terminal 55 SARS2 S1R 9mut- linker- SARS-CoV-2 S1R 9mut linked with SARS1 S1 with g g4239-112474-0281 SpyT-linker-SARS2 S1R SARS-CoV-2 S1R with 9mut SpyTag at the N-terminal 9mut 82 SpyT-linker-SARS2 S1R SARS-CoV-2 S1R with 10mut SpyTag at the N-terminal t e e e at4239-112474-02108 SpyT-linker-MERS S1- MERS S1 linked with SARS2 S1R 9mut with SpyTag at SARS2 S1R 10mut the N-terminal 109 SpyT-linker-MERS S1- MERS S1 linked with SARS2 S1R 9mut with SpyTag at he e e e t g g g gProtein sequences 1. SARS2 S1R (SEQ ID NO: 1) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN4239-112474-02DLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKP FERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 2. SARS2 S1R-8mut4 (SEQ ID NO: 2) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 3. SARS2 S1R-9mut (SEQ ID NO: 3) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 4. SARS2 S1R-10mut (SEQ ID NO: 4) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 5. SARS2 S1R-11mut (SEQ ID NO: 5) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 6. SARS2 S1R-8mut4-linker-SARS1 S1 (SEQ ID NO: 6) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPF KDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFN CTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRA ILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVV PSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVY ADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNV PFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLT GTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVST AIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHT 7. SARS2 S1R-9mut-linker-SARS1 S1 (SEQ ID NO: 7) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF4239-112474-02QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPF KDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFN CTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRA ILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVV PSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVY ADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNV PFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLT GTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVST AIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHT 8. SARS2 S1R-10mut-linker-SARS1 S1 (SEQ ID NO: 8) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPF KDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFN CTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRA ILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVV PSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVY ADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNV PFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLT GTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVST AIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHT 9. SARS2 S1R-11mut-linker-SARS1 S1 (SEQ ID NO: 9) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPF KDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFN CTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRA ILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVV PSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVY ADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNV PFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLT GTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVST AIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHT 10. SARS2 S1R-8mut4-linker-MERS S1 (SEQ ID NO: 10) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSA GHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRF FNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTF4239-112474-02MYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFY VYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLL SGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPI SQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGD YYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGR GVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQY SRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPR 11. SARS2 S1R-9mut-linker-MERS S1 (SEQ ID NO: 11) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSA GHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRF FNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTF MYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFY VYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLL SGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPI SQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGD YYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGR GVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQY SRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPR 12. SARS2 S1R-10mut-linker-MERS S1 (SEQ ID NO: 12) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSA GHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRF FNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTF MYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFY VYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLL SGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPI SQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGD YYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGR GVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQY SRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPR 13. SARS2 S1R-11mut-linker-MERS S1 (SEQ ID NO: 13) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSA GHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRF FNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTF MYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFY VYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLL SGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPI SQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGD4239-112474-02YYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGR GVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQY SRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPR 14. SARS2 S1 (SEQ ID NO: 14) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKP FERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS 15. SARS2 S1-8mut4 (SEQ ID NO: 15) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS 16. SARS2 S1-9mut (SEQ ID NO: 16) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS 17. SARS2 S1-10mut (SEQ ID NO: 17) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS 18. SARS2 S1-11mut (SEQ ID NO: 18) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS4239-112474-0219. SARS2 S1-8mut4-linker-SARS1 S1 (SEQ ID NO: 19) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGGSD LDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGIYFA ATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYIS DAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTAFLP AQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVR FPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVK GDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGK PCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTP SSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQL TPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHT 20. SARS2 S1-9mut-linker-SARS1 S1 (SEQ ID NO: 20) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGGSD LDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGIYFA ATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYIS DAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTAFLP AQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVR FPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVK GDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGK PCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTP SSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQL TPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHT 21. SARS2 S1-10mut-linker-SARS1 S1 (SEQ ID NO: 21) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGGSD LDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGIYFA ATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYIS DAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTAFLP AQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVR FPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVK GDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGK PCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTP SSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQL TPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHT 22. SARS2 S1-11mut-linker-SARS1 S1 (SEQ ID NO: 22)4239-112474-02QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGGSD LDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGIYFA ATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYIS DAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTAFLP AQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVR FPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVK GDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGK PCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTP SSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQL TPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHT 23. SARS2 S1-8mut4-linker-MERS S1 (SEQ ID NO: 23) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGVDV GPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTT PQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVL LPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNIT EDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPL TFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQV YNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQ SFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLS PLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCT AVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSM LKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPR 24. SARS2 S1-9mut-linker-MERS S1 (SEQ ID NO: 24) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGVDV GPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTT PQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVL LPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNIT EDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPL TFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQV YNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQ SFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLS PLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCT AVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSM LKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPR4239-112474-0225. SARS2 S1-10mut-linker-MERS S1 (SEQ ID NO: 25) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGVDV GPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTT PQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVL LPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNIT EDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPL TFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQV YNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQ SFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLS PLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCT AVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSM LKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPR 26. SARS2 S1-11mut-linker-MERS S1 (SEQ ID NO: 26) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGVDV GPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTT PQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVL LPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNIT EDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPL TFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQV YNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQ SFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLS PLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCT AVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSM LKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPR 27. SARS2 RBD-8mut4 (SEQ ID NO: 27) RVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKPFERDI STEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 28. SARS2 S2P 8mut4 (SEQ ID NO: 28) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRLFRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM4239-112474-02QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQ 29. SARS2 S2P 9mut (SEQ ID NO: 29) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRLFRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQ 30. SARS2 S2P 10mut (SEQ ID NO: 30) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRFLRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQ 31. SARS2 S2P 11mut (SEQ ID NO: 31) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRFLRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN4239-112474-02CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQ 32. SARS2 S2P 8mut4-TM (SEQ ID NO: 32) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRLFRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 33. SARS2 S2P 9mut-TM (SEQ ID NO: 33) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRLFRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 34. SARS2 S2P 10mut-TM (SEQ ID NO: 34) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRFLRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 35. SARS2 S2P 11mut-TM (SEQ ID NO: 35)4239-112474-02MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRFLRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 36. SARS1 S1 (SEQ ID NO: 36) GSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGI YFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFE YISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTA FLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGD VVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSF VVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSP DGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGV LTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHA DQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHT 37. MERS S1 (SEQ ID NO: 37) VDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHAT GTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHT LVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTY NITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKL QPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTP PQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFN YKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRK QLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQ NCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRST RSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPR 38. SARS2_S1R 8mut4-linker-SpyT (SEQ ID NO: 38) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGSGGAHIVMVDAYKPTK 39. SARS2_S1R 9mut-linker-SpyT (SEQ ID NO: 39) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGSGGAHIVMVDAYKPTK4239-112474-0240. SARS2_S1R 10mut-linker-SpyT (SEQ ID NO: 40) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGSGGAHIVMVDAYKPTK 41. SARS2_S1R 11mut-linker-SpyT (SEQ ID NO: 41) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGSGGAHIVMVDAYKPTK 42. SARS2_S18mut4-linker-SpyT (SEQ ID NO: 42) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGSGG AHIVMVDAYKPTK 43. SARS2_S19mut-linker-SpyT (SEQ ID NO: 43) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGSGG AHIVMVDAYKPTK 44. SARS2_S110mut-linker-SpyT (SEQ ID NO: 44) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGSGG AHIVMVDAYKPTK 45. SARS2_S111mut-linker-SpyT (SEQ ID NO: 45) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY4239-112474-02QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGSGG AHIVMVDAYKPTK 46. SARS2_RBD 8mut4-linker-SpyT (SEQ ID NO: 46) RVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKPFERDI STEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGSGGS GGAHIVMVDAYKPTK 47. SARS2_RBD 9mut-linker-SpyT (SEQ ID NO: 47) RVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKPFERDI STEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGSGGS GGAHIVMVDAYKPTK 48. SARS2_RBD 10mut-linker-SpyT (SEQ ID NO: 48) RVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKPFERDI STEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGSGGS GGAHIVMVDAYKPTK 49. SARS2_RBD 11mut-linker-SpyT (SEQ ID NO: 49) RVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKPFERDI STEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGSGGS GGAHIVMVDAYKPTK 50. SARS2 S2P 8mut4-linker-SpyT (SEQ ID NO: 50) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRLFRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQGGSGGSGGAHIVMVDAYKPTK 51. SARS2 S2P 9mut-linker-SpyT (SEQ ID NO: 51) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRLFRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS4239-112474-02NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQGGSGGSGGAHIVMVDAYKPTK 52. SARS2 S2P 10mut-linker-SpyT (SEQ ID NO: 52) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRFLRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQGGSGGSGGAHIVMVDAYKPTK 53. SARS2 S2P 11mut-linker-SpyT (SEQ ID NO: 53) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRFLRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQGGSGGSGGAHIVMVDAYKPTK 54. SARS2 S1R-8mut4-linker-SARS1 S1-linker-SpyT (SEQ ID NO: 54) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPF KDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFN CTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRA ILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVV PSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVY ADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNV PFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLT4239-112474-02GTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVST AIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSGGSGGAHIVMVDAYKPTK 55. SARS2 S1R-9mut-linker-SARS1 S1-linker-SpyT (SEQ ID NO: 55) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPF KDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFN CTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRA ILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVV PSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVY ADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNV PFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLT GTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVST AIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSGGSGGAHIVMVDAYKPTK 56. SARS2 S1R-10mut-linker-SARS1 S1-linker-SpyT (SEQ ID NO: 56) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPF KDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFN CTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRA ILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVV PSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVY ADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNV PFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLT GTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVST AIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSGGSGGAHIVMVDAYKPTK 57. SARS2 S1R-11mut-linker-SARS1 S1-linker-SpyT (SEQ ID NO: 57) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPF KDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFN CTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRA ILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVV PSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVY ADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNV PFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLT GTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVST AIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSGGSGGAHIVMVDAYKPTK 58. SARS2 S1R-8mut4-linker-MERS S1-linker-SpyT (SEQ ID NO: 58) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF4239-112474-02QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSA GHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRF FNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTF MYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFY VYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLL SGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPI SQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGD YYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGR GVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQY SRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRGGSGGSGGAHIVMVDA YKPTK 59. SARS2 S1R-9mut-linker-MERS S1-linker-SpyT (SEQ ID NO: 59) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSA GHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRF FNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTF MYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFY VYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLL SGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPI SQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGD YYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGR GVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQY SRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRGGSGGSGGAHIVMVDA YKPTK 60. SARS2 S1R-10mut-linker-MERS S1-linker-SpyT (SEQ ID NO: 60) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSA GHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRF FNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTF MYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFY VYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLL SGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPI SQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGD YYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGR GVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQY SRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRGGSGGSGGAHIVMVDA YKPTK 61. SARS2 S1R-11mut-linker-MERS S1-linker-SpyT (SEQ ID NO: 61) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY4239-112474-02QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSA GHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRF FNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTF MYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFY VYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLL SGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPI SQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGD YYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGR GVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQY SRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRGGSGGSGGAHIVMVDA YKPTK 62. SARS2 S1R-linker-SpyT (SEQ ID NO: 62) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKP FERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GSGGSGGAHIVMVDAYKPTK 63. SARS2 S1-8mut4-linker-SARS1 S1-linker-SpyT (SEQ ID NO: 63) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGGSD LDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGIYFA ATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYIS DAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTAFLP AQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVR FPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVK GDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGK PCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTP SSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQL TPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSGGSGGAHIVMVDAYKPTK 64. SARS2 S1-9mut-linker-SARS1 S1-linker-SpyT (SEQ ID NO: 64) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGGSD LDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGIYFA ATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYIS DAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTAFLP AQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVR FPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVK GDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGK4239-112474-02PCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTP SSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQL TPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSGGSGGAHIVMVDAYKPTK 65. SARS2 S1-10mut-linker-SARS1 S1-linker-SpyT (SEQ ID NO: 65) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGGSD LDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGIYFA ATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYIS DAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTAFLP AQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVR FPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVK GDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGK PCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTP SSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQL TPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSGGSGGAHIVMVDAYKPTK 66. SARS2 S1-11mut-linker-SARS1 S1-linker-SpyT (SEQ ID NO: 66) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGGSD LDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGIYFA ATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEYIS DAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTAFLP AQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDVVR FPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFVVK GDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGK PCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTP SSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHADQL TPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSGGSGGAHIVMVDAYKPTK 67. SARS2 S1-8mut4-linker-MERS S1-linker-SpyT (SEQ ID NO: 67) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGVDV GPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTT PQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVL LPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNIT EDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPL TFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQV YNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQ SFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLS4239-112474-02PLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCT AVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSM LKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRGGSGGSGGAHIVMVDAYKPTK 68. SARS2 S1-9mut-linker-MERS S1-linker-SpyT (SEQ ID NO: 68) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGVDV GPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTT PQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVL LPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNIT EDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPL TFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQV YNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQ SFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLS PLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCT AVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSM LKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRGGSGGSGGAHIVMVDAYKPTK 69. SARS2 S1-10mut-linker-MERS S1-linker-SpyT (SEQ ID NO: 69) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGVDV GPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTT PQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVL LPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNIT EDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPL TFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQV YNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQ SFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLS PLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCT AVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSM LKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRGGSGGSGGAHIVMVDAYKPTK 70. SARS2 S1-11mut-linker-MERS S1-linker-SpyT (SEQ ID NO:70) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGSGGVDV GPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTT PQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVL LPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYNIT EDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQPL TFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQV4239-112474-02YNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQ SFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLS PLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQNCT AVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTRSM LKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPRGGSGGSGGAHIVMVDAYKPTK 71. SARS1 S1-linker-SpyT (SEQ ID NO: 71) GSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGI YFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFE YISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTA FLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGD VVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSF VVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSP DGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGV LTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHA DQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSGGSGGAHIVMVDAYKPTK 72. SARS2 RBD-8mut4-linker-SARS1 S1-linker-SpyT (SEQ ID NO: 72) RVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKPFERDI STEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGSGGG SDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGIY FAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEY ISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTAF LPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDV VRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFV VKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPD GKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVL TPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHAD QLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHT 73. SARS2 RBD-9mut-linker-SARS1 S1-linker-SpyT (SEQ ID NO: 73) RVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKPFERDI STEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGSGGG SDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGIY FAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEY ISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTAF LPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDV VRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFV VKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPD GKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVL TPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHAD QLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHT 74. SARS2 RBD-10mut-linker-SARS1 S1-linker-SpyT (SEQ ID NO: 74) RVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKPFERDI STEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGSGGG SDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGIY FAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEY ISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTAF LPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDV VRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFV VKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPD GKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVL TPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHAD QLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHT 75. SARS2 RBD-11mut-linker-SARS1 S1-linker-SpyT (SEQ ID NO: 75)4239-112474-02RVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKPFERDI STEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGSGGG SDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNVTGFHTINHTFDNPVIPFKDGIY FAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVSKPMGTQTHTMIFDNAFNCTFEY ISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTLKPIFKLPLGIKITNFRAILTAF LPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVKSFEIDKGIYQTSNFRVVPSGDV VRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKCYGVSATKLNDLCFSNVYADSFV VKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPD GKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVL TPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNASSEVAVLYQDVNCTDVSTAIHAD QLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHT 76. SARS2 RBD-8mut4-linker-MERS S1-linker-SpyT (SEQ ID NO: 76) RVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKPFERDI STEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGSGGV DVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATG TTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTL VLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYN ITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQ PLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPP QVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNY KQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQ LSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQN CTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTR SMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPR 77. SARS2 RBD-9mut-linker-MERS S1-linker-SpyT (SEQ ID NO: 77) RVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKPFERDI STEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGSGGV DVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATG TTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTL VLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYN ITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQ PLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPP QVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNY KQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQ LSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQN CTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTR SMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPR 78. SARS2 RBD-10mut-linker-MERS S1-linker-SpyT (SEQ ID NO: 78) RVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKPFERDI STEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGSGGV DVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATG TTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTL VLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYN ITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQ PLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPP QVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNY KQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQ LSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQN CTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTR SMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPR 79. SARS2 RBD-11mut-linker-MERS S1-linker-SpyT (SEQ ID NO: 79) RVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKPFERDI4239-112474-02STEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGSGGV DVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQGLFPYQGDHGDMYVYSAGHATG TTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTL VLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTYN ITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDRKAWAAFYVYKLQ PLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPP QVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNY KQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQ LSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRGVFQN CTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLFGSVACEHISSTMSQYSRSTR SMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTPR 80. SpyT-linker-SARS2 S1R-8mut4 (SEQ ID NO: 80) AHIVMVDAYKPTKGGSGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHA IHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLP QGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLD SKASGNYNYRYRLFRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFEL LHAPATVCGPKKSTNLVKNK 81. SpyT-linker-SARS2 S1R-9mut (SEQ ID NO: 81) AHIVMVDAYKPTKGGSGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHA IHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLP QGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLD SKASGNYNYRYRLFRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFEL LHAPATVCGPKKSTNLVKNK 82. SpyT-linker-SARS2 S1R-10mut (SEQ ID NO: 82) AHIVMVDAYKPTKGGSGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHA IHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLP QGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLD SKASGNYNYRYRFLRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFEL LHAPATVCGPKKSTNLVKNK 83. SpyT-linker-SARS2 S1R-11mut (SEQ ID NO: 83) AHIVMVDAYKPTKGGSGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHA IHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLP QGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLD SKASGNYNYRYRFLRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFEL LHAPATVCGPKKSTNLVKNK 84. SpyT-linker-SARS2 S1R (SEQ ID NO: 84) AHIVMVDAYKPTKGGSGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHA IHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLP QGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLD4239-112474-02SKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFEL LHAPATVCGPKKSTNLVKNK 85. SpyT-linker-SARS2 S1-8mut4 (SEQ ID NO: 85) AHIVMVDAYKPTKGGSGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHA IHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLP QGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLD SKASGNYNYRYRLFRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFEL LHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSF GGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIG AGICASYQTQTNS 86. SpyT-linker-SARS2 S1-9mut (SEQ ID NO: 86) AHIVMVDAYKPTKGGSGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHA IHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLP QGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLD SKASGNYNYRYRLFRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFEL LHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSF GGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIG AGICASYQTQTNS 87. SpyT-linker-SARS2 S1-10mut (SEQ ID NO: 87) AHIVMVDAYKPTKGGSGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHA IHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLP QGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLD SKASGNYNYRYRFLRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFEL LHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSF GGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIG AGICASYQTQTNS 88. SpyT-linker-SARS2 S1-11mut (SEQ ID NO: 88) AHIVMVDAYKPTKGGSGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHA IHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLP QGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLD SKASGNYNYRYRFLRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFEL LHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSF GGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIG AGICASYQTQTNS 89. SpyT-linker-SARS2 S1 (SEQ ID NO: 89) AHIVMVDAYKPTKGGSGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHA IHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLP QGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLD SKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFEL LHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSF4239-112474-02GGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIG AGICASYQTQTNS 90. SpyT-linker-SARS2_RBD 8mut4 (SEQ ID NO: 90) AHIVMVDAYKPTKGGSGGSGGRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSA SFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASG NYNYRYRLFRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPA TVCGPKKSTNLVKNK 91. SpyT-linker-SARS2_RBD 9mut (SEQ ID NO: 91) AHIVMVDAYKPTKGGSGGSGGRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSA SFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASG NYNYRYRLFRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPA TVCGPKKSTNLVKNK 92. SpyT-linker-SARS2_RBD 10mut (SEQ ID NO: 92) AHIVMVDAYKPTKGGSGGSGGRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSA SFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASG NYNYRYRFLRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPA TVCGPKKSTNLVKNK 93. SpyT-linker-SARS2_RBD 11mut (SEQ ID NO: 93) AHIVMVDAYKPTKGGSGGSGGRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSA SFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASG NYNYRYRFLRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPA TVCGPKKSTNLVKNK 94. SpyT-linker-SARS1 S1-SARS2 S1R-8mut4 (SEQ ID NO: 94) AHIVMVDAYKPTKGGSGGSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNV TGFHTINHTFDNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVS KPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTL KPIFKLPLGIKITNFRAILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVK SFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKC YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLS TDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNAS SEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSG GQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFN DGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSS ANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITR FQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGI YQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL NDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLK PFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 95. SpyT-linker-SARS1 S1-SARS2 S1R-9mut (SEQ ID NO: 95) AHIVMVDAYKPTKGGSGGSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNV TGFHTINHTFDNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVS KPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTL KPIFKLPLGIKITNFRAILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVK SFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKC YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLS TDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNAS SEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSG GQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFN DGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSS ANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITR FQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGI YQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL4239-112474-02NDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLK PFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 96. SpyT-linker-SARS1 S1-SARS2 S1R-10mut (SEQ ID NO: 96) AHIVMVDAYKPTKGGSGGSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNV TGFHTINHTFDNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVS KPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTL KPIFKLPLGIKITNFRAILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVK SFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKC YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLS TDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNAS SEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSG GQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFN DGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSS ANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITR FQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGI YQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL NDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLK PFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 97. SpyT-linker-SARS1 S1-SARS2 S1R-11mut (SEQ ID NO: 97) AHIVMVDAYKPTKGGSGGSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNV TGFHTINHTFDNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVS KPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTL KPIFKLPLGIKITNFRAILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVK SFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKC YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLS TDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNAS SEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSG GQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFN DGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSS ANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITR FQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGI YQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL NDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLK PFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 98. SpyT-linker-SARS1 S1-SARS2 S1-8mut4 (SEQ ID NO: 98) AHIVMVDAYKPTKGGSGGSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNV TGFHTINHTFDNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVS KPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTL KPIFKLPLGIKITNFRAILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVK SFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKC YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLS TDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNAS SEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSG GQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFN DGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSS ANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITR FQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGI YQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL NDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLK PFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK CVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLY QDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS 99. SpyT-linker-SARS1 S1-SARS2 S1-9mut (SEQ ID NO: 99)4239-112474-02AHIVMVDAYKPTKGGSGGSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNV TGFHTINHTFDNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVS KPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTL KPIFKLPLGIKITNFRAILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVK SFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKC YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLS TDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNAS SEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSG GQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFN DGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSS ANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITR FQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGI YQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL NDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLK PFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK CVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLY QDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS 100. SpyT-linker-SARS1 S1-SARS2 S1-10mut (SEQ ID NO: 100) AHIVMVDAYKPTKGGSGGSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNV TGFHTINHTFDNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVS KPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTL KPIFKLPLGIKITNFRAILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVK SFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKC YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLS TDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNAS SEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSG GQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFN DGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSS ANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITR FQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGI YQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL NDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLK PFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK CVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLY QDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS 101. SpyT-linker-SARS1 S1-SARS2 S1-11mut (SEQ ID NO: 101) AHIVMVDAYKPTKGGSGGSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNV TGFHTINHTFDNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVS KPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTL KPIFKLPLGIKITNFRAILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVK SFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKC YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLS TDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNAS SEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSG GQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFN DGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSS ANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITR FQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGI YQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL NDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLK PFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK CVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLY QDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS 102. SpyT-linker-SARS1 S1-SARS2 RBD-8mut4 (SEQ ID NO: 102)4239-112474-02AHIVMVDAYKPTKGGSGGSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNV TGFHTINHTFDNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVS KPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTL KPIFKLPLGIKITNFRAILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVK SFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKC YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLS TDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNAS SEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSG GRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF TNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKPFERD ISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 103. SpyT-linker-SARS1 S1-SARS2 RBD-9mut (SEQ ID NO: 103) AHIVMVDAYKPTKGGSGGSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNV TGFHTINHTFDNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVS KPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTL KPIFKLPLGIKITNFRAILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVK SFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKC YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLS TDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNAS SEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSG GRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF TNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKPFERD ISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 104. SpyT-linker-SARS1 S1-SARS2 RBD-10mut (SEQ ID NO: 104) AHIVMVDAYKPTKGGSGGSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNV TGFHTINHTFDNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVS KPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTL KPIFKLPLGIKITNFRAILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVK SFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKC YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLS TDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNAS SEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSG GRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF TNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKPFERD ISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 105. SpyT-linker-SARS1 S1-SARS2 RBD-11mut (SEQ ID NO: 105) AHIVMVDAYKPTKGGSGGSGGGSDLDRCTTFDDVQAPNYTQHTSSMRGVYYPDEIFRSDTLYLTQDLFLPFYSNV TGFHTINHTFDNPVIPFKDGIYFAATEKSNVVRGWVFGSTMNNKSQSVIIINNSTNVVIRACNFELCDNPFFAVS KPMGTQTHTMIFDNAFNCTFEYISDAFSLDVSEKSGNFKHLREFVFKNKDGFLYVYKGYQPIDVVRDLPSGFNTL KPIFKLPLGIKITNFRAILTAFLPAQDTWGTSAAAYFVGYLKPTKFMLKYDENGTITDAVDCSQNPLAELKCSVK SFEIDKGIYQTSNFRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKRISNCVADYSVLYNSTSFSTFKC YGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLKDYGFYTTSGIGYQPYRVVVLSFELLNAPATVCGPKLS TDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCSFGGVSVITPGTNAS SEVAVLYQDVNCTDVSTAIHADQLTPAWRIYSTGNNVFQTQAGCLIGAEHVDTSYECDIPIGAGICASYHTGGSG GRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF TNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKPFERD ISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 106. SpyT-linker-MERS S1-SARS2 S1R-8mut4 (SEQ ID NO: 106) AHIVMVDAYKPTKGGSGGSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQ GLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAF MLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRN ASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIP HSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG4239-112474-02SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFS YPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQ YSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIA SQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHAT LFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTP RGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNP VLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEF RVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIG INITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFT VEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGV SPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFR KSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTN LVKNK 107. SpyT-linker-MERS S1-SARS2 S1R-9mut (SEQ ID NO: 107) AHIVMVDAYKPTKGGSGGSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQ GLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAF MLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRN ASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIP HSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFS YPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQ YSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIA SQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHAT LFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTP RGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNP VLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEF RVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIG INITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFT VEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGV SPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFR KSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTN LVKNK 108. SpyT-linker-MERS S1-SARS2 S1R-10mut (SEQ ID NO: 108) AHIVMVDAYKPTKGGSGGSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQ GLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAF MLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRN ASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIP HSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFS YPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQ YSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIA SQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHAT LFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTP RGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNP VLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEF RVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIG INITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFT VEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGV SPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLR KSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTN LVKNK 109. SpyT-linker-MERS S1-SARS2 S1R-11mut (SEQ ID NO: 109) AHIVMVDAYKPTKGGSGGSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQ GLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAF MLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRN ASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIP HSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFS4239-112474-02YPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQ YSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIA SQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHAT LFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTP RGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNP VLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEF RVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIG INITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFT VEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGV SPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLR KSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTN LVKNK 110. SpyT-linker-MERS S1-SARS2 S1-8mut4 (SEQ ID NO: 110) AHIVMVDAYKPTKGGSGGSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQ GLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAF MLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRN ASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIP HSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFS YPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQ YSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIA SQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHAT LFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTP RGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNP VLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEF RVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIG INITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFT VEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGV SPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFR KSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTN LVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQ VAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS 111. SpyT-linker-MERS S1-SARS2 S1-9mut (SEQ ID NO: 111) AHIVMVDAYKPTKGGSGGSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQ GLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAF MLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRN ASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIP HSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFS YPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQ YSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIA SQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHAT LFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTP RGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNP VLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEF RVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIG INITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFT VEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGV SPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFR KSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTN LVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQ VAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS 112. SpyT-linker-MERS S1-SARS2 S1-10mut (SEQ ID NO: 112) AHIVMVDAYKPTKGGSGGSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQ GLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAF MLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRN ASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIP HSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG4239-112474-02SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFS YPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQ YSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIA SQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHAT LFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTP RGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNP VLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEF RVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIG INITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFT VEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGV SPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLR KSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTN LVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQ VAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS 113. SpyT-linker-MERS S1-SARS2 S1-11mut (SEQ ID NO: 113) AHIVMVDAYKPTKGGSGGSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQ GLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAF MLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRN ASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIP HSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFS YPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQ YSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIA SQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHAT LFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTP RGGSGGQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNP VLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEF RVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIG INITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFT VEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGV SPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLR KSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTN LVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQ VAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNS 114. SpyT-linker-MERS S1-SARS2 RBD-8mut4 (SEQ ID NO: 114) AHIVMVDAYKPTKGGSGGSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQ GLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAF MLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRN ASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIP HSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFS YPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQ YSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIA SQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHAT LFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTP RGGSGGRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL NDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLK PFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 115. SpyT-linker-MERS S1-SARS2 RBD-9mut (SEQ ID NO: 115) AHIVMVDAYKPTKGGSGGSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQ GLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAF MLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRN ASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIP HSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFS YPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQ YSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIA SQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHAT4239-112474-02LFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTP RGGSGGRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL NDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLK PFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 116. SpyT-linker-MERS S1-SARS2 RBD-10mut (SEQ ID NO: 116) AHIVMVDAYKPTKGGSGGSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQ GLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAF MLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRN ASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIP HSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFS YPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQ YSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIA SQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHAT LFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTP RGGSGGRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL NDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLK PFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 117. SpyT-linker-MERS S1-SARS2 RBD-11mut (SEQ ID NO: 117) AHIVMVDAYKPTKGGSGGSGGVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQGRTYSNITITYQ GLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRIGAAANSTGTVIISPSTSATIRKIYPAF MLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRAFYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRN ASLNSFKEYFNLRNCTFMYTYNITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIP HSIRSIQSDRKAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSG SVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFS YPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRFLSDDRTEVPQLVNANQ YSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIA SQLGNCVEYSLYGVSGRGVFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHAT LFGSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSLCALPDTPSTLTP RGGSGGRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKL NDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLK PFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 118. SARS2 S1R-8mut4-15In-SpyT (SEQ ID NO: 118) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GGGSGGGGSGGGGSAHIVMVDAYKPTK 119. SARS2 S1R-9mut-15In-SpyT (SEQ ID NO: 119) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GGGSGGGGSGGGGSAHIVMVDAYKPTK 120. SARS2 S1R-10mut-15In-SpyT (SEQ ID NO: 120) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN4239-112474-02DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GGGSGGGGSGGGGSAHIVMVDAYKPTK 121. SARS2 S1R-11mut-15In-SpyT (SEQ ID NO: 121) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKG GGGSGGGGSGGGGSAHIVMVDAYKPTK 122. SARS2 S1-8mut4-15In-SpyT (SEQ ID NO: 122) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGGGSGGG GSGGGGSAHIVMVDAYKPTK 123. SARS2 S1-9mut-15In-SpyT (SEQ ID NO: 123) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRLFRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGGGSGGG GSGGGGSAHIVMVDAYKPTK 124. SARS2 S1-10mut-15In-SpyT (SEQ ID NO: 124) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGGGSGGG GSGGGGSAHIVMVDAYKPTK 125. SARS2 S1-11mut-15In-SpyT (SEQ ID NO: 125) QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIY QTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYNYRYRFLRKSKLKP FERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC VNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSGGGGSGGG GSGGGGSAHIVMVDAYKPTK4239-112474-02126. EnDS-Linker-SpyCatcher (SEQ ID NO: 126) MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGGGGGHHHHHHGGGGGPYGWEYAAHPLCEVEVLSD ENEVVKWGLRKSLPLIELRATFTLLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEE RKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIED ALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTMLLKFGSGSGSVTTLSGLSGEQGPSGDMTTEEDSA THIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQ VTVNGEATKGDAHTGSSGS 127. SARS2 Spike 8mut4 (SEQ ID NO: 127) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRLFRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 128. SARS2 Spike 9mut (SEQ ID NO: 128) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRLFRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 129. SARS2 Spike 10mut (SEQ ID NO: 129) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRFLRKSKLKPFERDISTEIYQAGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS4239-112474-02VLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 130. SARS2 Spike 11mut (SEQ ID NO: 130) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATTFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKASGNYN YRYRFLRKSKLKPFERDISTEIYQRGSTPCNGVAGPNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 131. SARS2 Spike (NCBI Ref. No. YP_009724390.1) (SEQ ID NO: 131) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGN CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0227] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described examples. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

4239-112474-02It is claimed:

1. A recombinant SARS-CoV-2 Spike protein or fragment thereof comprising the receptor binding domain (RBD) of the recombinant SARS-CoV-2 Spike protein, wherein the RBD of the recombinant SARS-CoV-2 Spike protein comprises: threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively; and optionally further comprises one, two, or three of phenylalanine, leucine, and arginine amino acids at positions 455, 456, and 475, respectively; and wherein the amino acid numbering is according to the reference SARS-CoV-2 Spike sequence set forth as SEQ ID NO:

131.

2. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 1, comprising the arginine at position 475.

3. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 1, comprising the phenylalanine and leucine at positions 455 and 456.

4. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 1, comprising the phenylalanine, leucine and arginine at positions 455, 456, and 475.

5. The recombinant SARS-CoV-2 Spike protein or fragment thereof of any one of claims 1-4, wherein: the threonine, asparagine, alanine, serine, arginine, lysine, alanine, and proline amino acids at positions 346, 417, 445, 446, 452, 460, 484, and 486, respectively, are due to one or more of R346T, K417N, V445A, G446S, L452R, N460K, E484A, and F486P amino acid substitutions; and the one, two, or three of arginine, phenylalanine, and leucine amino acids at positions 475, 455, and 456, respectively, are due to one or more of A475R, L455F, and F456L amino acid substitutions; and wherein the amino acid numbering is according to the reference SARS-CoV-2 Spike sequence set forth as SEQ ID NO:

131.

6. The fragment of the recombinant SARS-CoV-2 Spike protein of any one of claims 1-5, wherein the fragment is the S1 subunit of the SARS-CoV-2 Spike protein.4239-112474-027. The fragment of the recombinant SARS-CoV-2 Spike protein of claim 6, wherein the S1 subunit comprises about SARS-CoV-2 Spike residues 14-537 or 14-680 according to the reference SARS-CoV-2 Spike sequence set forth as SEQ ID NO:

131.

8. The fragment of the recombinant SARS-CoV-2 Spike protein of claim 6 or claim 7, comprising the amino acid sequence set forth as residues 14-537 or 14-680 of any one of SEQ ID NOs: 127-130, or an amino acid sequence at least 80% identical thereto and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids.

9. The fragment of the recombinant SARS-CoV-2 Spike protein of any one of claims 1-5, wherein the fragment is the ectodomain of the SARS-CoV-2 Spike protein.

10. The fragment of the recombinant SARS-CoV-2 Spike protein of claim 9, wherein the ectodomain comprises residues 14-1208 of the SARS-CoV-2 Spike according to the reference SARS-CoV-2 Spike sequence set forth as SEQ ID NO:

131.

11. The fragment of the recombinant SARS-CoV-2 Spike protein of claim 9 or claim 10, comprising the amino acid sequence set forth as residues 14-1208 of any one of SEQ ID NOs: 127-130, or an amino acid sequence at least 80% identical thereto and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids.

12. The recombinant SARS-CoV-2 Spike protein of any one of claims 1-5.

13. The recombinant SARS-CoV-2 Spike protein of claim 12, comprising the amino acid sequence set forth as any one of SEQ ID NOs: 127-130, or an amino acid sequence at least 80% identical thereto and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids.

14. The recombinant SARS-CoV-2 Spike protein or fragment thereof of any one of claims 1-5 or 9-13, further comprising proline substitutions at positions 986 and 987 of the4239-112474-02S2 subunit, wherein the amino acid numbering is according to the reference SARS-CoV-2 Spike sequence set forth as SEQ ID NO:

131.

15. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 14, wherein the proline substitutions at positions 986 and 987 are due to K986P and V987P amino acid substitutions.

16. The recombinant SARS-CoV-2 Spike protein or fragment thereof of any one of claims 1-5 or 9-15, wherein a S1 / S2 protease cleavage site of the S ectodomain is mutated to inhibit protease cleavage.

17. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 16, wherein the mutation to inhibit protease cleavage is a RRAR682-685GSAS substitution.

18. The fragment of the recombinant SARS-CoV-2 Spike protein of any one of claims 1-5, wherein the fragment is the RBD of the SARS-CoV-2 Spike protein.

19. The fragment of the recombinant SARS-CoV-2 Spike protein of claim 18, wherein the RBD comprises about residues 319-537 of the SARS-CoV-2 Spike according to the reference SARS-CoV-2 Spike sequence set forth as SEQ ID NO:

131.

20. The fragment of the recombinant SARS-CoV-2 Spike protein of claim 18 or claim 19, comprising the amino acid sequence set forth as residues 319-537 of any one of SEQ ID NOs: 127-130, or an amino acid sequence at least 80% identical thereto and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids.

21. The recombinant SARS-CoV-2 Spike protein or fragment thereof of any one of the prior claims, fused directly or indirectly via a peptide linker to a heterologous protein.

22. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 21, wherein the heterologous protein is a S1 subunit of a Spike protein of a heterologous coronavirus.4239-112474-0223. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 22, wherein the heterologous protein is a S1 subunit of a SARS-CoV-1 Spike protein.

24. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 23, comprising an amino acid sequence set forth as any one of SEQ ID NOs: 6-9, 19-22, 54-57, 63-66, 72-75, or 94-105, or an amino acid sequence at least 80% identical thereto and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids.

25. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 22, wherein the heterologous protein is a S1 subunit of a MERS-CoV Spike protein.

26. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 25, comprising an amino acid sequence set forth as any one of SEQ ID NOs: 10-13, 23-26, 58- 61, 67-70, 76-79, or 106-117, or an amino acid sequence at least 80% identical thereto and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids.

27. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 21, wherein the heterologous protein is a SpyTag moiety.

28. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 27, comprising an amino acid sequence set forth as any one of SEQ ID NOs: 38-61, 63-70, 72- 83, 85-88, or 90-125, or an amino acid sequence at least 80% identical thereto and having the 346T, 417N, 445A, 446S, 452R, 460K, 484A, and F486P amino acids and optionally the one or more of the 475R, 455F, and 456L amino acids.

29. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 21, wherein the heterologous protein is a trimerization domain.

30. The recombinant SARS-CoV-2 Spike protein or fragment thereof of claim 21, wherein the heterologous protein is a self-assembling protein nanoparticle subunit.4239-112474-0231. A nanoparticle, comprising the recombinant SARS-CoV-2 Spike protein or fragment thereof of any one of the prior claims.

32. The nanoparticle of claim 31, wherein the nanoparticle is a self-assembling protein nanoparticle, and comprising self-assembling protein nanoparticle subunits linked to the recombinant SARS-CoV-2 Spike protein or fragment thereof using an isopeptide bond conjugation system.

33. The recombinant SARS-CoV-2 Spike protein or fragment thereof, or nanoparticle, of any one of the prior claims, conjugated to a heterologous carrier.

34. A virus-like particle comprising the recombinant SARS-CoV-2 Spike protein or fragment thereof of any one of claims 1-29.

35. An isolated nucleic acid molecule encoding the recombinant SARS-CoV-2 Spike protein or fragment thereof of any one of claim 1-33.

36. The nucleic acid molecule of claim 35, operably linked to a promoter.

37. The nucleic acid molecule of claim 35 or claim 36, wherein the nucleic acid molecule is an mRNA molecule.

38. A vector comprising the nucleic acid molecule of claim 35.

39. The vector of claim 38, wherein the vector is a viral vector.

40. An immunogenic composition comprising the recombinant SARS-CoV-2 Spike protein or fragment thereof, nanoparticle, virus-like particle, nucleic acid molecule, or vector of any one of the prior claims, and a pharmaceutically acceptable carrier.4239-112474-0241. A method of producing a recombinant SARS-CoV-2 Spike protein or fragment thereof, comprising: expressing the nucleic acid molecule or vector of any one of claims 35-39 in a host cell to produce the recombinant SARS-CoV-2 Spike protein or fragment thereof; and purifying the recombinant SARS-CoV-2 Spike protein or fragment thereof.

42. A method for generating an immune response to SARS-CoV-2 Spike in a subject, comprising administering to the subject an effective amount of the recombinant SARS-CoV-2 Spike protein or fragment thereof, protein nanoparticle, virus-like particle, nucleic acid molecule, vector, or immunogenic composition of any one of claims 1-40 to generate the immune response.

43. A method for generating an immune response to a SARS-CoV-2 Spike in a subject, comprising administering to the subject an effective amount of a mRNA molecule encoding the recombinant SARS-CoV-2 Spike protein or fragment thereof of any one of claims 1-33 to generate the immune response.

44. The method of claim 42 or claim 43, wherein the immune response treats or inhibits infection with SARS-CoV-2.

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