CA3157810A1 - Vaccines against coronaviruses - Google Patents

Abstract

Disclosed herein are nucleic acid molecules comprising a polynucleotide, the polynucleotide encoding a polypeptide comprising a receptor-binding domain (RBD) of a spike (S) protein of a coronavirus. Also disclosed are nucleic acid molecules comprising a polynucleotide, the polynucleotide encoding a polypeptide comprising a plurality of antigenic coronavirus peptides interspersed in an ankyrin repeat scaffold, the ankyrin repeat scaffold comprising a plurality of ankyrin repeat motifs. Methods and uses of these nucleic acid molecules for inducing an immune response against a coronavirus are also disclosed.

Description

TITLE: VACCINES AGAINST CORONAVIRUSES
FIELD
[0001] The present disclosure generally relates to vaccine compositions and more particularly to nucleic acid vaccines against coronaviruses as well as methods and uses thereof.
INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing "P64662CA00_5T25_Sequence_Listing" (203, 141 bytes) created on April 29, 2022, is herein incorporated by reference.
BACKGROUND

[0003] Nanoparticle vaccine (NP) platforms involve the attachment of an antigen to the surface of a particle, either inorganic or organic in nature, to promote an immune response through enhanced trafficking and recognition by cellular receptors (Nguyen, B., Tolia, N.H.
Protein-based antigen presentation platforms for nanoparticle vaccines. npj Vaccines 6, 70 (2021)). Displaying antigens in a particulate array on NPs or, alternatively, VLPs can be used to increase their immunogenicity. Multivalent antigens that can mimic the repetitive and well-ordered antigenic structures found on many pathogens cross-link B-cell receptors (BCRs) and activate B cells more efficiently than their monovalent counterparts (Brouwer, P.J.M., Antanasijevic, A., Berndsen, Z. et al. Enhancing and shaping the immunogenicity of native-like HIV-1 envelope trimers with a two-component protein nanoparticle. Nat Commun 10, 4272 (2019)). In addition, such antigens can be taken up by antigen-presenting cells and trafficked to lymph nodes more efficiently, leading to improved formation of germinal centers (Brouwer, P.J.M., Antanasijevic, A., Berndsen, Z. et al. Enhancing and shaping the immunogenicity of native-like HIV-1 envelope trimers with a two-component protein nanoparticle.
Nat Commun 10, 4272 (2019)). One of the additional mechanisms of enhanced immune induction is a DC-mediated priming of T helper cells.

[0004] In Liu Xingjian et al. Self-assembling SARS-CoV-2 nanoparticle vaccines targeting the S protein induces protective immunity in mice (2021).
https://doi.org/10.1101/2021.02.05.428685. developed an NP vaccine against SARS-CoV-2, where Helicobacter ferritin was used as a self-assembling protein. The RBD, S1, and ectodomain (ECD) sequences of S protein were fusion expressed with Helicobacter pylori ferritin at N-terminal, respectively. Neutralizing antibodies (nAb) collected in ECD-Nano mice were able to inhibit SARS-CoV-2 infection in cells with a nAb titer of 1:160.
However, the neutralization activity of nAbs induced by RBD-Nano and S1-Nano could not be detected after 1:40 dilution.

[0005] In Tan, T.K., Rijal, P., Rahikainen, R. et al. A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain Date Recue/Date Received 2022-04-29 induces potent neutralising antibody responses. Nat Commun 12, 542 (2021), a vaccine was designed using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain and was found to elicit anti-RBD antibodies and neutralizing antibodies. The virus like platform is based on engineered aldolase from thermophilic bacteria that spontaneously assembles into a hollow 36-nanometre dodecahedral cage with subunits.

[0006] Ma X. et al. 2020 (Ma X, et al. Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses. Immunity. 2020 Dec 15;53(6):1315-1330.e9) designed nanoparticle vaccines that covalently conjugate 24 copies of RBD or RBD-HR protein subunits to the self-assembled Helicobacter pylori non-haem ferritin. This RBD and RBD heptad repeat (HR) NP
platform elicited higher IgG antibodies titer than corresponding monomers. The HR subunit within S2 of the S protein was also able to induce neutralizing antibodies.
The neutralizing antibody titer of RBD-HR nanoparticle was higher than in the monomer vaccine and the RBD-nanoparticle vaccine. The RBD-HR nanoparticle was also successful in neutralizing other coronaviruses.

[0007] It remains desirable to develop a nucleic acid vaccine that is effective for inducing humoral and T-cell responses to coronaviruses such as SARS-CoV-2.
SUMMARY

[0008] It is an object of the present disclosure to obviate or mitigate at least one disadvantage of previous vaccine compositions against coronaviruses, for example against SARS-CoV-2.

[0009] Accordingly, an aspect herein disclosed relates to a nucleic acid molecule comprising a polynucleotide, the polynucleotide encoding a polypeptide comprising:
a receptor-binding domain (RBD) of a spike (S) protein of a coronavirus, and one or more of:
a heptad repeat 2 (HR2) domain of the S protein;
a potato virus X capsid protein (PVXCP); and a trimerization domain.

[0010] In an embodiment, the coronavirus is a human beta coronavirus, optionally SARS-CoV-2 or a variant or mutant thereof.

[0011] In an embodiment, wherein the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of any one of SEQ ID NOs: 2 to 14, or is or comprises the sequence of any one of SEQ ID
NOs: 2 to 14.

Date Recue/Date Received 2022-04-29

[0012] In an embodiment, the polypeptide comprises the HR2 domain, the domain optionally having an amino acid sequence having the sequence of SEQ ID
NO: 15 or at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 15, or has or comprises the sequence of SEQ ID NO:15.

[0013] In an embodiment, the polypeptide comprises the PVXCP, the PVXCP
optionally having a sequence of SEQ ID NO: 16 or at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 16, or has or comprises the sequence of SEQ ID NO:16.

[0014] In an embodiment, the polypeptide comprises the trimerization domain, optionally a GCN4 leucine zipper or a T4 foldon domain.

[0015] In an embodiment, the polypeptide further comprises a leader peptide, an influenza virus hemagglutinin (HA) leader peptide or a leader peptide of the human immunoglobulin E (IgE) or a leader peptide of the SARS-CoV-2 spike protein.

[0016] In an embodiment, the polypeptide comprises the leader peptide, the RBD, the GCN4 leucine zipper and the HR2 domain (from N- to C- terminus).

[0017] In an embodiment, the polypeptide has or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity to any one of SEQ ID NOs: 23 to 28.

[0018] In an embodiment, the polypeptide comprises one or more additional RDBs and/or one or more additional trimerization domains.

[0019] In an embodiment, the polypeptide comprises three RBDs, optionally 3 linear tandem repeats of the RBD. For example, the three RBDs are from the same coronavirus variant or strain. For example, the three RBDs are from different variants or strains.

[0020] Another aspect herein disclosed relates to a nucleic acid molecule comprising a polynucleotide, the polynucleotide encoding a polypeptide comprising a plurality of antigenic coronavirus peptides interspersed in an ankyrin repeat scaffold, the ankyrin repeat scaffold comprising a plurality of ankyrin repeat motifs.

[0021] In an embodiment, the ankyrin repeat scaffold is a designed ankyrin repeat protein (DARPin).

[0022] In an embodiment, the polypeptide further comprises a leader peptide, for example an influenza virus hemagglutinin (HA) leader peptide, a leader peptide of the human immunoglobulin E (IgE) or a leader peptide of the SARS-CoV-2 spike protein.

[0023] In an embodiment, the polypeptide further comprising a Toll-Like receptor 2 (TLR2) agonist peptide, optionally having at least 80% sequence identity to the sequence of Date Recue/Date Received 2022-04-29 SEQ ID NO: 107 and capable of activating a toll receptor, or having or comprising the sequence of SEQ ID NO: 107.

[0024] In an embodiment, the polypeptide further comprising a FMS-like tyrosine kinase 3 ligand (FLT3L), optionally having at least 80% sequence identity to the sequence of SEQ ID NO: 29, or having or comprising the sequence of SEQ ID NO: 29.

[0025] In an embodiment, the polypeptide further comprising an a-helix dimerization domain (dHLX) or a CH3 dimerization domain, the dHLX optionally having at least 80%
sequence identity to the sequence of SEQ ID NO: 30, or having or comprising the sequence of SEQ ID NO: 30.

[0026] In an embodiment, the polypeptide further comprising a PVXCP, the PVXCP
optionally having a sequence of SEQ ID NO: 16 or at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 16, or has or comprises the sequence of SEQ ID NO:16.

[0027] In an embodiment, wherein the polypeptide comprises non-structural antigenic coronavirus peptides, optionally wherein each of the non-structural coronavirus peptides has or comprises an amino acid sequence selected from Table 1 or 2 or has at least 80% sequence identity to the amino acid sequence selected from Table 1 or 2.

[0028] In an embodiment, the polypeptide comprises structural antigenic coronavirus peptides, optionally wherein each of the structural coronavirus peptides has or comprises amino acid sequence selected from Table 1 or 3 or at least 80% sequence identity to the amino acid sequence selected from Table 1 or 3.

[0029] In an embodiment, the polypeptide has or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity to any one of SEQ ID NOs: 83 to 94 or 108 or formulas Ito VII, including any one or more of SEQ ID Nos: 95 to 101 or 109 to 167, 170 and/or 171.

[0030] A short tail or long tail such as the ones in SEQ ID NO: 170 or 171 can be used. Other sequences can also be used after the last immune peptide.

[0031] Another aspect disclosed herein includes polypeptides as well as vectors comprising the nucleic acid molecule disclosed herein.

[0032] Also provided in further aspects include compositions and pharmaceutical compositions comprising the nucleic acid molecules, vectors and polypeptides disclosed herein, as well as methods and uses thereof.

[0033] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure Date Recue/Date Received 2022-04-29 are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Fig. 1 is a schematic representation of Component 1 vaccine constructs. (IgE
and HA: leader peptides; RBD: receptor-binding protein; HR2: heptad repeat 2 domain of the S protein of SARS-CoV-2; COV1: scaffold protein with viral epitopes; GCN4:
trimerization domain GCN4 leucine zipper; PVXCP: potato virus X capsid protein; tFliC:
truncated Salmonella typhimurium flagellin; TLR: scaffold protein with TLR2 agonist peptides).

[0035] Fig. 2 is a schematic representation of variants of the Component 2 vaccine design (IgE, HA: leader peptides; COV-1, COV-2: scaffold protein with viral epitopes; TLR:
scaffold protein with TLR2 agonist peptides; FLT3L: FMS-like tyrosine kinase 3 ligand; dHLX:
a-helix dimerization domain; PVXCP - potato virus X capsid protein; 00: TLR2 agonist peptides (as shown in constructs #50 and #9)).

[0036] Fig. 3A and 3B are images showing alignments of homological spike (Fig. 3A) and RNA- dependent RNA polymerase (RdRp) protein (Fig. 3B) regions of beta coronaviruses epitope regions.

[0037] Fig. 4 is a schematic of an ankyrin scaffold structure.

[0038] Fig. 5A is a 3D model of the Component 2 COV2 scaffold structure with MHC
epitopes being identified therein. Average structure from 300 ns Molecular Dynamics.

[0039] Fig. 5B is a 3D model of the Component 2 COV1 scaffold structure with MHC
epitopes being identified therein. Average structure from 300 ns Molecular Dynamics.

[0040] Fig. 6A is a graph showing the average root-mean-square deviation (RMSD) of heavy atomic coordinates of ankyrin repeat cores comprised in COV-1 and COV-constructs.

[0041] Fig. 6B is a graph showing total RMSD of the heavy atoms of COV-1 (top line) and COV-2 (lower line) constructs during the Molecular Dynamics simulation.

[0042] Fig. 7 is a graph showing the solvent-accessible surface area (SASA) of COV-2 (top line) and COV-1 (lower line) constructs during the Molecular Dynamics simulation.

[0043] Fig. 8 is a 3D structure of the Component 1 RBD-HR2-PVXCP vaccine protein (average structure from molecular dynamics simulation; prepared in UCSF
Chimera 1.15).

[0044] Fig. 9 is a graph showing the distance (in angstroms) between outermost atoms in the RBD-HR2-PVXCP protein during the Molecular Dynamics simulation.

Date Recue/Date Received 2022-04-29

[0045] Fig. 10 is a 3D structure of the RBD-HR2-PVXCP formed virus-like particle (VLP).

[0046] Fig. 11 is an image of a Western Blot analysis of the supernatant of H EK293F
cells 5 days after transfection with the DNA plasmids variants 15, 16, 39, 39D, 40 and 40D
(detected using anti-S-protein polyclonal antibodies).

[0047] Fig. 12A and 12B are bar graphs showing dynamic light scattering (DLS) data of purified His6-RBD-PVXCP (Fig. 12A) and His6-RBD-HR2-PVXCP (Fig. 12B) constructs.

[0048] Fig. 13 is a graph showing anti-RBD IgG antibody responses in mice having received vaccine constructs #14,_#15 or #16 via electroporation_(EP) or polyethylenimine (PEI) transfection.

[0049] Fig 14 is a graph showing anti-RBD IgG antibody endpoint titers in mice having received vaccine constructs #15 or #16 via EP or PEI transfection.

[0050] Fig. 15A and 15B are graphs showing IFNy secretion using ELISPOT
assay (Fig. 15A) and average ELISPOT spot area per well (Fig. 15B) for vaccine constructs #10 and #11 (each comprising both component 1 and 2 vaccine portions).

[0051] Fig. 16A and 16B are graphs showing IFNy secretion using ELISPOT
assay (Fig. 16A) and average ELISPOT spot area per well (Fig. 16B) for vaccine construct #9.

[0052] Fig. 17A and Fig. 17B are graphs showing the endpoint titers of anti-RBD IgG
antibodies in rabbits (Fig. 17A) and mice (Fig. 17B).

[0053] Fig. 17 C is a graph of IFN-y prod uction of mouse splenocytes by several tested constructs.

[0054] Fig. 18 is a western blot image depicting the expression level of PVXCP
protein.

[0055] Fig. 19 is a schematic of a vaccine construct comprising trimeric receptor binding domain (RBD) on the surface of each PVXCP molecule.
DETAILED DESCRIPTION OF THE DISCLOSURE

[0056] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

Date Recue/Date Received 2022-04-29

[0057] The term "SARS-CoV-2" as used herein refers to severe acute respiratory syndrome coronavirus 2 and includes the wild type virus as well as variants and mutants thereof, including, without limitation, B.1.1.7 (alpha), P1, P2, B.1.351, B.1.617.2.1(delta plus), B.1.617.2 (delta), C.37 (lambda), B.1.621 (Mu) and B.1.1.529 (omicron).

[0058] The term "spike protein", "spike (S) protein" or "spike (S) glycoprotein" as used herein refers to a protein that forms a spike or peplomer projecting from the surface of an enveloped virus such as a coronavirus, e.g. SARS-CoV-2. The sequence of the spike protein of wild type SARS-CoV-2 is provided in for example NCB! Reference Sequence:
YP_009724390.1 and having SEQ ID NO: 1.

[0059] The term "receptor-binding domain" or "RBD" as used herein refers to a fragment of the spike protein that binds particular receptor(s) (e.g.
angiotensin-converting enzyme 2) on host cells in order to initiate the infection process. "RBD" as used herein can refer to the whole RBD sequence, or to a portion, fragment, variant or mutant thereof that is capable of binding the host receptor. For example, in the context of SARS-CoV-2 (NCB!
Reference Sequence: YP_009724390.1), the "RBD" can include the entire RBD domain (e.g.
corresponding to residues 319-541 of the SARS-CoV-2 spike protein) or a portion, fragment, variant or mutant thereof such as corresponding to residues 437 to 508 . The "RBD" can also include additional contiguous residues outside of residues 319-541 of the SARS-CoV-2 spike protein. For example, the RBD of the wild type SARS-CoV-2 spike protein is or comprises amino acid residues 319-541, 329-579, 437-508, 319-526 or 331-521 of the SARS-CoV-2 spike protein.

[0060] As used herein the term "ankyrin repeat scaffold" refers to a polypeptide that is composed of ankyrin repeat motifs (or tandem repeats) of a sequence of about 33 amino acid residues. Each repeat motif forms two antiparallel a-helices followed by a p-turn. One or more antigenic coronavirus peptides interspersed in the ankyrin repeat scaffold. As used herein, "interspersed" means that the one or more antigenic coronavirus peptides is/are inserted into or grafted onto the ankyrin repeat scaffold, for example at the loop formed between two adjacent repeats. "Interspersed" also means that the one or more antigenic coronavirus peptides replace a number of native residues of the ankyrin repeat scaffold.
The ankyrin repeat motifs stack together in a modular fashion to form a scaffold that exposes the antigenic coronavirus peptides and can be used to present these antigenic coronavirus peptides. The ankyrin repeat scaffold comprises at least 2 ankyrin repeat motifs, for example 5, 6, 7, 10, 12, 15, 20 and up to 34 ankyrin repeat motifs. For example, the ankyrin repeat scaffold is a designed ankyrin repeat protein (DARPin). For example, the ankyrin repeat scaffold is a DARPin scaffold sequence corresponding to Protein Data Bank accession number (DOI :10.1038/s41598-017-11472-x).

Date Recue/Date Received 2022-04-29

[0061] The term "nucleic acid molecule" as used herein refers to a linked series of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages, including for example cDNA, vectors and recombinant polynucleotides. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof, which function similarly.
Such modified or substituted nucleic acid molecules may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability in the presence of nucleases. The term also includes chimeric nucleic acid molecules that contain two or more chemically distinct regions. For example, chimeric nucleic acid molecules may contain at least one region of modified nucleotides that confer beneficial properties (e.g.
increased nuclease resistance, increased uptake into cells), or two or more nucleic acid molecules described herein may be joined to form a chimeric nucleic acid molecule. The polynucleotides may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine.
Also, the term "nucleic acid" can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid" includes the complementary nucleic acid sequences.

[0062] The term "isolated nucleic acid" as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA
techniques, or chemical precursors, or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences which naturally flank the nucleic acid (i.e. sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived. The term "nucleic acid" is intended to include DNA and RNA
and can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid" includes the complementary nucleic acid sequences, for example cDNA.

[0063] The term "polypeptide" as used herein refers to a polymer consisting of a large number of amino acid residues bonded together in a chain. The polypeptide may be arranged in a long, continuous and unbranched peptide chain. The polypeptide may also be arranged in a biologically functional way. The polypeptide may be folded into a specific three dimensional structure that confers it a defined activity.

[0064] The term "isolated polypeptide", also referred to as "isolated protein" refers to a polypeptide substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.

Date Recue/Date Received 2022-04-29

[0065] The term "amino acid" includes all of the naturally occurring amino acids as well as modified amino acids.

[0066] The term "variant" means a molecule such as a nucleic acid molecule or polypeptide that differs from but substantially retains the same function as the original molecule.
Changes to the molecule include a substitution, deletion or addition of one or a few to a plurality of nucleotides in a nucleic acid molecule or amino acids in a polypeptide, and includes particularly conservatively substituted molecules such as a conservative amino acid substitution. For example, the potato virus X capsid protein (PVXCP) can comprise up to 50, up to 40, up to 30, up to 20 or up to 10 amino acid deletion and/or conservative substitutions.
Similarly variants of other molecules or domains can comprise up to 50, up to 40, up to 30, up to 20 or up to 10 amino acid deletion and/or conservative substitutions.

[0067] A "conservative amino acid substitution" as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Examples of conservative amino acid substitutions include:
Conservative Substitutions Type of Amino Acid Substitutable Amino Acids Hydrophilic Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr Sulphydryl Cys Aliphatic Val, Ile, Leu, Met Basic Lys, Arg, His Aromatic Phe, Tyr, Trp

[0068] The term "sequence identity" as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding Date Recue/Date Received 2022-04-29 position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (Le., % identity=number of identical overlapping positions/total number of positions×100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.
Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci.
U.S.A. 90:5873-5877. Such an algorithm is incorporated into the N BLAST and XBLAST
programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present application. BLAST protein searches can be performed with the XBLAST
program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST
and NBLAST) can be used (see, e.g., the NCB! website). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN
program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0069] The expression "at least 80% sequence identity" used herein means at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% (or any percentage between 80% and 100% e.g. 87%, 88%, 89%).

[0070] By "at least moderately stringent hybridization conditions" it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm = 81 .5 C - 16.6 (Log10 [Na+]) + 0.41 Date Recue/Date Received 2022-04-29 (%(G+C) - 600/I), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1 % mismatch may be assumed to result in about a 1 C decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5 C.
Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride/sodium citrate (SSC)/5x Denhardt's solution/1.0% SDS at Tm - 5 C based on the above equation, followed by a wash of 0.2x SSC/0.1 SDS at 60 C. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42 C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in: Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0071] The term "linker" as used herein means a short polypeptide sequence interposed between any two neighboring peptide sequences as described herein.
In an embodiment, the linker is a polypeptide linker of Ito 10 amino acids, preferably 1, 2, 3, 4 or 5 naturally or non-naturally occurring amino acids. In an embodiment, the polypeptide can comprises one or more peptidic or polypeptidic linker(s) together with one or more other non-peptidic or non-polypeptidic linker(s). Further, different types of linkers, peptidic or non-peptidic, may be incorporated in the same polypeptide as deemed appropriate. In the event that a peptidic or polypeptidic linker is used to join two respective peptide sequences, the linker will be advantageously incorporated such that its N-terminal end is bound via a peptide bond to the C-terminal end of the one peptide sequence, and its C-terminal end via a peptide bond to the N-terminal end of the other peptide sequence. The individual peptide sequences within the polypeptide may also have one or more amino acids added to either or both ends, preferably to the C-terminal end. Thus, for example, linker or spacer amino acids may be added to the N-or C-terminus of the peptides or both, to link the peptides and to allow for convenient coupling of the peptides to each other and/or to a delivery system such as a carrier molecule serving as an anchor.

[0072] The term "subject" as used herein refers to any member of the animal kingdom, preferably a mammal, more preferably a human being, a rabbit or a rodent such as a rat or a mouse.

[0073] The term "prevention" or "preventing" as used herein means administering to a subject a nucleic acid molecule, immunogenic composition or vaccine herein disclosed. The Date Recue/Date Received 2022-04-29 administering may consist of a single administration, or alternatively comprise a series of administrations. The "prevention" or "preventing" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, prevention of infection by a coronavirus (e.g. SARS-CoV-2) or alleviation or amelioration of one or more symptoms or conditions related to coronavirus infection, diminished extent of infection, stabilized (i.e. not worsening) of symptom(s) related to the infection, preventing spread of infection or amelioration of symptom(s) related to the infection. It will be understood that "prevention" and "preventing" encompass treatment of a subject.

[0074] The term "composition" as used herein, refers to a mixture comprising two or more components. For example, the composition is a composition of two nucleic acid molecules disclosed herein. For example, the composition is a composition of two vectors disclosed herein. For example, the composition is a composition of two polypeptides disclosed herein.
The composition can further comprise a pharmaceutically acceptable carrier, diluent or excipient.

[0075] The term "immunogenic composition" as used herein means a composition that, when administered to a subject, is capable of eliciting an immune response. In some embodiments, the immunogenic composition is capable of eliciting a humoral response or a T-cell response. In some embodiments, the immunogenic composition is capable of eliciting a humoral and a T-cell response.

[0076] The expression "pharmaceutically acceptable" as used herein means acceptable for use in the pharmaceutical and veterinary arts, i.e. not being unacceptably toxic or otherwise unsuitable.

[0077] The term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.

[0078] As used herein, the phrase "effective amount", "therapeutically effective amount" or a "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired result. An effective"
amount of the nucleic acid molecule, vector, polypeptide or composition (together referred to as "compounds") Date Recue/Date Received 2022-04-29 disclosed herein may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compounds to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compounds are outweighed by the therapeutically beneficial effects A "prophylactically effective amount"
refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0079] In understanding the scope of the present disclosure, the term "comprising"
and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[0080] The term "consisting" and its derivatives, as used herein, are intended to be closed ended terms that specify the presence of stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.

[0081] Further, terms of degree such as "substantially", "about" and "approximately"
as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least 5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0082] More specifically, the term "about" means plus or minus 0.1 to 50%, 5-50%, or 10-40%, 10-20%, 10%-15%, preferably 5-10%, most preferably about 5% of the number to which reference is being made.

[0083] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing "a compound" includes a mixture of two or more compounds. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.

[0084] The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.

Date Recue/Date Received 2022-04-29

[0085] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. Ito 5 includes 1, 1.5, 2, 2/5, 3, 3.90, 4, and 5).
It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about"

[0086] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

[0087] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0088] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0089] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be under-stood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0090] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of methods and materials are now described.

[0091] The present disclosure relates generally to nucleic acid molecules, vectors, polypeptides and compositions useful in vaccines for the prevention of coronaviruses such as SARS-CoV-2. The inventors have developed vaccines aimed at inducing a predominantly humoral immune response (referred to herein as Component 1 vaccines) as well as vaccines aimed at inducing a predominantly cellular immune response (referred to herein as Component 2 vaccines). While each vaccine construct may be used separately, it was found that when both vaccine constructs are used together, they may provide an improved immune response against a coronavirus.
Component 1 vaccines Date Recue/Date Received 2022-04-29

[0092] The inventors have developed vaccines aimed at inducing a predominantly humoral immune response. These vaccines are also referred to herein as Component 1 vaccines.

[0093] Accordingly, a first aspect disclosed herein relates to a nucleic acid molecule comprising a polynucleotide, the polynucleotide encoding a polypeptide comprising a receptor-binding domain (RBD) of a spike (S) protein of a coronavirus.

[0094] Another aspect relates to a polypeptide comprising a receptor-binding domain (RBD) of a spike (S) protein of a coronavirus.

[0095] While the full RBD sequence may be used, it will be understood that a portion, variant or mutant thereof may also be used as long as the portion, variant or mutant is able to elicit an immune response, for example upon being introduced into and expressed in a host cell.

[0096] In an embodiment, the coronavirus is SARS-CoV-2 and the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity to the sequence of SEQ ID NO: 2. In an embodiment, the RBD is or comprises the sequence of SEQ ID NO: 2.

[0097] In an embodiment, the coronavirus is SARS-CoV-2 and the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity to the sequence of SEQ ID NO: 3. In an embodiment, the RBD is or comprises the sequence of SEQ ID NO: 3.

[0098] In an embodiment, the coronavirus is SARS-CoV-2 and the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity to the sequence of SEQ ID NO: 4. In an embodiment, the RBD is or comprises the sequence of SEQ ID NO: 4.

[0099] In an embodiment, the coronavirus is SARS-CoV-2 and the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity to the sequence of SEQ ID NO: 5. In an embodiment, the RBD is or comprises the sequence of SEQ ID NO: 5.

[00100] In another embodiment, the coronavirus is SARS-CoV-2 variant B.1.1.7(alpha) and the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 6. In an embodiment, the RBD has or comprises the sequence of SEQ ID NO: 6.

[00101] In another embodiment, the coronavirus is SARS-CoV-2 variant P.1 and the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%
or at least 99%

Date Recue/Date Received 2022-04-29 sequence identity to the sequence of SEQ ID NO: 7. In an embodiment, the RBD
has or comprises the sequence of SEQ ID NO: 7.

[00102] In another embodiment, the coronavirus is SARS-CoV-2 variant P.2 and the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%
or at least 99%
sequence identity to the sequence of SEQ ID NO: 8. In an embodiment, the RBD
has or comprises the sequence of SEQ ID NO: 8.

[00103] In another embodiment, the coronavirus is SARS-CoV-2 variant B.1.351 (beta) and the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 9. In an embodiment, the RBD has or comprises the sequence of SEQ ID NO: 9.

[00104] In another embodiment, the coronavirus is SARS-CoV-2 variant B.1.617.2 (delta) and the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 10. In an embodiment, the RBD
has or comprises the sequence of SEQ ID NO: 10.

[00105] In another embodiment, the coronavirus is SARS-CoV-2 variant B.1.617.2.1 (delta plus) and the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 11. In an embodiment, the RBD has or comprises the sequence of SEQ ID NO: 11.

[00106] In another embodiment, the coronavirus is SARS-CoV-2 variant C.37 (lambda) and the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 12. In an embodiment, the RBD has or comprises the sequence of SEQ ID NO: 12.

[00107] In another embodiment, the coronavirus is SARS-CoV-2 variant B.1.621 (Mu) and the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 13. In an embodiment, the RBD has or comprises the sequence of SEQ ID NO: 13.

[00108] In another embodiment, the coronavirus is SARS-CoV-2 variant B.1.1.529 (omicron) and the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%
or at least 99% sequence identity to the sequence of SEQ ID NO: 14. In an embodiment, the RBD has or comprises the sequence of SEQ ID NO: 14.

[00109] The polypeptide further comprises one or more of a heptad repeat 2 (HR2) domain of the S protein; a potato virus X capsid protein (PVXCP); and a trimerization domain.

[00110] In an embodiment, the polypeptide comprises the HR2 domain, the PVXCP
and the trimerization domain.

Date Recue/Date Received 2022-04-29

[00111] As referred to herein, "HR2 domain" means the entire heptad receptor sequence 2 (HR2) or a portion thereof e.g. any portion minimally comprising 10, 11 or 12 amino acids that may be used as an immunogen for eliciting neutralizing antibodies.
The HR2 in the SARS-CoV-2 spike protein (corresponding to amino acid residues 1163-1213 of SEQ ID NO:
1) is a conserved domain and is effective in eliciting neutralizing antibodies, as described in Elshabrawy HA et al. (2012) Human Monoclonal Antibodies against Highly Conserved HR1 and HR2 Domains of the SARS-CoV Spike Protein Are More Broadly Neutralizing. PLOS
ONE
7(11): e50366, herein incorporated by reference in its entirety.

[00112] In some embodiments, the HR2 domain and the RBD are of the same coronavirus strain or variant. In other embodiments, the HR2 domain and the RBD are of different coronavirus strain or variant.

[00113] In an embodiment, the polypeptide comprises an HR2 domain. In some embodiments, the HR2 domain has or comprises the sequence of SEQ ID NO:15 (corresponding to residues 1168-1203 of SARS-CoV-2 spike protein). In other embodiments, the HR2 domain is or comprises at least 10 continuous amino acids in SEQ ID
NO: 15. In some embodiments, the HR2 domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 15.

[00114] In an embodiment, the polypeptide comprises a PVXCP. As referred to herein, "PVXCP" or Potato virus X coat protein or PVX-gp5 means an entire PVXCP
protein sequence or a portion or variant thereof that maintains its ability to oligomerize and form oligomeric disks.
Various PVXCPs are known including but not limited to the sequence in Gene Bank Sequence ID: X65015.1, as well as related strains including but not limited to X3 (Gene ID: 7065758 ), HB, UK3, XC, XA, XC, KP, N11 or N14.PVXCP N14 for example includes an 11 amino acid insertion. In an embodiment, the PVXCP is or comprises the sequence of SEQ ID
NO: 16. In some embodiments, the PVXCP has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 16.
Sequence variants that maintain PVXCPs ability to oligomerize and form oligomeric disks are contemplated. Portions of the PVXCP, e.g. up to 29 amino acid residues, may be in some embodiments truncated or substituted with a different sequence, as described in Grinzato, A et al. Atomic structure of potato virus X, the prototype of the Alphaflexiviridae family. Nat Chem Biol 16, 564-569 (2020), herein incorporated by reference in its entirety.

[00115] In an embodiment, the polypeptide comprises a trimerization domain, optionally located at or near the C-terminus of the polypeptide.

[00116] In an embodiment, the trimerization domain is or comprises a GCN4 leucine zipper. For example the GCN4 leucine zipper has or comprises the sequence of SEQ ID NO:
17. In some embodiments, the GCN4 leucine zipper has at least 80%, at least 85%, at least Date Recue/Date Received 2022-04-29 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID
NO: 17.

[00117] In another embodiment, the trimerization domain is or comprises a T4 foldon domain (corresponding to the C-terminal residues of fibritin from bacteriophage T4). In some embodiments, the T4 foldon domain has or comprises the sequence of SEQ ID NO:
18. In some embodiments, the T4 foldon domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:
18.

[00118] In another embodiment, the polypeptide further comprises a truncated Salmonella typhimurium flagellin (tFliC). In some embodiments, the tFliC has or comprises the sequence of SEQ ID NO: 19. In some embodiments, the tFliC has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 19.

[00119] A linker can be inserted between one or more portions of the vaccine constructs using well understood techniques in the art.

[00120] For example, a linker can be inserted between a leader peptide and the RBD.
In an embodiment, the linker is or comprises GSGA (SEQ ID NO: 20).

[00121] In an embodiment, the polypeptide comprises a leader peptide. For example, the leader peptide is the leader peptide of the influenza virus hemagglutinin (HA), optionally having or comprising the sequence of SEQ ID NO: 21, or having at least 80%
sequence identity to the sequence of SEQ ID NO: 21.

[00122] For example, the leader peptide is the leader peptide of the human immunoglobulin E protein (IgE), optionally having or comprising the sequence of SEQ ID NO:
22, or having at least 80% sequence identity to the sequence of SEQ ID NO: 22.

[00123] It will be understood that other leader peptides known in the art that are used for secretion may be used such as IL2 and tPA.

[00124] As shown in the Examples and illustrated at Fig. 1, the inventors have developed Component 1 vaccines, examples of which include constructs 14, 15, 16, 39, 39D, 40 and 40D.

[00125] In one embodiment, the polypeptide comprises a leader peptide (optionally an HA leader peptide), an RBD, a GCN4 leucine zipper and an HR2 domain (from N-to C-terminus).

[00126] For example, the polypeptide has or comprises a sequence of SEQ
ID NO: 23 (corresponding to Construct #15). For example, the polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 23.

Date Recue/Date Received 2022-04-29

[00127] In another embodiment, the polypeptide comprises a leader peptide (optionally an HA leader peptide), an RBD and a PVXCP.

[00128] For example, the polypeptide has or comprises a sequence of SEQ
ID NO: 24 (corresponding to Construct #16). For example, the polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 24.

[00129] For example, the polypeptide has or comprises a sequence of SEQ
ID NO: 25 (corresponding to Construct #39). For example, the polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 25.

[00130] For example, the polypeptide has or comprises a sequence of SEQ
ID NO: 26 (corresponding to Construct #39D). For example, the polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:
26.

[00131] For example, the polypeptide has or comprises a sequence of SEQ
ID NO: 27 (corresponding to Construct #40). For example, the polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 27.

[00132] For example, the polypeptide has or comprises a sequence of SEQ
ID NO: 28 (corresponding to Construct #40D). For example, the polypeptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO:
28.

[00133] The inventors have also developed trimeric vaccines as described in the Examples. Accordingly, another aspect herein described is a nucleic acid molecule comprising a polynucleotide which encodes a polypeptide that comprises three receptor-binding domains (RBD) of a spike (S) protein of a coronavirus, three trimerization domains and a potato virus X
capsid protein.

[00134] In an embodiment, the polypeptide comprises a linear tandem repeat of three RBDs.

[00135] The three RBDs may in one embodiment be from the same coronavirus variant or strain. In another embodiment, the three RBDs are from 2 or 3 different RBDs. For example, a first RBD is from wild type SARS-CoV-2, a second RBD is from the SARS-CoV-2 delta variant, and a third RVD is from the SARS-CoV-2 omicron variant.

[00136] In an embodiment, the polypeptide comprises three RBDs, three trimerization domains, a PVXCP and two cleavage sites, optionally in the following N- to C-terminal order:
signal peptide ¨ RBD ¨ trimerization domain ¨ cleavage site ¨ RBD ¨
trimerization domain ¨
cleavage site ¨ RVD ¨ trimerization domain ¨ PVXCP.

Date Recue/Date Received 2022-04-29

[00137] In an embodiment, the trimerization domain is a T4 foldon domain or a GCN4 leucin zipper. In an embodiment, the trimerization domain is a type IX
collagen NC2 hetero-trimerization domain (domain 1, domain 2 or domain 3).

[00138] In an embodiment, the cleavage site is a furin cleavage site or a C1r-like protease cleavage site.

[00139] In an embodiment, the components of the construct are linked via a linker, for example a flexible linker e.g. GSGA (SEQ ID NO: 20).

[00140] In an embodiment, the polypeptide comprises the sequence of SEQ
ID NO:
77. In an embodiment, the polypeptide comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 77.

[00141] The RBD and the HR2 domains are viral antigens used for inducing an antibody response and are specific for a particular virus (e.g. SARS-CoV-2).
The RBD domain of the S-protein of SARS-CoV-2 can be from the wildtype virus but also from different variants, including but not limited to mutant strains: B.1.1.7 (alpha), P1, P2, B.1.351, B.1.617.2.1 (delta plus), B.1.617.2 (delta), C.37 (lambda), B.1.621 (Mu) and B.1.1.529 (omicron).

[00142] The nucleic acid molecule of the present disclosure can be linked to another nucleic acid so as to be expressed under control of a suitable promoter.
Examples of the promoter include a promoter that constitutively promotes the expression of a gene or operatively linked construct, a promoter that induces the expression of a gene or operatively linked construct by the action of a drug or the like (e.g. tetracycline or doxorubicin). The nucleic acid molecule of the present disclosure can be also linked to, in order to attain efficient transcription of the nucleic acid, other regulatory elements that cooperate with a promoter or a transcription initiation site, for example, a nucleic acid comprising an enhancer sequence or a terminator sequence. In addition to the nucleic acid of the present disclosure, a gene that can be a marker for confirming expression of the nucleic acid (e.g. a drug resistance gene, a gene encoding a reporter enzyme, or a gene encoding a fluorescent protein) may be incorporated.

[00143] "Operatively linked" as used herein means that the nucleic acid molecule is linked to regulatory sequences in a manner which allows expression of the nucleic acid molecule. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropriate regulatory sequences is dependent on the host cell chosen and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction Date Recue/Date Received 2022-04-29 sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.
Cornponent 2 vaccines

[00144] The inventors have developed vaccines aimed at inducing a predominantly cellular immune response. These vaccines are also referred to herein as Component 2 vaccines. The inventors also designed nucleic acid constructs that encode a polypeptides that comprises an ankyrin repeat scaffold which is modified to comprise a plurality of antigenic coronavirus peptides. The peptides are immunogenic peptides intended for MHC-II and MHC-I presentation to T-lymphocytes. The ankyrin repeats form a non-immunogenic scaffold protein and the polypeptide is configured such that the viral epitopes are exposed.

[00145] Accordingly, an aspect disclosed herein relates to a nucleic acid molecule comprising a polynucleotide, the polynucleotide encoding a polypeptide comprising a plurality of antigenic coronavirus peptides interspersed in an ankyrin repeat scaffold.

[00146] Another aspect provided relates to a polypeptide comprising a plurality of antigenic coronavirus peptides interspersed in an ankyrin repeat scaffold.

[00147] In an embodiment, the ankyrin repeat scaffold is a designed ankyrin repeat protein (DARPin).

[00148] The antigenic coronavirus peptides are peptides identified as being the most immunogenic. As described herein and shown in Table 1, pan-coronavirus T-cell epitopes were identified. These include peptides from structural proteins such as from capsid proteins (referred to herein as "COV-1" or COV1 group) and peptides from non-structural protein peptides which are predominantly considered epitopes of viral replicative proteins (referred to herein as" COV-2" or COV2 group).

[00149] "COV1_Scaffold' for example refers to the construct that includes peptides from the spike protein (S), the membrane glycoprotein (M) as for example indicated in Table 3, as well as proteins referred to in Table 1 such as the nucleocapsid protein.
These peptides may be used in the COV-X1 constructs.

[00150] COV-1 or COV-2 group epitopes were ranked based on their Grand Average of Hydropathy (GRAVY) index and isoelectric point. Selected peptides were incorporate onto the ankyrin scaffold protein.

[00151] Principles such as those described in Li et al 2006 (Li, J. Ankyrin Repeat: A Unique Motif Mediating Protein-Protein Interactions / J. Li, A. Mahajan, M.-D. Tsai // Biochemistry. -2006. - Vol. 45, Ng 51. - P. 15168-15178), can be used for identifying the sites for epitope insertions. The peptide(s)/epitope(s) were inserted into or replaced or partially replaced one or more of the flexible loop portions of the ankyrin protein. Each ankyrin repeat exhibits a helix-Date Recue/Date Received 2022-04-29 turn-helix conformation, and strings of such tandem repeats are packed in a nearly linear "bundle" to form helix-turn-helix bundles with relatively flexible loops.
These loops can have a variable composition and variable lengths. To define the beginning and end of the loops software such as UCSF Chimera software which is based on the algorithm described in Gibrat et al. 1987, (Gibrat, J.-F. Further developments of protein secondary structure prediction using information theory / J.-F. Gibrat, J. Gamier, B. Robson // J. Mol. Biol. ¨
1987.¨ Vol. 198, Ng 3.
¨ P. 425-443), can be used.

[00152] As demonstrated in the Examples, the flexible loops are parts thereof were replaced with desired peptides containing MHC epitopes. The peptide or peptides inserted can for example be about the same length as the flexible loop being replaced. The inserted peptides may be shorter, for example 1 or 2 or 3 amino acids shorter than a native loop or for example 1 or 3 or 3 amino acids longer than a native loop.

[00153] The polypeptide can in some embodiments include a plurality of different antigenic coronavirus peptides. In other embodiments, the polypeptide includes a plurality of identical coronavirus peptides.

[00154] As used herein, "pan-coronavirus" in the context of a vaccine means a vaccine that comprises antigenic coronavirus peptides or nucleic acid molecules encoding such peptides that have homology across various coronaviruses such as and without limitation to SARS-CoV-2, SARS-CoV-1, MERS, HKU1, NL63,0C4,229E.

[00155] In an embodiment, the coronavirus peptides have a length of about 10 to 25 amino acids, 12 to 23 amino acids, 15 to 25 amin acids or 15 to 20 amino acids.

[00156] For example, the plurality of coronavirus peptides is selected from peptides in Tables 2 and 3.

[00157] For example, the coronavirus peptide has at least 80% sequence identity to the peptide of Table 2 or 3.

[00158] In an embodiment, the antigenic coronavirus peptides are non-structural proteins, for example replicative proteins. For example, the antigenic coronavirus peptides are selected from Table 1 or 2.

[00159] For example, each of the non-structural coronavirus peptides has or comprises an amino acid sequence selected from Table 1 or 2 or has at least 80% sequence identity to the amino acid sequence selected from Table 1 or 2.

[00160] In an embodiment, the antigenic coronavirus peptides are from structural proteins such as the spike (S) protein, the nucleocapsid (N), the membrane (M) protein or the envelope (E) protein. For example, the antigenic coronavirus peptides are selected from Table 1 or 3.

Date Recue/Date Received 2022-04-29

[00161] For example, each of the structural coronavirus peptides has or comprises amino acid sequence selected from Table 1 or 3 or at least 80% sequence identity to the amino acid sequence selected from Table 1 or 3

[00162] For example, the polypeptide comprising a plurality of antigenic coronavirus peptides interspersed in an ankyrin repeat scaffold (or COV1 or COV2 scaffold) is or comprises a sequence as set out in Table 5.

[00163] For example, the polypeptide comprising a plurality of antigenic coronavirus peptides interspersed in an ankyrin repeat scaffold (or COV1 or COV2 scaffold) is or comprises a sequence of any one of formulas Ito VII, including for example components thereof as shown in any one of SEQ ID NOs: 95 to 101, 109-167, 170 and/or 171.

[00164] For example, the COV1 or COV2 scaffold is or comprises as sequence as set out in Table 6.

[00165] In an embodiment, the polypeptide further comprises a leader peptide, for example IgE or HA.

[00166] In an embodiment, the leader peptide is an influenza virus hemagglutinin (HA) leader peptide, optionally having at least 80% sequence identity to the sequence of SEQ ID
NO: 21, or having or comprising the sequence of SEQ ID NO: 21.

[00167] In another embodiment, the leader peptide is a leader peptide of the human immunoglobulin E protein (IgE), optionally having at least 80% sequence identity to the sequence of SEQ ID NO: 22, or having or comprising the sequence of SEQ ID NO:
22.

[00168] The polypeptide described herein can comprise one or more linkers. For example, the linker is a flexible linker, a rigid linker or a cleavable linker.

[00169] Linkers can be incorporated to the polypeptide to flank one or more of the antigenic coronavirus peptides (or MHC-epitopes) to enhance proteasomal cleavage in cells (e.g. mammalian cells). In some embodiments, the polypeptide comprises linkers flanking each antigenic coronavirus peptide.

[00170] In an embodiment, the linker is AAY. The AAY linker has been used in a multi-epitope vaccine design for efficient cleavage in the proteasomes in mammalian cells, to help form natural epitopes and prevent the formation of 'junctional epitopes' and to enhance epitope presentation (Yang Yet al. In silico design of a DNA-based HIV-1 multi-epitope vaccine for Chinese populations. Hum Vaccin I mmunother. 2015;11(3):795-805).

[00171] In another embodiment, the linker is GSGA (SEQ ID NO: 20), EAAAK
(SEQ ID
NO: 102), RVRR (SEQ ID NO: 103), GGGS (SEQ ID NO: 104), GPGPG (SEQ ID NO:
105), HEYGAEALERAG (SEQ ID NO: 106) Date Recue/Date Received 2022-04-29

[00172] In an embodiment, the polypeptide further comprising a Toll-Like receptor 2 (TLR2) agonist peptide, optionally having at least 80% sequence identity to the sequence of SEQ ID NO: 107 and able to activate a Toll receptor, or having or comprising the sequence of SEQ ID NO: 107. As used herein, "TLR2 agonist peptide" also includes a plurality thereof. For example, the TLR2 agonist peptide of SEQ ID NO: 107 consists of two different TLR2 peptide agonists ALSGCTGLTSITIPN (SEQ ID NO: 168) and GESAFKGCSGLKSIT (SEQ ID NO:
169), separated by GGS spacer. The TLR2 agonist peptide may comprise ALSGCTGLTSITIPN
SEQ
ID NO: 168) or GESAFKGCSGLKSIT (SEQ ID NO: 169)or fragments thereof that are able to activate a Toll receptor, or the combination, optionally spaced by a spacer such as glycine serine linkers.

[00173] The polypeptide disclosed herein may further comprise a FMS-like tyrosine kinase 3 ligand (FLT3L). FLT3L is a ligand for the FLT3 receptor which is expressed on the surface of conventional dendritic cells 1 and 2, which are known for their for their cross-priming activity of T-cells (CD4+ and CD8+ T-cells) and NK-cells. Accordingly, by targeting conventional dendritic cells, the immunogenicity and antiviral activity of the vaccine may be increased as seen with the hepatitis B virus (HBV) DNA vaccine described in Wang Y. et al., 2016 (Wang Y, Wu S, Wang ZC, Zhu XM, Yin XT, Gao K, Du ZY, Chen GZ, Yu JY. Enhanced immunity and antiviral effects of an HBV DNA vaccine delivered by a DC-targeting protein. J
Viral Hepat.
2016 Oct;23(10):798-804). Methods of augmenting an immune response by administering an amount of FLT3L are described in PCT Publication No. WO/1997/012633, incorporated herein by reference in its entirety.

[00174] In an embodiment, the polypeptide further comprising a FMS-like tyrosine kinase 3 ligand (FLT3L), optionally having at least 80% sequence identity to the sequence of SEQ ID NO: 29, or having or comprising the sequence of SEQ ID NO: 29.

[00175] In an embodiment, the polypeptide further comprising an a-helix dimerization domain (dHLX) or a CH3 dimerization domain, the dHLX optionally having at least 80%
sequence identity to the sequence of SEQ ID NO: 30, or having or comprising the sequence of SEQ ID NO: 30.

[00176] In an embodiment, the polypeptide further comprising a PVXCP, the PVXCP
optionally having a sequence of SEQ ID NO: 16 or at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 16, or has or comprises the sequence of SEQ ID NO:16

[00177] In an embodiment, the polypeptide comprises a plurality of linkers flanking one or more of the antigenic coronavirus peptides, optionally wherein the linker is or comprises AAY, GSGA (SEQ ID NO: 20), EAAAK (SEQ ID NO: 102), RVRR (SEQ ID NO: 103), GGGS

(SEQ ID NO: 104), GPGPG (SEQ ID NO: 105), HEYGAEALERAG (SEQ ID NO: 106).

Date Recue/Date Received 2022-04-29

[00178] In an embodiment, the polypeptide has or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity to any one of SEQ ID NOs: 83 to 94, 108 and formulas Ito VII, including one or more of components 95- 101, 109 to 167, 170 and/or 171.

[00179] In an embodiment, the polypeptide has or comprises the amino acid sequence of SEQ ID NO: 83. In an embodiment, the polypeptide has or comprises the amino acid sequence of SEQ ID NO: 84. In an embodiment, the polypeptide has or comprises the amino acid sequence of SEQ ID NO: 85. In an embodiment, the polypeptide has or comprises the amino acid sequence of SEQ ID NO: 86. In an embodiment, the polypeptide has or comprises the amino acid sequence of SEQ ID NO: 87. In an embodiment, the polypeptide has or comprises the amino acid sequence of SEQ ID NO: 88. In an embodiment, the polypeptide has or comprises the amino acid sequence of SEQ ID NO: 89. In an embodiment, the polypeptide has or comprises the amino acid sequence of SEQ ID NO: 90, In an embodiment, the polypeptide has or comprises the amino acid sequence of SEQ ID NO: 91. In an embodiment, the polypeptide has or comprises the amino acid sequence of SEQ ID NO: 92. In an embodiment, the polypeptide has or comprises the amino acid sequence of SEQ ID
NO: 93. In an embodiment, the polypeptide has or comprises the amino acid sequence of SEQ
ID NO: 94.
In an embodiment, the polypeptide has or comprises the amino acid sequence of formula I, for example comprising SEQ ID NOs: 95, 109, 110, 111 112, 113, 114 and/or 115. In an embodiment, the polypeptide has or comprises the amino acid sequence of formula II, for example comprising SEQ ID NOs: 96, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125. 126, 127 128, 170 and/or 171. In an embodiment, the polypeptide has or comprises the amino acid sequence of formula III, for example comprising SEQ ID NOs: 97, 129, 130, 131, 132, 133, 134 and/or 135. In an embodiment, the polypeptide has or comprises the amino acid sequence of formula IV, for example comprising SEQ ID NOs: 98,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, and/or 148. In an embodiment, the polypeptide has or comprises the amino acid sequence of formula V, for example comprising SEQ ID NOs: 99, 149, 150, 151, 152, 153, 154, and/or 155. In an embodiment, the polypeptide has or comprises the amino acid sequence of formula VI, for example comprising SEQ ID NOs: 100,156, 157, 158, and/or 159.
In an embodiment, the polypeptide has or comprises the amino acid sequence of formula VII, for example comprising SEQ ID NOs: 101, 160, 161, 162, 163, 164, 165, 166 and/or 167. In an embodiment, the polypeptide has or comprises the amino acid sequence of SEQ ID
NO: 108.

[00180] Also provided is a nucleic acid encoding any polypeptide described herein, including for example any one of SEQ ID Nos: 83 to 94, 108, or formulas 1-VII, comprising for example one or more components such as those described in SEQ ID Nos: 95-101 and 109 to 167, 170 and/or 171.

Date Recue/Date Received 2022-04-29

[00181] In another aspect there is provided a vector comprising a nucleic acid molecule disclosed herein.

[00182] The nucleic acid molecule of the present disclosure can be inserted into a vector, and the vector can be introduced into a cell. For example, a virus vector such as a retrovirus vector (including an oncoretrovirus vector, a lentivirus vector, and a pseudotyped vector), an adenovirus vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector or a sendai virus vector, an Epstein-Barr virus (EBV) vector, and a HSV
vector can be used. For example, a virus vector lacking the replicating ability so as not to self-replicate in an infected cell can be used.

[00183] In an embodiment, the virus is a dead or attenuated virus. In another embodiment the virus is a live virus.

[00184] In an embodiment, the vector is a plasmid vector.

[00185] In addition, a non-virus vector can also be used in the present disclosure in combination with a liposome or a condensing agent such as a cationic lipid as described in WO
96/10038, WO 97/18185, WO 97/25329, WO 97/30170 and WO 97/31934 (which are incorporated herein by reference in their entirety).

[00186] The nucleic acid molecule of the present disclosure can be also introduced into a cell by calcium phosphate transduction, DEAE-dextran, electroporation, or particle bombardment.

[00187] For example, when a retrovirus vector is used, the process can be carried out by selecting a suitable packaging cell based on a LTR sequence and a packaging signal sequence possessed by the vector and preparing a retrovirus particle using the packaging cell.
Examples of the packaging cell include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm-12 (US Patent No. 5,278,056), and Psi-Crip [Proceedings of the National Academy of Sciences of the United States of America, vol. 85, pp.
6460-6464 (1988)].
A retrovirus particle can also be prepared using a 293 cell or a 293T cell having high transfection efficiency. Many kinds of retrovirus vectors produced based on retroviruses and packaging cells that can be used for packaging of the retrovirus vectors are widely commercially available from many companies.

[00188] In an embodiment, the nucleic acid molecule is associated with a lipid, optionally wherein the nucleic acid molecule or vector is encapsulated in a liposome, interspersed within a liposome lipid bilayer or attached to a liposome.

[00189] Compositions comprising the nucleic acid molecule disclosed herein, the vector disclosed herein, or the polypeptide disclosed herein are also provided.

Date Recue/Date Received 2022-04-29

[00190] In an embodiment, the composition comprises a nucleic acid molecule encoding a Component 1 polypeptide and a nucleic acid molecule encoding a Component 2 polypeptide. As mentioned above, Component 1 is aimed at inducing a predominantly humoral immune response and Component 2 is aimed at inducing a predominantly cellular immune response.

[00191] In an embodiment, the composition is a pharmaceutical composition.

[00192] In an embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, buffer, diluent or adjuvant.

[00193] Another aspect includes a method of eliciting an immune response in a subject, comprising administering an effective amount of the nucleic acid molecule the polypeptide, the composition or the pharmaceutical composition disclosed herein.

[00194] In an embodiment, the nucleic acid molecule, the vector, the composition or the pharmaceutical composition is administered by electroporation, injection, optionally needleless injection, using a virus or using living bacteria.

[00195] The effective amount can be determined using known methods. The effective amount may for example be calculated on a mass/mass basis (e.g. micrograms or milligrams per kilogram of subject), or may be calculated on a mass/volume basis (e.g.
concentration, micrograms or milligrams per milliliter). Using a mass/volume unit, an antibody may be present at an amount from about 0.1 pg/ml to about 20 mg/ml, or any amount therebetween, for example 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000, 1500, 2000, 5000, 10000, 20000 pg/ml, or any amount therebetween;
or from about 1 pg/ml to about 2000 pg/ml, or any amount therebetween, for example 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 50.0 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000, 1500, 2000, pg/ml or any amount therebetween; or from about 10 ug/ml to about 1000 ug/ml or any amount therebetween, for example 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 50.0 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000 pg/ml, or any amount therebetween; or from about 30 ug/ml to about 1000 ug/ml or any amount therebetween, for example 30.0, 35.0, 40.0, 50.0 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000 pg/ml.

[00196] Quantities and/or concentrations may be calculated on a mass/mass basis (e.g. micrograms or milligrams per kilogram of subject), or may be calculated on a mass/volume basis (e.g. concentration, micrograms or milligrams per milliliter). Using a mass/volume unit, an antibody or peptide may be present at an amount from about 0.1 pg/ml to about 20 mg/ml, or any amount therebetween, for example 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000, 1500, 2000, 5000, 10000, 20000 pg/ml, or any amount therebetween; or from about 1 pg/ml to about 2000 pg/ml, or any amount Date Recue/Date Received 2022-04-29 therebetween, for example 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 50.0 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000, 1500, 2000, pg/ml or any amount therebetween; or from about 10 ug/ml to about 1000 ug/ml or any amount therebetween, for example 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 50.0 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000 pg/ml, or any amount therebetween; or from about 30 ug/ml to about 1000 ug/ml or any amount therebetween, for example 30.0, 35.0, 40.0, 50.0 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160 180, 200, 250, 500, 750, 1000 pg/ml.

[00197] A further aspect relates to a use of the nucleic acid molecule the polypeptide, the composition or the pharmaceutical composition disclosed herein for eliciting an immune response in a subject.

[00198] In an embodiment, the immune response is against a coronavirus, optionally a human beta coronavirus.

[00199] In an embodiment, the subject is a mouse, a rabbit or a human.

[00200] Also provided herein in an aspect is a use of the nucleic acid molecule the polypeptide, the composition or the pharmaceutical composition disclosed herein in the manufacture of a medicament.

[00201] In an embodiment, the medicament is a vaccine.

[00202] Another aspect includes a method of producing a polypeptide in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising the nucleic acid molecule, the vector, the composition or the pharmaceutical composition herein disclosed.

[00203] The above disclosure generally describes the present application.
A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[00204] The following non-limiting examples are illustrative of the present disclosure:
EXAMPLES

DESIGN OF VACCINE CONSTRUCTS

[00205] A vaccine platform comprising two vaccine components was designed.
Component 1 is aimed at inducing a predominantly humoral immune response and Component Date Recue/Date Received 2022-04-29 2 is aimed at inducing a predominantly cellular immune response. Each component is comprised in a plasmid ( or other genetic construct). For each plasmid/genetic construct, several alternative variants were tested. Both components were found to act synergistically to provide improved antiviral immunity. However each of Component 1 and 2 may be used separately, for example as a B-cell or T-cell response booster.
Component 1 vaccine

[00206] Vaccine Component 1 comprises at least RBD alone or RBD with one or several of the others listed below:
- Leader peptide: for example the leader peptide of the influenza virus hemagglutinin (HA) or the leader peptide of the human immunoglobulin E protein (IgE).
- One or more Receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, or portion or variant thereof;
- Linker: inserted between the leader peptide and the RBD;
- Heptad repeat 2 (HR2) domain of the SARS-CoV-2 spike protein or a portion thereof (e.g. minimally 10 consecutive amino acids within said sequence);
- Trimerization domain: for example GCN4 leucine zipper or foldon domain (corresponding to the C-terminal residues of fibritin from bacteriophage T4);
- TLR scaffold protein with TLR2 agonist peptide(s) (e.g. scaffold protein of ankyrin repeats protein comprising TLR2-agonistic peptides exposed instead of non-structured loops);
- Potato virus X capsid protein (PVXCP);
- Truncated Salmonella typhimurium flagellin (tFliC); and

[00207] Viral antigens (RBD, HR2) for inducing an antibody response are specific for a particular virus (in this case, SARS-CoV-2). These regions of the protein are designed to induce virus neutralizing antibodies. For example, the RBD domain of the S-protein of SARS-CoV-2 can be from the wildtype SARS-CoV-2 virus but also from different variants, including but not limited to mutant strains: B.1.1.7 (alpha), P1, P2, B.1.351, B.1.617.2.1 (delta plus), B.1.617.2 (delta), C.37 (lambda), B.1.621 (Mu) and B.1.1.529 (omicron). Various combinations of protein domains detailed above have been tested in Component 1 vaccines, as shown in Fig. 1.
Component 2 vaccine

[00208] Vaccine Component 2 is a synthetic construct comprising fragments of viral proteins ("viral epitopes") interspersed in a scaffold protein such as an ankyrin repeat scaffold.

Date Recue/Date Received 2022-04-29 Component 2 of the vaccine comprises of scaffold/epitopes protein (COV1, COV2, or their modifications), and one or several additional domains from the list:

[00209]
- Leader peptide: IgE or HA;
- COV1 or COV2 scaffold protein with viral epitopes;
- TLR scaffold with TLR2 agonist peptide;
- FMS-like tyrosine kinase 3 ligand (FLT3L);
- a-helix dimerization domain (dHLX) or a CH3 dimerization domain;
- Potato virus X capsid protein (PVXCP).

[00210] Viral epitopes are immunogenic peptides (for example, with a length of 15 to 20 amino acids) for presentation by MHC-II and MHC-I classes to T-lymphocytes.
Peptides are included in a compact protein structure, which consists of several ankyrin repeats connected by a flexible linker (hereinafter referred to as "scaffold protein") in such a way that the viral epitopes are exposed on the outside, and the scaffold forms a carrying non-immunogenic framework of the protein. "COV1" (interchangeably COV-1) refers to an ankyrin scaffold that comprises viral epitopes derived from viral structural proteins, for example, epitopes of the virus capsid proteins. "COV2" (interchangeably COV-2) refers to an ankyrin scaffold that comprises viral epitopes derived from viral non-structural proteins, for example, conserved epitopes of viral replicative proteins

[00211] Several alternative constructs for the Component 2 vaccine are shown in Fig.
2.

[00212] While neutralizing antibodies is a primary component of a vaccine's broad protective immunity, inducing virus-specific CD8 + T cells may also significantly enhance antibody-based protection. A strong T-cell immune response, including CD8 cytotoxic lymphocytes and Th1 assistance, can provide elimination of infected cells, a milder course of the disease in a case of a low titer of neutralizing antibodies, and a long immunological memory.
Moreover, in contrast to B-cell epitopes, which are restricted to the spike protein, T-lymphocytes can target a broad range of epitopes across SARS-CoV-2 structural and non-structural proteins, parts of which are conservative and constrained from mutations in circulating virus strains.

[00213] The selection of T-cell epitopes was carried out as follows Date Recue/Date Received 2022-04-29 1) Sequence homology

[00214] The concept of a pan-coronavirus vaccine implies the selection and inclusion in the vaccine of strong MHC epitopes in conservative regions of the proteins among some or all human Betacoronavirus, or among Sarbecoviruses. The homology of structural and non-structural proteins of the most common coronaviruses that cause human disease has been assessed: SARS-CoV-2, SARS-1, MERS, HKU1, NL63, 0C4, 229E. (Jaiswal et al.
Classical Coronaviruses. In: Saxena S. (eds) Coronavirus Disease 2019 (COVID-19).
Medical Virology:
From Pathogenesis to Disease Control. Springer, Singapore. doi: 10.1007/978-7_12). The highest priority in the choice of epitopes was given to sequences that were identical in all listed viruses. The second level of priority was given to sequences found homologous in beta-coronaviruses: SARS-CoV-2, SARS-1, MERS, HKU1, 0C4. The minimum homology level allowed when choosing epitopes is homology among sarbecoviruses (SARS-CoV-2, SARS-1).
Conservative amino acid substitutions were allowed.

[00215] The selected peptides were ranked according to the following categories (indicated in table 1) :
= grade 1 'homological' epitopes shared with other Betacoronaviruses, more than 80% homology = grade 2 'conservative' epitopes, differences in amino acids with other Betacoronoviruses are conservative, more than 80% of the conservative sequence = grade 3 Sarbecovirus only = grade 4 SARS-2 only (all strains)

[00216] An example of the location of the epitope on the aligned protein sequences of beta coronaviruses is shown in Fig. 3A and 3B.
2) T Cell Epitope Prediction

[00217] The selection of MHC-II and MHC-I epitopes among homologous regions of viral proteins was carried out using the predictive algorithms IEDB, SYFPEITHI, and NetMHCpan 4.1. Search analysis was performed for the most frequent alleles of human MHCI
class: HLA-A*01:01, HLA-A*02:01, HLA-A*24:02, HLA-A*03:01, HLA-B*07:02, and human MHC-II class: DRB1*13, DRB1*03, DRB1*07, DRB1*11, DQB1*03, DQB1*02.

[00218] Combined HLA binding and immunogenicity approach was applied for MHCII
epitope prediction by means of IEDB web tool, as described in Dhanda et al.
(2018) Predicting HLA CD4 Immunogenicity in Human Populations. Front. Immunol. 9:1369 (doi:
10.3389/fimmu.2018.01369), herein incorporated by reference. 15 mer peptides with an Date Recue/Date Received 2022-04-29 immunogenicity score parameter higher than 90 were assessed as 'highly immunogenic' and were selected for further consideration.

[00219] Basic MHCI epitopes screening was done using NetMHCpan EL 4.1 method as described in Reynisson et al., Nucleic Acids Research, Volume 48, Issue W1, 02 July 2020, Pages W449¨W454, (doi: 10.1093/nar/gkaa379)), herein incorporated by reference. 9 mer peptides with Percentile Rank higher than 90 were assessed as highly immunogenic. Other parameters, such as Proteasome Score, TAP Score, Processing Score were taking into account. Peptides with MHC IC50 less than 50 nM were included in a candidate list.
3) T Cell Epitope found in convalescent COVID-19 patients

[00220] A search for epitopes with the established T-cell response was performed in convalescent COVID-19 patients as described in Kared et al., Biorxiv : the Preprint Server for Biology. 2020 Oct. DOI: 10.1101/2020.10.08.330688; Quadeer et al., bioRxiv;
2020. DOI:
10.1101/2020.08.26.267724; Nathan et al., Cell. 2021 Aug;184(17):4401-4413.e10. DOI:
10.1016/j.ce11.2021.06.029. PMID: 34265281; PMCID: PMC8241654.; Poh et al., Nat Commun 11, 2806 (2020). https://doi.orq/10.1038/s41467-020-16638-2.

[00221] 10 peptides from those selected in Table 1 were found among the peptides of recovered patients. 10 more peptides were added in the candidates list from convalescent patients. These 10 peptides are also tested for homology between viruses, which is generally lower than the first list of peptides.

[00222] When selecting the final list of peptide candidates for inclusion in the pan-coronavirus vaccine, MHCI epitopes in homologous regions are selected.
Otherwise, preference is given to strong MHCI and MHCII epitopes located in homologous regions of viral proteins are used, so that the peptide includes a strong MHCI epitope, or a MHCI epitope placed within a longer MHCII epitope, or a peptide included two or more closely located MHCI epitopes, some of them found in convalescent patients. A list of identified peptides is presented in Table 1.
Table 1. List of selected peptides and their features # Peptide ID Amino acid sequence SEQ features grade Set ID
NO:
1 ORF1_3619 IAMSAFAMMFVKHKH 31 Convalescent, 4 -3633 strong MHCI, MHCII
2 ORF1_3794 FGLFCLLNRYFRLTLGVY 32 Two MHCII 3 COV2 -3813 DY epitopes Date Recue/Date Received 2022-04-29 3 ORF1_4028 AMQTMLFTMLRKLDN 33 Strong MHCI, 2 4 RdRp_330- VRKIFVDGVPFVVST 34 Convalescent, 2 COV2 344 strong MHCI, MHCII
RdRp_335- VDGVPFVVSTGYHFR 35 Strong MHCII 2 6 RdRp_370- ELLVYAADPAMHAAS 36 Strong MHCII 2 COV2 7 RdRp_540- TQMNLKYAISAKNRARTV 37 Two strong 1 COV2 epitopes 8 RdRp_575- LLKSIAATRGATVVI 38 Strong MHCII 2 9 RdRp_650- HRFYRLANECAQVLS 39 Strong MHCII 2 RdRp_745- FYAYLRKHFSMMILS 40 Strong MHCI, 3 COV2 11 RdRp_750- RKHFSMMILSDDAVV 41 Strong MHCII 1 12 RdRp_785- VLYYQNNVFMSEAKC 42 Strong MHCII 2 13 RdRp_855- MIERFVSLAIDAYPL 43 Strong MHCII, 1 COV2 14 H_141-155 TEETFKLSYGIATVR 44 Convalescent, 3 COV2 strong MHCI, MHCII
H_206-220 GDAVVYRGTTTYKLN 45 Convalescent, 3 COV2 strong MHCI, MHCII
16 H_211-225 YRGTTTYKLNVGDYF 46 Strong MHCII 3 17 H_221-235 VGDYFVLTSHTVMPL 47 Strong MHCII 3 18 H_286-300 TGKSHFAIGLALYYP 48 Strong MHCII 3 Date Recue/Date Received 2022-04-29 19 H_351-365 TLEQYVFCTVNALPE 49 Strong MHCII, 3 COV2 MHC-I
20 H_556-570 CNVNRFNVAITRAKV 50 Strong MHCII 2 21 USE_224- RYKLEGYAFEHIVYGDFS 51 Strong MHCI

22 USE_243- SQLGGLHLLIGLAKRFK 52 Strong MHCI, 3 COV2 23 USE_294- VIDLLLDDFVEIIKS 53 Strong MHCI, 3 COV2 24 USE_324- YTEISFMLWCKDGHV 54 Strong MHCI, 3 25 S_553-570 TESNKKFLPFQQFGRDIA 55 Convalescent 3 26 S_809-826 PSKPSKRSFIEDLLFNKV 56 Convalescent 3 27 S_863-877 PLLTDEMIAQYTSAL 57 Convalescent 3 28 S_265-279 YYVGYLQPRTFLLKY 58 Convalescent 3 29 S_814-828 KRSFIEDLLFNKVTL 59 Convalescent, 3 COV1 strong MHCII
30 S_902-916 MAYRFNGIGVTQNVL 60 Strong MHCII 3 COV1 31 S_912-926 TQNVLYENQKLIANQ 61 Convalescent, 3 COV1 strong MHCII
32 S_992-1006 QIDRLITGRLQSLQT 62 Convalescent, 2 COV1 strong MHCII
33 S_1211- KWPVVYIWLGFIAGLI 63 Strong MHCII 1 COV1 34 S_462-476 KPFERDISTEIYQAG 64 Strong MHCII 3 COV2 35 S_507-521 PYRVVVLSFELLHAP 65 Strong MHCII 3 COV2 36 M_44-58 RFLYIIKLIFLWLLWP 66 Strong MHC-I 2 COV1 Date Recue/Date Received 2022-04-29 37 M_91-110 MWLSYFIASFRLFARTRS 67 Strong MHCI, MW MHCII
38 N_216-230 DAALALLLLDRLNQL 68 Strong MHCI, 3 MHCII, convalescent 39 N_301-315 WPQIAQFAPSASAFF 69 Strong MHCI, 4 MHCII, convalescent 40 N_357-371 I DAYKTFPPTEPKKD 70 Strong MHCI, 3 convalescent 41 N_104-118 LSPRVVYFYYLGTGPE 71 Strong MHCI, 3 convalescent 42 orf3a_137- NPLLYDANYFLCWH 72 Strong MHCI, 4 150 MHCII, convalescent 43 orf3a_206- YFTSDYYQLYSTQLS 73 Strong MHCI, 4 220 MHCII, convalescent

[00223] In order to design a stable and soluble protein that contains a number of T-cell epitopes of SARS-CoV-2, various antibody scaffolds and alternative non-antibody scaffolds were investigated. Existing synthetic T cell epitope-containing domains, for example, amyloid proteins, T4 lysozyme, carboxymethylcellulose or immunoglobulin molecules have limited possibilities for a plurality of T-cell epitopes or represent a synthetic conjugate of matrix and peptide.

[00224] Ankyrin repeat proteins (e.g. DARPins) can be used as scaffolds as they can expose a number of variable non-structured loops (from 2 to 34) within the helix-loop-helix fold structure and are stable and water-soluble, as described by Mosavi et al, Protein Sci. 2004 Jun;
13(6): 1435-1448, herein incorporated by reference its entirety.

[00225] Pan-coronavirus T-cell epitopes from Table 1 were divided into two groups.
The COV-1 group contains peptides from SARS-CoV-2 structural proteins, and the group contains non-structural protein peptides. SARS-CoV-2 MHC epitopes were ranked based on their Grand Average of Hydropathy (GRAVY) index and isolectric point (pi) to alternate acidic Date Recue/Date Received 2022-04-29 and basic properties of the nearest peptides in the structure to decrease molecule entropy and make it more compact and soluble (Table 2 and Table 3).
Table 2. COV2 peptides from SARS-CoV-2 non-structural proteins with predicted properties No Peptide SEQ ID pl GRAVY
Hydrophobicity Index number in the NO: COV2 scaffold protein (from N- to C-terminus) of Fig.

2 ORF1_379 32 88 0.64 66.41 9* (included in 4-3813 linker) 35 65 78 0.81 43.13 2 S_507-521 15 H_206-220 45 96 -046 24.91 3 34 S_462-476 64 44 -0.91 28.37 4 6 RdRp_370- 36 41 0.59 30.98 5 22 USE_224- 52 117 042 45.5 6 23 USE_294- 53 3_S 1.16 62.61 7 19 H_351-365 49 7 -0.19 31.42 8 14 H_141-155 44 31 0.32 38.51 10 13 43 101 0.5 43.7 11 RdRp_855-40 41 0.93 47.79 12 RdRp_745-7 RdRp_540- 37 116 -048 26.72 13 4 RdRp_330- 34 10_I 1.05 37.27 14 Date Recue/Date Received 2022-04-29 Table 3. COV1 peptides from SARS-CoV-2 structural proteins with predicted properties No Peptide SEQ pl GRAVY Hydrophobicity Index number in the ID COV1 scaffold NO protein (from N- to C-terminus) of Fig.
27 S_912-926 57 6,90 -0,82 27,07 1 28 S_992-1006 58 10,90 -0,35 30,78 2 33 M_91-110 63 12,20 0,41 65,72 3 29 S_1211-1225 59 9,70 1,03 70,82 4 25 S_814-828 55 9,90 0,09 46,71 5 32 M_44-59 62 10,40 1,56 74,61 6 26 S_902-916 66 9,90 0,36 34,02 7

[00226] As the Ankyrin repeat structure proved to be stable, AAY linkers were incorporated to flank the MHC-epitopes to enhance proteasomal cleavage in mammalian cells.
A general ankyrin scaffold structure showing immune peptides interspersed therein is presented in Fig. 4.

[00227] A 3D model showing the molecule structure of the designed constructs was built using homology modeling, as shown in Fig. 5A (COV2 peptides) and Fig. 5B
(COV1 peptides).

[00228]
Subsequently, computer simulation using 100 nanosecond (ns) Molecular Dynamics (MD) simulation was conducted to assess structural features of the designed constructs: namely stability of ankyrin repeats and flexibility of MHC-peptides inside the loops.
Analysis of a root-mean-square deviation (RMSD) of heavy atomic (non-hydrogen atoms) positions against the first frame in dynamics demonstrated that the main structure-forming element - ankyrin repeats - had low RMSD for both COV-1 and COV-2 constructs in comparison with the crystal resolution of 3.2 A resolution of Ankyrin repeats protein [Yufan Wu et al, 2017 Scientific Reports volume 7, Article number: 11217 (2017)], and both COV-1 and constructs stabilized shortly after simulation, as shown in Fig. 6A.

Date Recue/Date Received 2022-04-29

[00229] The total RMSDs of the whole structures are increasing with time (Fig. 6B).
This increase can be explained by the increasing distances between ankyrin repeats cores inside the construct and may suggests that loops were constantly moving without breaking the protein structure.

[00230] These analyses show the high stability of the ankyrin core that should facilitate the stability of the ankyrin based constructs. But at the same time, it shows a flexible and movable connection of ankyrin repeat cores inside the construct to facilitate peptidase hydrolysis for better peptide presentation. It is clearly seen from the dynamic of the solvent-accessible surface area (SASA) that the constructs remain mostly constant during the whole simulation (Fig. 7).

1) In silico design and analysis

[00231] In order to assess the RBD-HR2-PVXCP vaccine protein and its behavior in a solvent, 500 ns Molecular Dynamics (MD) simulation in explicit solvent was carried out. Fig. 8 illustrates the 3D structure of the RBD-HR2-PVXCP protein.

[00232] The MD
trajectory of the RBD-HR2-PVXCP protein was analyzed and average distance between the outermost Ca-atoms in the construct was calculated (Fig.
9).

[00233] The Potato virus X particle is formed by repeated segments made of 8.8 PVXCP proteins, forming a left-handed helical structure. It is believed that protein may form a nanodisk conformation, as illustrated at Fig. 10, akin to the potato virus X
and, a molecular model of protein-based virus-like particles (VLPs) using for example Protein Data Bank: 6R7G as a template is thus envisaged. Taking into account the size of the molecule, the hydrodynamic radius of the PVXCP-fusion oligomer IS estimated to be 10-12 nm.
2) Vaccine generation and in vitro testing

[00234] The vaccine constructs are generated and their expression measured. Table 4 below lists the generated Component 1 vaccine constructs.
Table 4. Component 1 Vaccine constructs Vaccine Vaccine SEQ ID
construct construct Amino acid sequence NO:
No name IgE-RBD- MDWTWILFLVAAATRVHSGNITNLCPFGEVFNATRFASV 74 14 LeucineZip YAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLN
per-HR2- DLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPD
3xFLAG DFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFE

Date Recue/Date Received 2022-04-29 RDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGV
GYQPYRVVVLSFELLHAPGGGSGGGSGGAGSRMKQIE
DKIEEILSKIYHIENEIARIKKLIGERTSGGSGGTGGSGGT
GGAAAGGSGGDISGINASVVN IQKEI DRLNEVAKNLN ES
LIDLQELGNSDYKDDDDKDYKDDDDKDYKDDDDK
where:
MDVVTWILFLVAAATRVHS - IgE signal sequence NITN... HAP - SARS-COV2 RBD domain GSRMKQIEDKIEEILSKIYHIENEIARIKKLIGERT¨
trimerization domain (Leucine zipper) DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG ¨
SARS-COV2 HR2 domain DYKDDDDKDYKDDDDKDYKDDDDK ¨ 3xFLAG tag CPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSAS
FSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAP
GQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN
YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCY
FPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP
KKGGGSGGGSGGAGSRMKQIEDKIEEILSKIYHI EN EIAR
IKKLIGERTSGGSGGTGGSGGTGGAAAGGSGGDISGIN
ASVVNIQKEIDRLNEVAKNLNESLIDLQELGNSDYKDDD
DKDYKDDDDKDYKDDDDK
HA-RBD-15 LeucineZip where:
per-HR2-3xFLAG MNTQILVFALIAIIPTNADKI ¨ HA signal sequence RVQPT PKK ¨ SARS-CoV2 RBD domain GSRMKQIEDKIEEILSKIYHIENEIARIKKLIGERT¨
trimerization domain (Leucine zipper) DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG ¨
SARS-COV2 HR2 domain DYKDDDDKDYKDDDDKDYKDDDDK ¨ 3xFLAG tag CPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSAS
FSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAP
GQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN
YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCY
HA-RBD-FPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP

KKGSAAAGPGPMSAPASTTQATGSTTSTTTKTAGATPA
3xFLAG
TASGLFTIPDGDFFSTARAIVASNAVATNEDLSKIEAIWK
DMKVPTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPY
SNGISRARLAAAIKEVCTLRQFCMKYAPVVWNWMLTNN
SPPANWQAQGFKPEHKFAAFDFFNGVTNPAAIMPKEGL
I RPPS EAEMNAAQTAAFVKITKARAQSN DFAS LDAAVTR

Date Recue/Date Received 2022-04-29 GRITGTTTAEAVVTLPPPGNSDYKDDDDKDYKDDDDKD
YKDDDDK
where:
MNTQILVFALIAIIPTNADKI ¨ HA signal sequence RVQPT PKK ¨ SARS-CoV2 RBD domain SAPAST.....VVTLPPP ¨ potato X virus coat protein (PVXCP) DYKDDDDKDYKDDDDKDYKDDDDK - 3xFLAG tag CPFG EVFNATRFASVYAWN RKRI SNCVADYSVLYN SAS
FSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAP
GQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN
YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCY
FPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP
KKGSAAAGPGPMSAPASTTQATGSTTSTTTKTAGATPA
TASGLFTIPDGDFFSTARAIVASNAVATN EDLSKIEAIWK
DMKVPTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPY
SNG IS RARLAAAI KEVCTLRQFCMKYAPVVWNWMLTN N
SPPANWQAQGFKPEHKFAAFDFFNGVTNPAAIMPKEGL

PVXCP GRITGTTTAEAVVTLPPP
where:
MNTQILVFALIAIIPTNADKI (SEQ ID NO: 21) ¨ HA signal sequence RVQPT PKK ¨ SARS-CoV2 RBD domain SAPAST.....VVTLPPP ¨ potato X virus coat protein (PVXCP) CPFG EVFNATRFASVYAWN RKRI SNCVADYSVLYN SAS
FSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAP
GQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN
YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCY
FPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP
KKGSAAAGGSGGDISGINASVVNIQKEIDRLNEVAKNLN
HA-RBD- ESLI DLQELG NSGSGAG PG PMSAPASTTQATGSTTSTT

PVXCP DLSKIEAIWKDMKVPTDTMAQAAWDLVRHCADVGSSA
QTEM I DTGPYSNG IS RARLAAAI KEVCTLRQFCMKYAPV
VWNWMLTNNSPPANWQAQGFKPEHKFAAFDFFNGVT
NPAAI MPKEG LI RPPSEAE MNAAQTAAFVKITKARAQSN
DFASLDAAVTRGRITGTTTAEAVVTLPPP
where:

Date Recue/Date Received 2022-04-29 MNTQILVFALIAIIPTNADKI (SEQ ID NO: 21) ¨ HA signal sequence RVQPT PKK ¨ SARS-CoV2 RBD domain DISG DLQEL -SARS-CoV2 HR2 domain SAPAST.....VVTLPPP ¨ potato X virus coat protein (PVXCP) CPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSAS
FSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAP
GQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN
YRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCY
FPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP
KKGSAAAGPGPMSAPASTTQATGSTTSTTTKTAGATPA
TASGLFTIPDGDFFSTARAIVASNAVATNEDLSKIEAIWK
DMKVPTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPY
SNG IS RARLAAAI KEVCTLRQFCMKYAPVVWNWMLTN N
HA- SPPANWQAQGFKPEHKFAAFDFFNGVTNPAAIMPKEGL
RBD(B.1.6 IRPPSEAEMNAAQTAAFVKITKARAQSNDFASLDAAVTR

17.2)- GRITGTTTAEAVVTLPPP
PVXCP
where:
MNTQILVFALIAIIPTNADKI (SEQ ID NO: 21) ¨ HA signal sequence RVQPT PKK ¨ SARS-CoV2 RBD domain (B.1.617.2 strain) SAPAST.....VVTLPPP ¨ potato X virus coat protein (PVXCP) CPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSAS
FSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAP
GQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN
YRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCY
FPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP
KKGSAAAGGSGGDISGINASVVNIQKEIDRLNEVAKNLN
ESLI DLQELG NSGSGAG PG PMSAPASTTQATGSTTSTT
HA- TKTAGATPATASGLFTIPDGDFFSTARAIVASNAVATNE
40D RBD(B.1.6 DLSKIEAIWKDMKVPTDTMAQAAWDLVRHCADVGSSA
17.2)-HR2- QTEMIDTGPYSNGISRARLAAAIKEVCTLRQFCMKYAPV
PVXCP VWNWMLTNNSPPANWQAQGFKPEHKFAAFDFFNGVT
NPAAI MPKEG LI RPPSEAE MNAAQTAAFVKITKARAQSN
DFASLDAAVTRGRITGTTTAEAVVTLPPP
where:
MNTQILVFALIAIIPTNADKI (SEQ ID NO: 21) ¨ HA signal sequence Date Recue/Date Received 2022-04-29 RVQ PT PKK ¨ SARS-CoV2 RBD domain (B.1.617.2 strain) DISG DLQEL -SARS-CoV2 HR2 domain (B.1.617.2 strain) SAPAST.....VVTLPPP ¨ potato X virus coat protein (PVXCP)

[00235] Plasmid vectors were transiently transfected into HEK293 FreeStyle cells using polyethylenimine (PEI) transfection. 24 hours before transfection HEK 293 FreeStyle cell line was subcultured to final density 5*10^5 cells per ml of antibiotic-free FreeStyle medium to reach 1*10^6 cells per ml to the transfection day. A DNA and PEI had taken in amounts of 2 ptg and 3.2 tig respectively per well. DNA:PEI complexes were made by mixing of separately prepared solutions of 2 ptg of DNA and 3.2 ptg of PEI diluted by OptiMem to final volumes of 37 .1. After PEI solution adding to DNA, the mixture rested at room temperature for 25 minutes. The final mixture was added to the cells followed by placing them in an incubator (37C, 5% CO2) for 5 days with a constant shaking of 120 rpm.

[00236] Conditioned expressing medium was centrifuged to remove cell debris and then cell medium was analyzed by Western Blotting using anti-S-protein polyclonal antibodies (Fig. 11). As can be seen, all the vaccine candidates have a similar level of expression yielding ¨1-10 ug of a protein per ml of culture supernatant.

[00237] In order to confirm PVXCP fused protein multimerization status, a hexahistidine (Hs6) tag was added to the N-terminal part of constructs #39 and #40. These fusion proteins were purified from 100 ml HEK293 FreeStyle supernatant by means of metal-chelating affinity chromatography (Ni-NTA) and were analyzed using dynamic light scattering (DLS). The molecule radius was determined by the intensity of scattering of a given light. It was found that the majority of the proteins (96%) had a radius of 5.2 ¨ 13.1 nm (Fig. 12) with an average radius of 10 nm which corresponded to the size ranging between half a PVX disk and two PVX disks stacked together. The results confirmed the multimerization effect of PVXCP
protein in addition to its adjuvant properties.
3) in vivo testing of the potency of DNA vaccine component 1 to induce a humoral immune response in a murine model.

[00238] To monitor priming of humoral immunity, 5 mice in each group were vaccinated by electroporation (EP) with 50 ug of DNA encoding #15 and #16 variants or by i.m. injection of DNA: linear polyethyleneimine (PEI) complex (w/w = 1:2). Animals received 50 ug DNA
vaccines for the EP and 10 ug for the DNA: PEI via intramuscular route without adjuvant at week 0 and week 2. Serum samples were collected on day 28 and analyzed by ELISA for the Date Recue/Date Received 2022-04-29 presence of IgG specific for RBD. The anti-RBD IgG antibody responses are illustrated in Fig.
13 for groups #14_EP, #15_EP, #16_EP, #15_PEI and #16_PEI.

[00239] Endpoint titers values were determined for the groups #16_EP and #15_EP
and #16_PEI (Fig. 14). Electroporation of constructs #15 and #16 resulted in the highest anti-RBD titer exceeding 106. For DNA:PEI injection the titer was lower with one mouse not responding thus the dose may be adjusted on the large animals model.
4) in vivo testing of the potency of DNA vaccine component 1 to induce a humoral immune response in rabbits.

[00240] Testing was performed to measure the antibody response in rabbits vaccinated with plasmid DNA # 39 with the delivery by needle free injection.

[00241] In this experiment aqueous DNA solution, 100 pg /100 pl was delivered by needle free injection in 3 rabbits (N=3).

[00242] Vaccinations were performed three times with an interval of 14 days (0 days -first immunization, 14 days - second immunization, 42 days - third immunization). Serum samples were collected on days 0, 28, 56, 75 and 105 and analyzed by ELISA for the presence of IgG specific for RBD. Serum samples were heat inactivated and serially diluted from 300 to 1930178.906 times to measure endpoint titer (Fig. 17A). At week 4 (2 doses), the median RBD-specific end-point titers were 32 952 (range: 9 557-72 430). At week 8 (3 doses), the median RBD-specific end-point titers were 48 633 (range: 29 173-206 324). On day 105 (2 months after 3rd dose) the median RBD-specific end-point titers were - 65 699 (range:

663).
5) in vivo testing of the potency of DNA vaccine component 1 to induce a humoral and T-cell immune response in mice.

[00243] Testing was performed to compare different vaccine designs by the antibody and T-cell response in mice vaccinated with low-dose plasmid DNA (component 1) with a needle injection followed by electroporation as the best delivery method.

[00244] The experiment included six test groups of animals plus 1 control group:
= Group 1: Aqueous DNA solution of vector - 2.5 pg ¨ 6 BALB/c (female) = Group 2: Aqueous DNA solution of #15 -2.5 pg ¨6 BALB/c (female) = Group 3: Aqueous DNA solution of #16 -2.5 pg ¨6 BALB/c (female) = Group 4: Aqueous DNA solution of #39 - 2.5 pg ¨6 BALB/c (female) = Group 5: Aqueous DNA solution of #39D (#39 with t617.2 RBD) - 2.5 pg ¨6 BALB/c (female) = Group 6: Aqueous DNA solution of #40 -2.5 pg ¨6 BALB/c (female) Date Recue/Date Received 2022-04-29 = Group 7: Aqueous DNA solution of #40D (#40 with 1.617.2 RBD) -2.5 pg ¨6 BALB/c (female)

[00245]
Vaccinations were performed twice with an interval of 14 days. On day 28 after the second dose mice were sacrificed. Serum was taken and analyzed by ELISA to measure the antibody titer. Antibody titers were measured against Wuhan RBD variant (Wuhan) and B.1.617.2 variant (Delta). Splenocytes isolated from the spleen and used to measure T-cell response.

[00246] The anti-RBD IgG antibody endpoint titers are illustrated in Fig. 17B. On day 28 the median RBD-specific end-point titers for leading vaccine candidate #39 were - 383 360 (range: 565 ¨ 661 792) and 239 298 (range: 774 ¨ 301 212) for Wuhan RBD and Delta RBD
respectively.

[00247] Elispot analysis of IFNy production by splenocytes was done with SARS-CoV-2 51 scanning pool (Mabtech) using Mouse IFN-y ELISpot PLUS kit (ALP) (Mabtech). Antigen peptide free wells were used as negative controls, PHA stimulation as positive control. Groups #15, #16, #39 and #40 constructs showed significant (p<0.05) response relative to negative control. Positive responses were defined as number of spots/mln in the test SDEV above the negative control. Medium number of spots/mIn cells was #15 (306), #16 (122), #39 (167), #40 (142), #40D (43) (Fig. 17 C). Data shown as number of spots per million cells after the negative control was subtracted from the test value.

[00248] Despite minimal DNA dose (2.5 pg), vaccine variants with wild type RBD were able to achieve positive T-cell immune response. In the group vaccinated with #39 variant, all of the 6 mice significantly responded to the vaccine as seen in Fig. 17 C.

[00249] Constructs #10 having SEQ ID NO: 75 and #11 having SEQ ID NO: 76 were generated. These Component 1 constructs include a receptor binding domain (RBD) of the SARS-CoV-2 S-protein for eliciting an antibody response. In addition, as shown in Fig. 1, the constructs further comprise a COV1 scaffold protein for eliciting a T-cell response. In other words, these constructs combine Components 1 and 2.

[00250] Vaccination was performed in mice by intramuscular injection of 50 micrograms followed by electroporation on BTX AgilePulseTM apparatus and was repeated after 14 days. On day 28 after the first immunization, splenocytes were harvested from individual mice. To assess T-cell immune response, interferon-gamma (IFNy) secretion was Date Recue/Date Received 2022-04-29 determined using the BD ELISPOT mouse IFNy ELISPOT Set (BD Biosciences, USA).
Effector cells were plated in duplicate at 2.5 = 105 per well in 200 pl final volume with medium alone or pg/ml of the peptide library for COV1 for 24 h. Phytohemagglutinin 122 (Sigma, USA) (4 pg/ml) was used as a positive control. The plates were analyzed with an ELISPOT reader (AID
EliSpot Reader iSpot Spectrum). The mean number of spots from duplicate wells was calculated for each responder animal and adjusted to represent the mean number of spots per 106 spleen cells, as shown in Fig. 15A. The average ELISPOT spot area per well is shown in Fig. 15B. As can be seen, the combination of the RBD for the antibody response and COV1 scaffold protein for T-cell response did not provide the expected antibody response and moderate T-cell response. Therefore, Components 1 and 2 were separated antigens for antibody response were separated into truncated Component 1 vaccines (e.g.
constructs 15, 16, 40) were separated and tested separately from component 2.
Vaccine construct #9

[00251] Construct #9 (COV-2) was then tested. Vaccination was performed by intramuscular injection of 50 pg followed by electroporation on apparatus BTX
AgilePulse and was repeated after 14 days. On day 28 after first immunization splenocytes were harvested from individual mice. IFNy secretion was determined using the BD ELISPOT mouse IFNy ELISPOT Set (BD Biosciences, USA). Effector cells were plated in duplicate at 2.5 = 105 per well in 200 pl final volume with medium alone or 10 pg/ml of the peptide library for COV-2 for 24 h (Fig. 16).

[00252] It was found that RBD fragment [319-529] of the SARS-CoV-2 spike protein significantly enhanced the expression level of PVXCP protein. Results are shown in Fig. 18.
Surprisingly, the expression level of RBD[319-529] - PVXCP was much higher than RBD or PVXPC alone, or than RBD [331-521]-PVXCP fusion. As the immunogenicity of the DNA\ RNA
encoded protein correlates with its expression it is believed the RBD fragment [319-529] may result in a better outcome.

TRIMERIC VACCINES

[00253] A vaccine construct was developed to provide a trimeric receptor binding domain (RBD) on the surface of each PVXCP molecule, as illustrated in Fig. 19.
This was achieved by linking multimeric protein domains with a flexible linker. Upon introduction into the host cells, the constructed fusion protein RBD-RBD-RBD-PVXCP is cleaved in the endoplasmic reticulum (ER) by one or more proteases (e.g. furin or C1r-LP) and forms trimers on the basis Date Recue/Date Received 2022-04-29 of T4 foldon or other homo- or heterodimerization domain. The trimeric nature of the RBD is believed to enhance the efficacy of the vaccine to elicit neutralizing antibodies.

[00254] It also possible to use 1, 2 or 3 different RBDs such as RBDs from different variants, e.g. alpha, beta and delta variants, to enhance antibody titers to different variants of concern through the single vaccine application.

[00255] The trimeric RBD construct has the sequence:
MNTQIL VFALIAIIPTNADKIGSGARVQPTESIVRF PN ITN LCPFG EVFNATRFASVYAWN RKRI

NYKLPDDFTGCVIAWNSNN LDSKVGGNYNYLYRLFRKSN LKPFERDISTEIYQAGSTPCNGV
EGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKGGGSSGYIPEAPRDG
QAYVRKDGEWVLLSTFLGSYQTQTNSPRRARSVASQSIIAGAGRVQPTESIVRFPNITNLCP
FGEVFNATRFASVYAWN RKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADS

KPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPAT
VCGPKKGGGSSGYIPEAPRDGQAYVRKDGEWVLLSTFLGSYQTQTNSPRRARSVASQSII
AGSTRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTF

NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTN
GVGYQPYRVVVLSFELLHAPATVCGPKKGGGSSGYIPEAPRDGQAYVRKDGEWVLLSTFL
AAAG PG PMSAPASTTQATGSTTSTTTKTAGATPATASGLFTIPDGDFFSTARAIVASNAVAT
NEDLSKIEAIWKDMKVPTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPYSNGISRARLAA
AIKEVCTLRQFCMKYAPVVWNWMLTNNSPPANWQAQGFKPEHKFAAFDFFNGVINPAAI
MPKEGLIRPPSEAEMNAAQTAAFVKITKARAQSNDFASLDAAVIRGRITGTTTAEAVVTLP
PP (SEQ ID NO:77) where:
MNTQILVFALIAIIPTNADKI (SEQ ID NO: 21) ¨ HA signal peptide RVQ PT .... PKK ¨ SARS-CoV2 RBD domain GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 18) - T4 foldon homotrimerization domain or GCN4 leucin zipper GSRMKQIEDKIEEILSKIYHIENEIARIKKLIGER (SEQ ID NO:
17) SAPAST.....VVTLPPP ¨ potato X virus coat protein (PVXCP)

[00256] The T4 foldon homotrimerization domain or GCN4 leucin zipper may include (or be substituted with) type IX collagen NC2 hetero-trimerization domains in any order:
domainl: GKEASEQRIRELCGGMISEQIAQLAAHLRKPLAPG (SEQ ID NO: 78) domain2: GRDATDQHIVDVALKMLQEQLAEVAVSAKREALG (SEQ ID NO: 79) domain3: GRAPTDQHIKQVCMRVIQEHFAEMAASLKRPDSG (SEQ ID NO: 80)

[00257] The foregoing provides a construct for oligomerization of two RBD
fragments with a single RBD-PVXCP construct.
The construct includes a YQTQTNSPRRARSVASQSIIA (SEQ ID NO: 81) - furin cleavage site It will be understood that the furin cleavage may be substituted with for example:

Date Recue/Date Received 2022-04-29 VCGRPVTPIAQNQTTLGSSRAKLGNFPWQ (SEQ ID NO: 82) - C1r-like protease cleavage site.

[00258] Component 2 vaccine constructs were generated as detailed in Table 5 and illustrated in Fig. 2. The COV1/COV2 peptides were previously identified in Example 2 at Table 1.
Table 5: List of the Component 2 sequences:
Vaccine Vaccine Amino acid sequence SEQ ID
No constructs NO:

VTQNVLYENQKLIANQPLHLAAWEGHLGIVEVLLKNG
QIDRLITGRLQSLQTGHTPLHLAAYTGHLEIVEVLLKNG
MWLSYFIASFRLFARTRSMWGTAPLHLAAMWGHLEIV
EVLLKNGKWPVVYIWLGFIAGLITPKDLARDNGNEWIR
ELLEKAERKLKDLDRKLLEAARAGHRDEVEDLIKNGK
RSFIEDLLFNKVTLGFTPLHLAAWEGHLGIVEVLLKNG
RFLYIIKLIFLWLLWPGHTPLHLAAYTGHLEIVEVLLKN
GMAYRFNGIGVTQNVLGTAPLHLAAMWGHLEIVEVLL
KNGTRGCSGLTSITFPNSLTGKTPKDLARDNGNEWIR
ELLEKAERKLKDLDRKLLEAARAGHRDEVEDLIKNGG
ESAFKGCSGLKSITGFTPLHLAAWEGHLGIVEVLLKNG
AMQTMLFTMLRKLDNGHTPLHLAAYTGHLEIVEVLLK
NGALSGCTGLTSITIPNAPLHLAAMWGHLEIVEVLLKN
GALSGCTGLTSITIPNTPFDLAIDNGNEDIAEVLQKGSA
AAGNSGSGDYKDDDDKDYKDDDDKDYKDDDDK
where:
#19 COV1-TLR MDVVTWILFLVAAATRVHS - IgE leader G ¨ spacer DLGKKLLEAARAGQDDEVTQNVLYENQKLIANQPLHL
AAWEGH LGIVEVLLKNGQI DRLITGRLQSLQTGHTPLH
LAAYTGHLEIVEVLLKNGMWLSYFIASFRLFARTRSM
WGTAPLHLAAMWGHLEIVEVLLKNGKWPVVYIWLGFIA
GLITPKDLARDNGNEWIRELLEKAERKLKDLDRKLLEA
ARAGHRDEVEDLIKNGKRSFIEDLLFNKVTLGFTPLHL
AAWEGHLGIVEVLLKNGRFLYIIKLIFLWLLWPGHTPLH
LAAYTGHLEIVEVLLKNGMAYRFNGIGVTQNVLGTAPL
HLAAMWGHLEIVEVLLKNG ¨ 'COV1_Scaffold' TR - spacer GCSGLTSITFPNSLTGKTPKDLARDNGNEWIRELLEKA
ERKLKDLDRKLLEAARAGHRDEVEDLIKNGGESAFKG
CSGLKSITGFTPLHLAAWEGHLGIVEVLLKNGAMQTM
LFTMLRKLDNGHTPLHLAAYTGHLEIVEVLLKNGALSG

Date Recue/Date Received 2022-04-29 CTGLTSITIPNAPLHLAAMWGHLEIVEVLLKNGALSGC
TGLTSITIPNTPFDLAIDNGNEDIAEVLQKGS
- 'TLR scaffold with TLR2 agonist peptide' AAAGNSGSG ¨ spacer DYKDDDDKDYKDDDDKDYKDDDDK
- 3 FLAG tags I RELSDYLLQDYPVTVASN LQDE ELCGG LWRLVLAQR
WMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPP
SCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLE
LQCQPDSSTLPPPWSPRPLEATAPTAPQPGAANGAS
GGGGSGGGGTPLGDTTHTSGGELEELLKHLKELLKG
PRKGELEELLKHLKELLKGLSGALDLGKKLLEAARAG
QDDEVTQNVLYENQKLIANQPLHLAAWEGHLGIVEVL
LKNGQI DRLITGRLQSLQTGHTPLHLAAYTGHLEIVEVL
LKNGMWLSYFIASFRLFARTRSMWGTAPLHLAAMWG
HLEIVEVLLKNGKWPVVYIWLGFIAGLITPKDLARDNGN
EWIRELLEKAERKLKDLDRKLLEAARAGHRDEVEDLIK
NGKRSFIEDLLFNKVTLGFTPLHLAAWEGHLGIVEVLL
KNGRFLYIIKLIFLWLLWPGHTPLHLAAYTGHLEIVEVL
LKNGMAYRFNGIGVTQNVLGTAPLHLAAMWGHLEIVE
VLLKNGTRGCSGLTSITFPNSLTGKTPKDLARDNGNE
WI RELLEKAERKLKDLDRKLLEAARAGHRDEVEDLIKN
GGESAFKGCSGLKSITGFTPLHLAAWEGHLGIVEVLLK
hF"-dHLX- NGAMQTMLFTMLRKLDNGHTPLHLAAYTGHLEIVEVL
#20 COV1 - LKNGALSGCTGLTSITIPNAPLHLAAMWGHLEIVEVLL
TLR KNGALSGCTGLTSITI PNTPFDLAIDNGNEDIAEVLQKG
SAAAGNSGSGDYKDDDDKDYKDDDDKDYKDDDDK
where:
MDVVTWILFLVAAATRVHS - IgE leader G ¨ spacer TQDCSFQHSPISSDFAVKI RELSDYLLQDYPVTVASNL
QDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLE
RVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQ
LVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPL
EATAPTAPQP - human FLT3L ectodomain GAANGASGGGGSGGGG ¨ spacer TPLGDTTHTSG ¨ hinge GELEELLKHLKELLKGPRKGELEELLKHLKELLK ¨
dimerization domain Date Recue/Date Received 2022-04-29 GLSGAL ¨ spacer DLGKKLLEAARAGQDDEVTQNVLYENQKLIANQPLHL
AAWEGHLGIVEVLLKNGQIDRLITGRLQSLQTGHTPLH
LAAYTGHLEIVEVLLKNGMWLSYFIASFRLFARTRSM
WGTAPLHLAAMWGHLEIVEVLLKNGKWPVVYIWLGFIA
GLITPKDLARDNGNEWIRELLEKAERKLKDLDRKLLEA
ARAGHRDEVEDLIKNGKRSFIEDLLFNKVTLGFTPLHL
AAWEGHLGIVEVLLKNGRFLYIIKLIFLWLLWPGHTPLH
LAAYTGHLEIVEVLLKNGMAYRFNGIGVTQNVLGTAPL
HLAAMWGHLEIVEVLLKNG - 'COVLScaffold' TR ¨ spacer GCSGLTSITFPNSLTGKTPKDLARDNGNEWIRELLEKA
ERKLKDLDRKLLEAARAGHRDEVEDLIKNGGESAFKG
CSGLKSITGFTPLHLAAWEGHLGIVEVLLKNGAMQTM
LFTMLRKLDNGHTPLHLAAYTGHLEIVEVLLKNGALSG
CTGLTSITIPNAPLHLAAMWGHLEIVEVLLKNGALSGC
TGLTSITIPNTPFDLAIDNGNEDIAEVLQKGS
- 'TLR scaffold with TLR2 agonist peptides' AAAGNSGSG ¨ spacer DYKDDDDKDYKDDDDKDYKDDDDK -3 FLAG tags LAAWEGHLGIVEVLLKNGQIDRLITGRLQSLQTGHTPL
HLAAYTGHLEIVEVLLKNGMWLSYFIASFRLFARTRSM
WGTAPLHLAAMWGHLEIVEVLLKNGKWPVVYIWLGFIA
GLITPKDLARDNGNEWIRELLEKAERKLKDLDRKLLEA
ARAGHRDEVEDLIKNGKRSFIEDLLFNKVTLGFTPLHL
AAWEGHLGIVEVLLKNGRFLYIIKLIFLWLLWPGHTPLH
LAAYTGHLEIVEVLLKNGMAYRFNGIGVTQNVLGTAPL
HLAAMWGHLEIVEVLLKNGNSDYKDDDDKDYKDDDD
KDYKDDDDK
where:

M - start translation DLGKKLLEAARAGQDDEVTQNVLYENQKLIANQPLHL
AAWEGH LGIVEVLLKNGQI DRLITGRLQSLQTGHTPLH
LAAYTGHLEIVEVLLKNGMWLSYFIASFRLFARTRSM
WGTAPLHLAAMWGHLEIVEVLLKNGKWPVVYIWLGFIA
GLITPKDLARDNGNEWIRELLEKAERKLKDLDRKLLEA
ARAGHRDEVEDLIKNGKRSFIEDLLFNKVTLGFTPLHL
AAWEGHLGIVEVLLKNGRFLYIIKLIFLWLLWPGHTPLH
LAAYTGHLEIVEVLLKNGMAYRFNGIGVTQNVLGTAPL
HLAAMWGHLEIVEVLLKNG - 'COV1_Scaffold' NS ¨ spacer DYKDDDDKDYKDDDDKDYKDDDDK -3 FLAG tags Date Recue/Date Received 2022-04-29 DDEVTQNVLYENQKLIANQPLHLAAWEGHLGIVEVLL
KNGQIDRLITGRLQSLQTGHTPLHLAAYTGHLEIVEVLL
KNGMWLSYFIASFRLFARTRSMWGTAPLHLAAMWGH
LEIVEVLLKNGKWPVVYIWLGFIAGLITPKDLARDNGNE
WIRELLEKAERKLKDLDRKLLEAARAGHRDEVEDLIKN
GKRSFIEDLLFNKVTLGFTPLHLAAWEGHLGIVEVLLK
NGRFLYIIKLIFLWLLWPGHTPLHLAAYTGHLEIVEVLL
KNGMAYRFNGIGVTQNVLGTAPLHLAAMWGHLEIVEV
LLKNGNSDYKDDDDKDYKDDDDKDYKDDDDK
where:
HA-COV1 MNTQILVFALIAII PTNADKI - HA leader #24 (3xFLAG) GSGA ¨ spacer DLGKKLLEAARAGQDDEVTQNVLYENQKLIANQPLHL
AAWEGH LGIVEVLLKNGQI DRLITGRLQSLQTGHTPLH
LAAYTGHLEIVEVLLKNGMWLSYFIASFRLFARTRSM
WGTAPLHLAAMWGHLEIVEVLLKNGKWPVVYIWLGFIA
GLITPKDLARDNGNEWIRELLEKAERKLKDLDRKLLEA
ARAGHRDEVEDLIKNGKRSFIEDLLFNKVTLGFTPLHL
AAWEGHLGIVEVLLKNGRFLYIIKLIFLWLLWPGHTPLH
LAAYTGHLEIVEVLLKNGMAYRFNGIGVTQNVLGTAPL
HLAAMWGHLEIVEVLLKNG ¨ 'COV1_Scaffold"
NS ¨ spacer DYKDDDDKDYKDDDDKDYKDDDDK -3 FLAG tags I RELSDYLLQDYPVTVASN LQDE ELCGG LWRLVLAQR
WMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPP
SCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLE
LQCQPDSSTLPPPWSPRPLEATAPTAPQPGAANGAS
GGGGSGGGGTPLGDTTHTSGGELEELLKHLKELLKG
PRKGELEELLKHLKELLKGLSGALDLGKKLLEAARAG
QDDEVTQNVLYENQKLIANQPLHLAAWEGHLGIVEVL
LKNGQIDRLITGRLQSLQTGHTPLHLAAYTGHLEIVEVL
LKNGMWLSYFIASFRLFARTRSMWGTAPLHLAAMWG
HLEIVEVLLKNGKWPVVYIWLGFIAGLITPKDLARDNGN
hF"-d HLX- EWIRELLEKAERKLKDLDRKLLEAARAGHRDEVEDLIK
#28 COV1 NGKRSFIEDLLFNKVTLGFTPLHLAAWEGHLGIVEVLL
(3xFLAG) KNGRFLYIIKLIFLWLLWPGHTPLHLAAYTGHLEIVEVL
LKNGMAYRFNGIGVTQNVLGTAPLHLAAMWGHLEIVE
VLLKNGNSGSGDYKDDDDKDYKDDDDKDYKDDDDK
where:
MDVVTWILFLVAAATRVHS - IgE leader G - spacer TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNL
QDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLE
RVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQ
LVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPL

Date Recue/Date Received 2022-04-29 EATAPTAPQP - human FLT3L ectodomain GAANGASGGGGSGGGG - spacer TPLGDTTHTSG ¨ hinge GELEELLKHLKELLKGPRKGELEELLKHLKELLK ¨
dimerization domain GLSGAL ¨ spacer DLGKKLLEAARAGQDDEVTQNVLYENQKLIANQPLHL
AAWEGH LGIVEVLLKNGQI DRLITGRLQSLQTGHTPLH
LAAYTGHLEIVEVLLKNGMWLSYFIASFRLFARTRSM
WGTAPLHLAAMWGHLEIVEVLLKNGKWPVVYIWLGFIA
GLITPKDLARDNGNEWIRELLEKAERKLKDLDRKLLEA
ARAGHRDEVEDLIKNGKRSFIEDLLFNKVTLGFTPLHL
AAWEGHLGIVEVLLKNGRFLYIIKLIFLWLLWPGHTPLH
LAAYTGH LEIVEVLLKNG MAYRFNG I GVTQNVLGTAPL
HLAAMWGHLEIVEVLLKNG ¨ 'COV1_Scaffold' NSGSG ¨ spacer DYKDDDDKDYKDDDDKDYKDDDDK -3 FLAG tags I RELSDYLLQDYPVTVASN LQDE ELCGG LWRLVLAQR
WMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPP
SCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLE
LQCQPDSSTLPPPWSPRPLEATAPTAPQPGAANGAS
GGGGSGGGGTPLGDTTHTSGGELEELLKHLKELLKG
PRKGELEELLKHLKELLKGLSGALDLGKKLLEAARAG
QDDEVTQNVLYENQKLIANQPLHLAAWEGHLGIVEVL
LKNGQI DRLITGRLQSLQTGHTPLHLAAYTGHLEIVEVL
LKNGMWLSYFIASFRLFARTRSMWGTAPLHLAAMWG
HLEIVEVLLKNGKWPVVYIWLGFIAGLITPKDLARDNGN
EWIRELLEKAERKLKDLDRKLLEAARAGHRDEVEDLIK
NGKRSFIEDLLFNKVTLGFTPLHLAAWEGHLGIVEVLL
KNGRFLYIIKLIFLWLLWPGHTPLHLAAYTGHLEIVEVL
hF"-d HLX- LKNGMAYRFNGIGVTQNVLGTAPLHLAAMWGHLEIVE
#50 COV1-TLR VLLKNGTRSGSSALSGCTGLTSITIPNTGSSGESAFKG
(3xFLAG) CSGLKSITAAAGNSGSGDYKDDDDKDYKDDDDKDYK
DDDDK
where:
MDVVTWILFLVAAATRVHS - IgE leader G ¨ spacer TQDCSFQHSPISSDFAVKI RELSDYLLQDYPVTVASNL
QDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLE
RVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQ
LVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPL
EATAPTAPQP - human FLT3L ectodomain Date Recue/Date Received 2022-04-29 GAANGASGGGGSGGGG ¨ spacer TPLGDTTHTSG ¨ hinge GELEELLKHLKELLKGPRKGELEELLKHLKELLK ¨
dimerization domain GLSGAL ¨ spacer DLGKKLLEAARAGQDDEVTQNVLYENQKLIANQPLHL
AAWEGH LGIVEVLLKNGQI DRLITGRLQSLQTGHTPLH
LAAYTGHLEIVEVLLKNGMWLSYFIASFRLFARTRSM
WGTAPLHLAAMWGHLEIVEVLLKNGKWPVVYIWLGFIA
GLITPKDLARDNGNEWIRELLEKAERKLKDLDRKLLEA
ARAGHRDEVEDLIKNGKRSFIEDLLFNKVTLGFTPLHL
AAWEGHLGIVEVLLKNGRFLYIIKLIFLWLLWPGHTPLH
LAAYTGH LEIVEVLLKNG MAYRFNG I GVTQNVLGTAPL
HLAAMWGHLEIVEVLLKNG ¨ 'COV1_Scaffold"
TRSGSS ¨ spacer ALSGCTGLTSITI PNTGSSGESAFKGCSGLKSIT
- TLR agonist peptides sequence AAAGNSGSG ¨ spacer DYKDDDDKDYKDDDDKDYKDDDDK -3 FLAG tags DDEVTQNVLYENQKLIANQPLHLAAWEGHLGIVEVLL
KNGQI DRLITGRLQSLQTGHTPLHLAAYTGHLEIVEVLL
KNGMWLSYFIASFRLFARTRSMWGTAPLHLAAMWGH
LEIVEVLLKNGKWPVVYIWLGFIAGLITPKDLARDNGNE
WI RELLEKAERKLKDLDRKLLEAARAGHRDEVEDLIKN
GKRSFI EDLLFNKVTLGFTPLHLAAWEGHLGIVEVLLK
NGRFLYIIKLIFLWLLWPGHTPLHLAAYTGHLEIVEVLL
KNGMAYRFNGIGVTQNVLGTAPLHLAAMWGHLEIVEV
LLKNGAAAGPGPMSAPASTTQATGSTTSTTTKTAGAT
PATASG LFTI PDG DFFSTARAIVASNAVATN ED LSKI EA

#22 PVXCP TGPYSNGISRARLAAAIKEVCTLRQFCMKYAPVVWNW
(3xFLAG) MLTNNSPPANWQAQGFKPEHKFAAFDFFNGVTNPAA
I MPKEG LI RPPSEAEM NAAQTAAFVKITKARAQSN D FA
SLDAAVTRGRITGTTTAEAVVTLPPPGNSDYKDDDDK
DYKDDDDKDYKDDDDK
where:
MNTQILVFALIAIIPTNADKI ¨ HA signal sequence GSGA ¨ spacer DLGKKLLEAARAGQDDEVTQNVLYENQKLIANQPLHL
AAWEGH LGIVEVLLKNGQI DRLITGRLQSLQTGHTPLH
LAAYTGHLEIVEVLLKNGMWLSYFIASFRLFARTRSM

Date Recue/Date Received 2022-04-29 WGTAPLHLAAMWGHLEIVEVLLKNGKWPVVYIWLGFIA
GLITPKDLARDNGNEWIRELLEKAERKLKDLDRKLLEA
ARAGHRDEVEDLIKNGKRSFIEDLLFNKVTLGFTPLHL
AAWEGHLGIVEVLLKNGRFLYIIKLIFLWLLWPGHTPLH
LAAYTGHLEIVEVLLKNGMAYRFNGIGVTQNVLGTAPL
HLAAMWGHLEIVEVLLKNG ¨ 'COVLScaffold' AAGPGP ¨ linker MSAPASTTQATGSTTSTTTKTAGATPATASGLFTIPDG
DFFSTARAIVASNAVATNEDLSKIEAIWKDMKVPTDTM
AQAAWDLVRHCADVGSSAQTEMIDTGPYSNGISRAR
LAAAIKEVCTLRQFCMKYAPVVWNWMLTNNSPPANW
QAQGFKPEHKFAAFDFFNGVTNPAAIMPKEGLIRPPS
EAEMNAAQTAAFVKITKARAQSNDFASLDAAVTRGRI
TGTTTAEAVVTLPPP ¨ potato X virus coat protein (PVXCP) GNS ¨ spacer DYKDDDDKDYKDDDDKDYKDDDDK ¨ 3xFLAG tag LGIVEVLLKNGPYRVVVLSFELLHAPGHTPLHLAAYTG
HLEIVEVLLKNGGDAVVYRGTTTYKLNGTAPLHLAAM
WGHLEIVEVLLKNGKPFERDISTEIYQAGTPKDLARDN
GNVLNDILSRLDPPEAEVSFKELLVYAADPAMHAASY
VTQQLIRAAEIRASANLAATKMSECVGHRDEVEDLIKN
GSQLGGLHLLIGLAKRFKGFTPLHLAAWEGHLGIVEVL
LKNGVIDLLLDDFVEIIKSGHTPLHLAAYTGHLEIVEVLL
KNGTLEQYVFCTVNALPEGTAPLHLAAMWGHLEIVEV
LLKNGFGLFCLLNRYFRLTLGVYDYGKTPKDLARDNG
NTERLKLFAAETLKATEETFKLSYGIATVRGHRDEVED
LIKNGMIERFVSLAIDAYPLGFTPLHLAAWEGHLGIVEV
LLKNGFYAYLRKHFSMMILSGHTPLHLAAYTGHLEIVE
VLLKNGTQMNLKYAISAKNRARTVAGAPLHLAAMWG
#27 COV2 HLEIVEVLLKNGVRKIFVDGVPFVVSTTPKDLARDNGN
(3xFLAG) SDYKDDDDKDYKDDDDKDYKDDDDK
where:
M - start translation DLGKKLLEAARAGQDDEVRILLANGTPLHLAAWEGHL
GIVEVLLKNGPYRVVVLSFELLHAPGHTPLHLAAYTGH
LEIVEVLLKNGGDAVVYRGTTTYKLNGTAPLHLAAMW
GHLEIVEVLLKNGKPFERDISTEIYQAGTPKDLARDNG
NVLNDILSRLDPPEAEVSFKELLVYAADPAMHAASYVT
QQLIRAAEIRASANLAATKMSECVGHRDEVEDLIKNGS
QLGGLHLLIGLAKRFKGFTPLHLAAWEGHLGIVEVLLK
NGVIDLLLDDFVEIIKSGHTPLHLAAYTGHLEIVEVLLKN
GTLEQYVFCTVNALPEGTAPLHLAAMWGHLEIVEVLL
KNGFGLFCLLNRYFRLTLGVYDYGKTPKDLARDNGNT
ERLKLFAAETLKATEETFKLSYGIATVRGHRDEVEDLIK

Date Recue/Date Received 2022-04-29 NGMIERFVSLAIDAYPLGFTPLHLAAWEGHLGIVEVLL
KNGFYAYLRKHFSMMILSGHTPLHLAAYTGHLEIVEVL
LKNGTQMNLKYAISAKNRARTVAGAPLHLAAMWGHL
EIVEVLLKNGVRKIFVDGVPFVVSTTPKDLARDN
- '00V2 Scaffold' GNS ¨ spacer DYKDDDDKDYKDDDDKDYKDDDDK -3 FLAG tags DDEVRILLANGTPLHLAAWEGHLGIVEVLLKNGPYRVV
VLSFELLHAPGHTPLH LAAYTGH LEIVEVLLKNGG DAV
VYRGTTTYKLNGTAPLHLAAMWGHLEIVEVLLKNGKP
FERDISTEIYQAGTPKDLARDNGNVLNDILSRLDPPEA
EVSFKELLVYAAD PAM HAASYVTQQLI RAAE I RASAN L
AATKMSECVGHRDEVEDLIKNGSQLGGLHLLIGLAKR
FKGFTPLHLAAWEGHLGIVEVLLKNGVI DLLLDDFVE II
KSGHTPLHLAAYTGHLEIVEVLLKNGTLEQYVFCTVNA
LPEGTAPLHLAAMWGHLEIVEVLLKNGFGLFCLLNRY
FRLTLGVYDYGKTPKDLARDNGNTERLKLFAAETLKA
TEETFKLSYGIATVRGHRDEVEDLIKNGMIERFVSLAID
AYPLGFTPLHLAAWEGHLGIVEVLLKNGFYAYLRKHF
SMMILSGHTPLHLAAYTGHLEIVEVLLKNGTQMNLKYA
I SAKN RARTVAGAPLH LAAMWGH LEIVEVLLKNGVRKI
FVDGVPFVVSTTPKDLARDNGNSDYKDDDDKDYKDD
DDKDYKDDDDK
where:

#25 (3xFLAG) MNTQILVFALIAIIPTNADKI ¨ HA signal sequence GSGA - spacer DLGKKLLEAARAGQDDEVRILLANGTPLHLAAWEGHL
GIVEVLLKNGPYRVVVLSFELLHAPGHTPLHLAAYTGH
LEIVEVLLKNGGDAVVYRGTTTYKLNGTAPLHLAAMW
GHLEIVEVLLKNGKPFERDISTEIYQAGTPKDLARDNG
NVLN D I LSRLD PPEAEVSFKELLVYAADPAM HAASYVT
QQLIRAAEIRASANLAATKMSECVGHRDEVEDLIKNGS
QLGGLHLLIGLAKRFKGFTPLHLAAWEGHLGIVEVLLK
NGVIDLLLDDFVEIIKSGHTPLHLAAYTGHLEIVEVLLKN
GTLEQYVFCTVNALPEGTAPLHLAAMWGHLEIVEVLL
KNGFGLFCLLNRYFRLTLGVYDYGKTPKDLARDNGNT
ERLKLFAAETLKATEETFKLSYGIATVRG H RD EVEDLI K
NGMIERFVSLAIDAYPLGFTPLHLAAWEGHLGIVEVLL
KNGFYAYLRKHFSMMILSGHTPLHLAAYTGHLEIVEVL
LKNGTQMNLKYAISAKNRARTVAGAPLHLAAMWGHL
EIVEVLLKNGVRKIFVDGVPFVVSTTPKDLARDN
- '00V2 Scaffold' GNS ¨ spacer Date Recue/Date Received 2022-04-29 DYKDDDDKDYKDDDDKDYKDDDDK -3 FLAG tags I RELSDYLLQDYPVTVASN LQDE ELCGG LWRLVLAQR
WMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPP
SCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLE
LQCQPDSSTLPPPWSPRPLEATAPTAPQPGAANGAS
GGGGSGGGGTPLGDTTHTSGGELEELLKHLKELLKG
PRKGELEELLKHLKELLKGLSGALDLGKKLLEAARAG
QDDEVRILLANGTPLHLAAWEGHLGIVEVLLKNGPYR
VVVLSFELLHAPGHTPLHLAAYTGHLEIVEVLLKNGGD
AVVYRGTTTYKLNGTAPLHLAAMWGHLEIVEVLLKNG
KPFERDISTEIYQAGTPKDLARDNGNVLNDILSRLDPP
EAEVSFKELLVYAAD PAM HAASYVTQQLI RAAEI RASA
NLAATKMSECVGHRDEVEDLIKNGSQLGGLHLLIGLA
KRFKGFTPLHLAAWEGHLGIVEVLLKNGVIDLLLDDFV
El IKSGHTPLH LAAYTGHLEIVEVLLKNGTLEQYVFCTV
NALPEGTAPLHLAAMWGHLEIVEVLLKNGFGLFCLLN
RYFRLTLGVYDYGKTPKDLARDNGNTERLKLFAAETL
KATEETFKLSYGIATVRGHRDEVEDLIKNGMIERFVSL
Al DAYPLGFTPLH LAAWEGHLGIVEVLLKNGFYAYLRK
HFSMMI LSGHTPLHLAAYTGHLEIVEVLLKNGTQMN LK
YAISAKNRARTVAGAPLHLAAMWGHLEIVEVLLKNGV
RKIFVDGVPFVVSTTPKDLARDNGNSDYKDDDDKDYK
hF"-dHLX- DDDDKDYKDDDDK
#29 COV2 (3xFLAG) where:
MDVVTWILFLVAAATRVHS - IgE leader G ¨ spacer TQDCSFQHSPISSDFAVKI RELSDYLLQDYPVTVASNL
QDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLE
RVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQ
LVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPL
EATAPTAPQP - human FLT3L ectodomain GAANGASGGGGSGGGG - spacer TPLGDTTHTSG ¨ hinge GELEELLKHLKELLKGPRKGELEELLKHLKELLK ¨
dimerization domain GLSGAL ¨ spacer DLGKKLLEAARAGQDDEVRILLANGTPLHLAAWEGHL
GIVEVLLKNGPYRVVVLSFELLHAPGHTPLHLAAYTGH
LEIVEVLLKNGGDAVVYRGTTTYKLNGTAPLHLAAMW
GHLEIVEVLLKNGKPFERDISTEIYQAGTPKDLARDNG
NVLN D I LSRLD PPEAEVSFKELLVYAADPAM HAASYVT

Date Recue/Date Received 2022-04-29 QQLIRAAEIRASANLAATKMSECVGHRDEVEDLIKNGS
QLGGLHLLIGLAKRFKGFTPLHLAAWEGHLGIVEVLLK
NGVIDLLLDDFVEIIKSGHTPLHLAAYTGHLEIVEVLLKN
GTLEQYVFCTVNALPEGTAPLHLAAMWGHLEIVEVLL
KNGFGLFCLLNRYFRLTLGVYDYGKTPKDLARDNGNT
ERLKLFAAETLKATEETFKLSYGIATVRG H RD EVEDLI K
NGMIERFVSLAIDAYPLGFTPLHLAAWEGHLGIVEVLL
KNGFYAYLRKHFSMMILSGHTPLHLAAYTGHLEIVEVL
LKNGTQMNLKYAISAKNRARTVAGAPLHLAAMWGHL
EIVEVLLKNGVRKIFVDGVPFVVSTTPKDLARDN
- '00V2 Scaffold' GNS ¨ spacer DYKDDDDKDYKDDDDKDYKDDDDK -3 FLAG tags I RELSDYLLQDYPVTVASN LQDE ELCGG LWRLVLAQR
WMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPP
SCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLE
LQCQPDSSTLPPPWSPRPLEATAPTAPQPGAANGAS
GGGGSGGGGTPLGDTTHTSGGELEELLKHLKELLKG
PRKGELEELLKHLKELLKGLSGALDLGKKLLEAARAG
QDDEVRILLANGTPLHLAAWEGHLGIVEVLLKNGPYR
VVVLSFELLHAPGHTPLHLAAYTGHLEIVEVLLKNGGD
AVVYRGTTTYKLNGTAPLHLAAMWGHLEIVEVLLKNG
KPFERDISTEIYQAGTPKDLARDNGNVLNDILSRLDPP
EAEVSFKELLVYAAD PAM HAASYVTQQLI RAAEI RASA
NLAATKMSECVGHRDEVEDLIKNGSQLGGLHLLIGLA
KRFKGFTPLHLAAWEGHLGIVEVLLKNGVIDLLLDDFV
El IKSGHTPLHLAAYTGHLEIVEVLLKNGTLEQYVFCTV
NALPEGTAPLHLAAMWGHLEIVEVLLKNGFGLFCLLN
RYFRLTLGVYDYGKTPKDLARDNGNTERLKLFAAETL
KATEETFKLSYGIATVRGHRDEVEDLIKNGMIERFVSL
Al DAYPLGFTPLHLAAWEGHLGIVEVLLKNGFYAYLRK
hF"-dHLX-HFSMMI LSGHTPLHLAAYTGHLEIVEVLLKNGTQMN LK
#9 COV2-TLR
YAISAKNRARTVAGAPLHLAAMWGHLEIVEVLLKNGV
(3xFLAG) RKIFVDGVPFVVSTTPKDLARDNTRSGSSALSGCTGL
TSITIPNTGSSGESAFKGCSGLKSITAAAGNSGSGDYK
DDDDKDYKDDDDKDYKDDDDK
where:
MDVVTWILFLVAAATRVHS - IgE leader G ¨ spacer TQDCSFQHSPISSDFAVKI RELSDYLLQDYPVTVASNL
QDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLE
RVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQ
LVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPL
EATAPTAPQP - human FLT3L ectodomain GAANGASGGGGSGGGG ¨ spacer TPLGDTTHTSG ¨ hinge Date Recue/Date Received 2022-04-29 GELEELLKHLKELLKGPRKGELEELLKHLKELLK ¨
dimerization domain GLSGAL ¨ spacer DLGKKLLEAARAGQDDEVRILLANGTPLHLAAWEGHL
GIVEVLLKNGPYRVVVLSFELLHAPGHTPLHLAAYTGH
LEIVEVLLKNGGDAVVYRGTTTYKLNGTAPLHLAAMW
GHLEIVEVLLKNGKPFERDISTEIYQAGTPKDLARDNG
NVLN D I LSRLD PPEAEVSFKELLVYAADPAM HAASYVT
QQLIRAAEIRASANLAATKMSECVGHRDEVEDLIKNGS
QLGGLHLLIGLAKRFKGFTPLHLAAWEGHLGIVEVLLK
NGVIDLLLDDFVEIIKSGHTPLHLAAYTGHLEIVEVLLKN
GTLEQYVFCTVNALPEGTAPLHLAAMWGHLEIVEVLL
KNGFGLFCLLNRYFRLTLGVYDYGKTPKDLARDNGNT
ERLKLFAAETLKATEETFKLSYGIATVRG H RD EVEDLI K
NGMIERFVSLAIDAYPLGFTPLHLAAWEGHLGIVEVLL
KNGFYAYLRKHFSMMILSGHTPLHLAAYTGHLEIVEVL
LKNGTQMNLKYAISAKNRARTVAGAPLHLAAMWGHL
EIVEVLLKNGVRKIFVDGVPFVVSTTPKDLARDN
- '00V2 Scaffold' TRSGSS ¨ spacer ALSGCTGLTSITI PNTGSSGESAFKGCSGLKSIT
- TLR agonist peptides sequence AAAGNSGSG ¨ spacer DYKDDDDKDYKDDDDKDYKDDDDK -3 FLAG tags DDEVRILLANGTPLHLAAWEGHLGIVEVLLKNGPYRVV
VLSFELLHAPGHTPLH LAAYTGH LEIVEVLLKNGG DAV
VYRGTTTYKLNGTAPLHLAAMWGHLEIVEVLLKNGKP
FERDISTEIYQAGTPKDLARDNGNVLNDILSRLDPPEA
EVSFKELLVYAAD PAM HAASYVTQQLI RAAE I RASAN L
AATKMSECVGHRDEVEDLIKNGSQLGGLHLLIGLAKR
FKGFTPLHLAAWEGHLGIVEVLLKNGVI DLLLDDFVE II
KSGHTPLHLAAYTGHLEIVEVLLKNGTLEQYVFCTVNA
LPEGTAPLHLAAMWGHLEIVEVLLKNGFGLFCLLNRY

#23 PVXCP TEETFKLSYGIATVRGHRDEVEDLIKNGMIERFVSLAID
(3xFLAG) AYPLGFTPLHLAAWEGHLGIVEVLLKNGFYAYLRKHF
SMMILSGHTPLHLAAYTGHLEIVEVLLKNGTQMNLKYA
I SAKN RARTVAGAPLH LAAMWGH LEIVEVLLKNGVRKI
FVDGVPFVVSTTPKDLARDNAAAGPGPMSAPASTTQ
ATGSTTSTTTKTAGATPATASGLFTIPDGDFFSTARAIV
ASNAVATNEDLSKIEAIWKDMKVPTDTMAQAAWDLVR
HCADVGSSAQTEM I DTG PYSNG I SRARLAAAIKEVCTL
RQFCMKYAPVVWNWMLTNNSPPANWQAQGFKPEH
KFAAFDFFNGVTNPAAIMPKEGLIRPPSEAEMNAAQT
AAFVKITKARAQSNDFASLDAAVTRGRITGTTTAEAVV
TLPPPGNSDYKDDDDKDYKDDDDKDYKDDDDK

Date Recue/Date Received 2022-04-29 where:
MNTQILVFALIAIIPTNADKI ¨ HA signal sequence GSGA - linker DLGKKLLEAARAGQDDEVRILLANGTPLHLAAWEGHL
GIVEVLLKNGPYRVVVLSFELLHAPGHTPLHLAAYTGH
LEIVEVLLKNGGDAVVYRGTTTYKLNGTAPLHLAAMW
GHLEIVEVLLKNGKPFERDISTEIYQAGTPKDLARDNG
NVLNDILSRLDPPEAEVSFKELLVYAADPAMHAASYVT
QQLIRAAEIRASANLAATKMSECVGHRDEVEDLIKNGS
QLGGLHLLIGLAKRFKGFTPLHLAAWEGHLGIVEVLLK
NGVIDLLLDDFVEIIKSGHTPLHLAAYTGHLEIVEVLLKN
GTLEQYVFCTVNALPEGTAPLHLAAMWGHLEIVEVLL
KNGFGLFCLLNRYFRLTLGVYDYGKTPKDLARDNGNT
ERLKLFAAETLKATEETFKLSYGIATVRGHRDEVEDLIK
NGMIERFVSLAIDAYPLGFTPLHLAAWEGHLGIVEVLL
KNGFYAYLRKHFSMMILSGHTPLHLAAYTGHLEIVEVL
LKNGTQMNLKYAISAKNRARTVAGAPLHLAAMWGHL
EIVEVLLKNGVRKIFVDGVPFVVSTTPKDLARDN
- '00V2 Scaffold' AAAGPGP ¨ linker MSAPASTTQATGSTTSTTTKTAGATPATASGLFTIPDG
DFFSTARAIVASNAVATNEDLSKIEAIWKDMKVPTDTM
AQAAWD LVRHCADVGSSAQTEM I DTG PYSNG I SRAR
LAAAIKEVCTLRQFCMKYAPVVWNWMLTNNSPPANW
QAQGFKPEHKFAAFDFFNGVTNPAAIMPKEGLIRPPS
EAEMNAAQTAAFVKITKARAQSNDFASLDAAVTRGRI
TGTTTAEAVVTLPPP ¨ potato X virus coat protein (PVXCP) NS ¨ spacer DYKDDDDKDYKDDDDKDYKDDDDK ¨ 3xFLAG tag LGIVEVLLKNGPYRVVVLSFELLHAPGHTPLHLAAYTG
HLEIVEVLLKNGGDAVVYRGTTTYKLNGTAPLHLAAM
WGHLEIVEVLLKNGKPFERDISTEIYQAGTPKDLARDN
GNVLN DI LSRLDPPEAEVSFKELLVYAADPAM HAASY
VTQQLIRAAEIRASANLAATKMSECVGHRDEVEDLIKN
GSQLGGLHLLIGLAKRFKGFTPLHLAAWEGHLGIVEVL

LKNGVIDLLLDDFVEIIKSGHTPLHLAAYTGHLEIVEVLL
#21 PVXCP
KNGTLEQYVFCTVNALPEGTAPLHLAAMWGHLEIVEV
(3xFLAG) LLKNGFGLFCLLNRYFRLTLGVYDYGKTPKDLARDNG
NTERLKLFAAETLKATEETFKLSYGIATVRGHRDEVED
LIKNGMIERFVSLAIDAYPLGFTPLHLAAWEGHLGIVEV
LLKNGFYAYLRKHFSMMILSGHTPLHLAAYTGHLEIVE
VLLKNGTQMNLKYAISAKNRARTVAGAPLHLAAMWG
HLEIVEVLLKNGVRKIFVDGVPFVVSTTPKDLARDNAA
AGPGPMSAPASTTQATGSTTSTTTKTAGATPATASGL

Date Recue/Date Received 2022-04-29 FTIPDGDFFSTARAIVASNAVATNEDLSKIEAIWKDMKV
PTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPYSNG
I SRARLAAAI KEVCTLRQFCMKYAPVVWNWMLTN N SP
PANWQAQGFKPEHKFAAFDFFNGVTNPAAIMPKEGLI
RPPSEAEMNAAQTAAFVKITKARAQSNDFASLDAAVT
RGRITGTTTAEAVVTLPPPGNSDYKDDDDKDYKDDDD
KDYKDDDDK
where:
M - start translation DLGKKLLEAARAGQDDEVRILLANGTPLHLAAWEGHL
GIVEVLLKNGPYRVVVLSFELLHAPGHTPLHLAAYTGH
LEIVEVLLKNGGDAVVYRGTTTYKLNGTAPLHLAAMW
GHLEIVEVLLKNGKPFERDISTEIYQAGTPKDLARDNG
NVLN D I LSRLDPPEAEVSFKE LLVYAAD PAM HAASYVT
QQLIRAAEIRASANLAATKMSECVGHRDEVEDLIKNGS
QLGGLHLLIGLAKRFKGFTPLHLAAWEGHLGIVEVLLK
NGVIDLLLDDFVEIIKSGHTPLHLAAYTGHLEIVEVLLKN
GTLEQYVFCTVNALPEGTAPLHLAAMWGHLEIVEVLL
KNGFGLFCLLNRYFRLTLGVYDYGKTPKDLARDNGNT
ERLKLFAAETLKATEETFKLSYGIATVRGHRDEVEDLIK
NGMIERFVSLAIDAYPLGFTPLHLAAWEGHLGIVEVLL
KNGFYAYLRKHFSMMILSGHTPLHLAAYTGHLEIVEVL
LKNGTQMNLKYAISAKNRARTVAGAPLHLAAMWGHL
EIVEVLLKNGVRKIFVDGVPFVVSTTPKDLARDN
- '00V2 Scaffold' AAAGPGP ¨ linker MSAPASTTQATGSTTSTTTKTAGATPATASGLFTIPDG
DFFSTARAIVASNAVATNEDLSKIEAIWKDMKVPTDTM
AQAAWD LVRHCADVGSSAQTEM I DTG PYSNG I SRAR
LAAAIKEVCTLRQFCMKYAPVVWNWMLTNNSPPANW
QAQGFKPEHKFAAFDFFNGVTNPAAI MPKEGLIRPPS
EAEMNAAQTAAFVKITKARAQSN DFASLDAAVTRG RI
TGTTTAEAVVTLPPP ¨ potato X virus coat protein (PVXCP) NS ¨ spacer DYKDDDDKDYKDDDDKDYKDDDDK ¨ 3xFLAG tag

[00259] Based on the foregoing sequences in Table 5, generic sequences of the COV1 and COV2 scaffolds were determined, and are shown in Table 6.
Table 6: List of generic COV1 and COV2 scaffold sequences:

Date Recue/Date Received 2022-04-29 Vaccine Amino acid sequence SEQ ID
NO:
DLGKKLLEAARAGQDDEVRILLAMMPLHLAAWEGHLGIVEVLLK 95, 109, NGUMA2GHTPLHLAAYTGHLEIVEVLLKNGUMA2GTAPLHLAA 110, 111 MWGHLEIVEVLLKNGX2M2TPKDLARDNGNEWIRELLEKAERKLK 112, DLDRKLLEAARAGHRDEVEDLIKNGanaGFTPLHLAAWEGHLGI 113, VEVLLKNGaLlaGHTPLHLAAYTGH LE IVEVLLKNGaLlaGTAP 114, 115 FDLAIDNGNEDIAEVLQK
Formula I
COV-X1 where:
X1 is independently chosen from any one of the peptides from Table 1 or a flexible linker optionally chosen from GSGA, (GGGGS)n or other flexible hydrophilic linker (e.g. SEQ ID NO: 20); and X2 is a linker providing proteolytic cleavage site, independently chosen from AAY, EAAAK (SEQ ID NO: 102), RVRR (SEQ ID NO: 103), GGGS
(SEQ ID NO: 104), GPGPG (SEQ ID NO: 105), HEYGAEALERAG
(SEQ ID NO: 106) or absent.
DLGKKLLEAARAGQDDEVRILLAMMTPLHLAAWEGHLGIVEVLL 96, 116, KNGUMA2GHTPLHLAAYTGHLEIVEVLLKNGX2M2GTAPLHLAA 117, MWGHLEIVEVLLKNGX2M2TPKDLARDNGNUMA2SFKX2Xla 118, GHRDEVEDLIKNGanaGFTPLHLAAWEGHLGIVEVLLKNGan 119, X2AAYTGHLEIVEVLLKNGX2M2GTAPLH LAAMWGH LEIVEVLLKN 120, GanaGKTPKDLARDNGNanaGHRDEVEDLIKNGUtiaGF 121, TPLHLAAWEGHLGIVEVLLKNGanaGHTPLHLAAYTGHLEIVEVL 122, LKNGUtiaGTAPLHLAAMWGHLEIVEVLLKNGX2M2TPX3 123, 124, Formula II 125.
126, COV-X2 where: 127, 128 X1 is independently chosen from any one of the peptides from Table 1 or a flexible linker optionally chosen from GSGA, (GGGGS)n or other flexible hydrophilic linker (e.g. SEQ ID NO: 20); and X2 is a linker providing proteolytic cleavage site, independently chosen from AAY, EAAAK (SEQ ID NO: 102), RVRR (SEQ ID NO: 103), GGGS
(SEQ ID NO: 104), GPGPG (SEQ ID NO: 105), HEYGAEALERAG
(SEQ ID NO: 106) or absent; and X3 is either a short tail - KDLARDN (SEQ ID NO: 170) or a long tail -FDLAIDNGNEDIAEVLQK (SEQ ID NO: 171) DLGKKLLEAARAGQDDEVRILLAMMPLHLAAWEGHLGIVEVLLK 97, 129, COV-X3 NGUMA2GHTPLHLAAYTGHLEIVEVLLKNGUMA2GTAPLHLAA 130, MWGHLEIVEVLLKNGUtiaTEDLAKDNGNQW1AQKLEEAKKEDL 131, DRKLLEAARAGHRDEVEDLIKNGanaGFTPLHLAAWEGHLGIVE 132, VLLKNGanaGHTPLHLAAYTGHLEIVEVLLKNGUMA2GTAPFD 133, Date Recue/Date Received 2022-04-29 LAID NGN EDIAEVLQK 134, 135 Formula III
where:
X1 is independently chosen from any one of the peptides from Table 1 or a flexible linker optionally chosen from GSGA, (GGGGS)n or other flexible hydrophilic linker (e.g. SEQ ID NO: 20); and X2 is a linker providing proteolytic cleavage site, independently chosen from AAY, EAAAK (SEQ ID NO: 102), RVRR (SEQ ID NO: 103), GGGS
(SEQ ID NO: 104), GPGPG (SEQ ID NO: 105), HEYGAEALERAG
(SEQ ID NO: 106) or absent.
DLGKKLLEAARAGQDDEVRI LLAUtiaTPLH LAAWEGH LGIVEVLL 98, 137, KNGUMA2GHTPLHLAAYTGHLEIVEVLLKNGX2M2GTAPLH LAA 138, MWGHLEIVEVLLKNGX2M2TPKDLARDNGN EWI RELLEKAERKLK 139, DLDRKLLEAARAGH RD EVEDLIKNGUMA2G FTPLH LAAWEGHLGI 140, VEVLLKNG=1,12GHTPLHLAAYTGH LE IVEVLLKNGUMA2GTAP 141, LH LAAMWG H LEIVEVLLKNanaGKTPKDLARD NG N EWI RELLEK 142, AERKLKDLDRKLLEAARAGHRDEVEDLIKNGanaGFTPLHLAAW 143, EGH LGIVEVLLKNGX2M2GHTPLH LAAYTGH LE IVEVLLKNGaal 144, aGTAPLH LAAMWGHLEIVEVLLKNGanaGTAPFDLAI DN GN EDI 145.
AEVLQK 146, 147, 148 COV-X4 Formula IV
where:
X1 is independently chosen from any one of the peptides from Table 1 or a flexible linker optionally chosen from GSGA, (GGGGS)n or other flexible hydrophilic linker (e.g. SEQ ID NO: 20); and X2 is a linker providing proteolytic cleavage site, independently chosen from AAY, EAAAK (SEQ ID NO: 102), RVRR (SEQ ID NO: 103), GGGS
(SEQ ID NO: 104), GPGPG (SEQ ID NO: 105), HEYGAEALERAG
(SEQ ID NO: 106) or absent.
DLGKKLLEAARAGQDDEVX2M2PLHLAAWEGHLGIVEVLLKNGX2 99, 149, LIA2G HTPLH LAAYTGH LEIVEVLLKNGX2M2GTAPLH LAAMWGH 150, LEIVEVLLKN GanaTPKDLARD NGN EWI RE LLEKAERKLKDLDR 151, KLLEAARAG H RD EVEDLI KNGX2M2G FTPLH LAAWEGH LGIVEVL 152, LKNGUtiaGHTPLHLAAYTGHLEIVEVLLKNGX2M2GTAPLHLA 153, AMWGHLEIVEVLLKNG 154, 155 Formula V
where:
X1 is independently chosen from any one of the peptides from Table 1 or a flexible linker optionally chosen from GSGA, (GGGGS)n or other flexible hydrophilic linker (e.g. SEQ ID NO: 20); and X2 is a linker providing proteolytic cleavage site, independently chosen Date Recue/Date Received 2022-04-29 from AAY, EAAAK (SEQ ID NO: 102), RVRR (SEQ ID NO: 103), GGGS
(SEQ ID NO: 104), GPGPG (SEQ ID NO: 105), HEYGAEALERAG
(SEQ ID NO: 106) or absent.
DLGKKLLEAARAGQDDEVRILLAMMTPLHLAAWEGHLGIVEVLL 100, KNGUMA2GHTPLHLAAYTGHLEIVEVLLKNGX2M2GTAPLHLAA 156, MWGHLEIVEVLLKNGanaTAFDISIDNGNEDLAEI LQKLN 157, 158, 159 Formula VI
where:
COV-X6 X1 is independently chosen from any one of the peptides from Table 1 or a flexible linker optionally chosen from GSGA, (GGGGS)n or other flexible hydrophilic linker (e.g. SEQ ID NO: 20); and X2 is a linker providing proteolytic cleavage site, independently chosen from AAY, EAAAK (SEQ ID NO: 102), RVRR (SEQ ID NO: 103), GGGS
(SEQ ID NO: 104), GPGPG (SEQ ID NO: 105), HEYGAEALERAG
(SEQ ID NO: 106) or absent.
DLGKKLLEAARAGQDDEVRILLAMMTPLHLAAWEGHLGIVEVLL 101, KNGUMA2GHTPLHLAAYTGHLEIVEVLLKNGX2M2GTAPLHLAA 160, MWGHLEIVEVLLKNGX2M2TPKDLARDNGNEWIRELLEKAERKLK 161, DLDRKLLEAARAGHRDEVEDLIKNGanaGFTPLHLAAWEGHLGI 162, VEVLLKNG=1,12GHTPLHLAAYTGH LE IVEVLLKNGUMA2GTAP 163, LHLAAMWGHLEIVEVLLKNGanaGKTAFDISIDNGNEDLAEI LQK 164, LN 165, 166, 167 Formula VII

where:
X1 is independently chosen from any one of the peptides from Table 1 or a flexible linker optionally chosen from GSGA, (GGGGS)n or other flexible hydrophilic linker (e.g. SEQ ID NO: 20); and X2 is a linker providing proteolytic cleavage site, independently chosen from AAY, EAAAK (SEQ ID NO: 102), RVRR (SEQ ID NO: 103), GGGS
(SEQ ID NO: 104), GPGPG (SEQ ID NO: 105), HEYGAEALERAG
(SEQ ID NO: 106) or absent.
LIST OF SEQUENCES
Amino acid sequence SEQ
ID NO:

Date Recue/Date Received 2022-04-29 Spike protein of MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPD 1 SARS-CoV-2 KVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFD
NPVLPFN DGVYFASTEKSNI I RGWI FGTTLDSKTQSLLIVN
NATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY
SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKN I DG
YFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLL
ALHRSYLTPGDSSSGVVTAGAAAYYVGYLQPRTFLLKYNE
NGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQP
TESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVA
DYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIR
G DEVRQIAPGQTGKIADYNYKLPDD FTGCVIAWN SN N LDS
KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV
EGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPA
TVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPF
QQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTN
TSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNV
FQTRAGCLIGAEHVNNSYECDI PIGAGICASYQTQTNSPR
RARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTT
El LPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLN

LPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAA
RDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGVVT
FGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQF
NSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLS
SNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVT
QQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHL
MSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAH
FPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDV
VIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI
SGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK
WPVVYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCG
SCCKFDEDDSEPVLKGVKLHYT
RBD_wt portion RVQPTESIVRFPN ITN LCPFG EVFNATRFASVYAWN RKRI S 2 corresponding NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS
to residues 328- FVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSN
579 of SARS- NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP
CoV-2 spike CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL
protein HAPATVCGPKK
RBD_wt portion NSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAG 3 corresponding STPCNGVEGFNCYFPLQSYGFQPTNGVGYQPY
to residues 437-508 of SARS-CoV-2 spike protein RBD_wt portion RVQPTESIVRFPN ITN LCPFG EVFNATRFASVYAWN RKRI S 4 corresponding NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS
to residues 319- FVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSN
526 of SARS- NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP
CoV-2 spike CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL
protein HAPATVCG

Date Recue/Date Received 2022-04-29 RBD_wt portion NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYN 5 corresponding SASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQI
to residues 331- APGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNY
521 of SARS- NYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCY
CoV-2 spike FPLQSYGFQPTNGVGYQPYRVVVLSFELLHAP
protein RBD_ RVQPTESIVRFPN ITN LCPFGEVFNATRFASVYAWNRKRIS 6 B.1.1.7(alpha) NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS
SARS-CoV-2 FVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSN
NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP
CNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELL
HAPATVCGPKK
RBD_P.1 RVQPTESIVRFPN ITN LCPFGEVFNATRFASVYAWNRKRIS 7 SARS-CoV-2 NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS
FVIRGDEVRQIAPGQTGTIADYNYKLPDDFTGCVIAWNSN
NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP
CNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELL
HAPATVCGPKK
RBD_P.2 RVQPTESIVRFPN ITN LCPFGEVFNATRFASVYAWNRKRIS 8 SARS-CoV-2 NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS
FVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSN
NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP
CNGVKGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL
HAPATVCGPKK
RBD_ B.1.351 RVQPTESIVRFPN ITN LCPFGEVFNATRFASVYAWNRKRIS 9 (beta) NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS
SARS-CoV-2 FVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSN
NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP
CNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELL
HAPATVCGPKK
RBD_B.1.617.2 RVQPTESIVRFPN ITN LCPFGEVFNATRFASVYAWNRKRIS 10 (delta) NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS
SARS-CoV-2 FVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSN
NLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKP
CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL
HAPATVCGPKK
RBD_B.1.617.2. RVQPTESIVRFPN ITN LCPFGEVFNATRFASVYAWNRKRIS 11 1 (delta plus) NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS
SARS-CoV-2 FVIRGDEVRQIAPGQTGTIADYNYKLPDDFTGCVIAWNSN
NLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKP
CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL
HAPATVCGPKK
RBD_C.37 RVQPTESIVRFPN ITN LCPFGEVFNATRFASVYAWNRKRIS 12 (lambda) NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS
SARS-CoV-2 FVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSN
NLDSKVGGNYNYQYRLFRKSNLKPFERDISTEIYQAGSTP
CNGVEGFNCYSPLQSYGFQPTNGVGYQPYRVVVLSFELL
HAPATVCGPKK

Date Recue/Date Received 2022-04-29 B.1.621 (Mu) RVQPTESIVRFPN ITN LCPFGEVFNATKFASVYAWNRKRI S 13 SARS-CoV-2 NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS
FVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSN
NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP
CNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELL
HAPATVCGPKK
B.1.1.529 RVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRIS 14 (omicron) NCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADS
SARS-CoV-2 FVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSN
KLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKP
CNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELL
HAPATVCGPKK

(corresponding to residues 1168-1203 of SARS-CoV-2 spike protein potato virus X FSTARAIVASNAVATNEDLSKIEAIWKDMKVPTDTMAQAA
WDLVRHCADVGSSAQTEM I DTGPYSN GI SRARLAAAI KEV
CTLRQFCMKYAPVVWNWMLTNNSPPANWQAQGFKPEH
KFAAFDFFNGVTN PAAI M PKEG LI RPPSEAEM NAAQTAAF
VKITKARAQSNDFASLDAAVTRGRITGTTTAEAVVTLPPP

Saccharomyces cerevisiae T4 foldon GYIPEAPRDGQAYVRKDGEVVVLLSTFL 18 domain Escherichia virus T4 tFliC AQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAK 19 Salmonella DDAAGQAIAN RFTAN IKG LTQASRNAN DG I SIAQTTEGALN
typhimurium El NNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEID
RVSGQTQFNGVKVLAQDNTLTIQVGANDGETI DIDLKQI NS
QTLGLDTLNVQQKYKVSDTAVKMSYTDNNGKTIDGGLAV
KVGDDYYSATQNKDGSI S I NTTKYTADDGTSKTALN KLGG
ADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTE
NPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNN
LTSARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQV
PQNVLSLLR
Linker GSGA 20 HA leader M NTQI LVFALIAI I PTNADKI 21 sequence Influenza virus IgE leader MDVVTWILFLVAAATRVHS 22 sequence human Date Recue/Date Received 2022-04-29 Construct #15 MNTQI LVFALIAI I PTNADKIGSGARVQPTESIVRFPN ITN LC 23 (Component 1) PFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFST
FKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG
KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF
RKSN LKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYG
FQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKGGGSG
GGSGGAGSRMKQI EDKI EEI LSKIYH I EN EIARIKKLIGERTS
GGSGGTGGSGGTGGAAAGGSGG DISG I NASVVNIQKEID
RLNEVAKN LNESLI DLQELGNSDYKDDDDKDYKDDDDKD
YKDDDDK
Construct #16 MNTQI LVFALIAI I PTNADKIGSGARVQPTESIVRFPN ITN LC 24 (Component 1) PFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFST
FKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG
KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF
RKSN LKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYG
FQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKGSAAAG
PG PM SAPASTTQATG STTSTTTKTAGATPATASG LFTI PD
GDFFSTARAIVASNAVATNEDLSKI EAIWKDMKVPTDTMA
QAAWD LVRHCADVGSSAQTEM I DTG PYSNG I SRARLAAAI
KEVCTLRQFCMKYAPVVWNWMLTNNSPPANWQAQGFK
PEHKFAAFDFFNGVTN PAAI M PKEG LI RPPSEAEM NAAQT
AAFVKITKARAQSN DFASLDAAVTRGRITGTTTAEAVVTLP
PPGNSDYKDDDDKDYKDDDDKDYKDDDDK
Construct #39 MNTQI LVFALIAI I PTNADKIGSGARVQPTESIVRFPN ITN LC 25 (Component 1) PFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFST
FKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG
KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF
RKSN LKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYG
FQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKGSAAAG
PG PM SAPASTTQATG STTSTTTKTAGATPATASG LFTI PD
GDFFSTARAIVASNAVATNEDLSKI EAIWKDMKVPTDTMA
QAAWD LVRHCADVGSSAQTEM I DTG PYSNG I SRARLAAAI
KEVCTLRQFCMKYAPVVWNWMLTNNSPPANWQAQGFK
PEHKFAAFDFFNGVTN PAAI M PKEG LI RPPSEAEM NAAQT
AAFVKITKARAQSN DFASLDAAVTRGRITGTTTAEAVVTLP
PP
Construct #39D MNTQI LVFALIAI I PTNADKIGSGARVQPTESIVRFPN ITN LC 26 (Component 1) PFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFST
FKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG
KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLF
RKSN LKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYG
FQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKGSAAAG
PG PM SAPASTTQATGSTTSTTTKTAGATPATASG LFTI PD
GDFFSTARAIVASNAVATNEDLSKI EAIWKDMKVPTDTMA
QAAWD LVRHCADVGSSAQTEM I DTG PYSNG I SRARLAAAI
KEVCTLRQFCMKYAPVVWNWMLTNNSPPANWQAQGFK
PEHKFAAFDFFNGVTN PAAI M PKEG LI RPPSEAEM NAAQT
AAFVKITKARAQSN DFASLDAAVTRGRITGTTTAEAVVTLP
PP
Construct #40 MNTQI LVFALIAI I PTNADKIGSGARVQPTESIVRFPN ITN LC 27 (Component 1) PFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFST

Date Recue/Date Received 2022-04-29 FKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG
KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF
RKSN LKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYG
FQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKGSAAAG
GSGGD I SG INASVVN IQKE ID RLN EVAKN LN ESLIDLQELG
NSGSGAGPGPMSAPASTTQATGSTTSTTTKTAGATPATA
SGLFTIPDGDFFSTARAIVASNAVATN EDLSKI EAIWKD MK
VPTDTMAQAAWDLVRH CADVGSSAQTEM I DTG PYSNG I S
RARLAAAIKEVCTLRQFCMKYAPVVWNWMLTN NSPPAN
WQAQGFKPEHKFAAFDFFNGVTNPAAI MPKEG LI RPPS EA
EMNAAQTAAFVKITKARAQSNDFASLDAAVTRGRITGTTT
AEAVVTLPPP
Construct #40D M NTQI LVFALIAI I PTNADKIGSGARVQPTESIVRFPN ITN LC 28 (Component 1) PFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFST
FKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG
KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLF
RKSN LKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYG
FQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKGSAAAG
GSGGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG
NSGSGAGPGPMSAPASTTQATGSTTSTTTKTAGATPATA
SGLFTIPDGDFFSTARAIVASNAVATN EDLSKI EAIWKD MK
VPTDTMAQAAWDLVRH CADVGSSAQTEM I DTG PYSNG I S
RARLAAAIKEVCTLRQFCMKYAPVVWNWMLTN NSPPAN
WQAQGFKPEHKFAAFDFFNGVTNPAAI MPKEG LI RPPS EA
EMNAAQTAAFVKITKARAQSNDFASLDAAVTRGRITGTTT
AEAVVTLPPP

human EELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEI
HFVTKCAFQPPPSCLRFVQTN I SRLLQETSEQLVALKPWIT
RQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQP
dHLX GELEELLKHLKELLKGPRKGELEELLKHLKELLK 30 Artificial ORF1_3619- IAMSAFAMMFVKHKH 31 SARS CoV-2 ORF1_3794- FGLFCLLNRYFRLTLGVYDY 32 SARS CoV-2 ORF 1_4028- AMQTMLFTMLRKLDN 33 SARS CoV-2 Rd Rp_330-344 VRKIFVDGVPFVVST 34 SARS CoV-2 RdRp_335-349 VDGVPFVVSTGYHFR 35 SARS CoV-2 RdRp_370-384 ELLVYAADPAMHAAS 36 SARS CoV-2 Date Recue/Date Received 2022-04-29 RdRp_540-559 SARS CoV-2 RdRp_575-589 LLKSIAATRGATVVI 38 SARS CoV-2 RdRp_650-664 HRFYRLANECAQVLS 39 SARS CoV-2 RdRp_745-759 FYAYLRKHFSMMILS 40 SARS CoV-2 Rd Rp_750-764 RKH FS MMI LS DDAVV 41 SARS CoV-2 RdRp_785-799 VLYYQNNVFMSEAKC 42 SARS CoV-2 RdRp_855-869 MIERFVSLAIDAYPL 43 SARS CoV-2 H_141-155 TEETFKLSYGIATVR 44 SARS CoV-2 H_206-220 GDAVVYRGTTTYKLN 45 SARS CoV-2 H_211-225 YRGTTTYKLNVGDYF 46 SARS CoV-2 H_221-235 VGDYFVLTSHTVMPL 47 SARS CoV-2 H_286-300 TGKSHFAIGLALYYP 48 SARS CoV-2 H_351-365 TLEQYVFCTVNALPE 49 SARS CoV-2 H_556-570 CNVNRFNVAITRAKV 50 SARS CoV-2 USE_224-241 RYKLEGYAFEHIVYGDFS 51 SARS CoV-2 USE_243-259 SQLGGLHLLIGLAKRFK 52 SARS CoV-2 USE_294-308 VI DLLLDDFVEI I KS 53 SARS CoV-2 USE_324-338 YTEISFMLWCKDGHV 54 SARS CoV-2 S_553-570 TESNKKFLPFQQFGRDIA 55 SARS CoV-2 Date Recue/Date Received 2022-04-29 S_809-826 PSKPSKRSFIEDLLFNKV 56 SARS CoV-2 S_863-877 PLLTDEMIAQYTSAL 57 SARS CoV-2 S_265-279 YYVGYLQPRTFLLKY 58 SARS CoV-2 S_814-828 KRSFIEDLLFNKVTL 59 SARS CoV-2 S_902-916 MAYRFNGIGVTQNVL 60 SARS CoV-2 S_912-926 TQNVLYENQKLIANQ 61 SARS CoV-2 S_992-1006 QIDRLITGRLQSLQT 62 SARS CoV-2 S_1211-1225 KWPVVYIWLGFIAGLI 63 SARS CoV-2 S_462-476 KPFERDISTEIYQAG 64 SARS CoV-2 S_507-521 PYRVVVLSFELLHAP 65 SARS CoV-2 M_44-59 RFLYIIKLIFLWLLWP 66 SARS CoV-2 M_91-110 MWLSYFIASFRLFARTRSMW 67 SARS CoV-2 N_216-230 DAALALLLLDRLNQL 68 SARS CoV-2 N_301-315 WPQIAQFAPSASAFF 69 SARS CoV-2 N_357-371 IDAYKTFPPTEPKKD 70 SARS CoV-2 N_104-118 LSPRVVYFYYLGTGPE 71 SARS CoV-2 orf3a_137-150 NPLLYDANYFLCWH 72 SARS CoV-2 orf3a_206-220 YFTSDYYQLYSTQLS 73 SARS CoV-2 Construct #14 (Component 1) AWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLC
FTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG
CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEI

Date Recue/Date Received 2022-04-29 YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV
VVLSFELLHAPGGGSGGGSGGAGSRMKQIEDKIEEILSKIY
HIENEIARIKKLIGERTSGGSGGTGGSGGTGGAAAGGSGG
DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGNSDYK
DDDDKDYKDDDDKDYKDDDDK
Construct #10 MDVVTWILFLVAAATRVHSGNITNLCPFGEVFNATRFASVY 75 (Combined AWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLC
Components 1 FTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG
and 2) CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEI
YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV
VVLSFELLHAPGGGSGGGSGGAGSRMKQIEDKIEEILSKIY
HI EN EIARIKKLIGERTSGGSGGTGGSGGTGGSADLGKKL
LEAARAGQDDEVTQNVLYENQKLIANQPLHLAAWEGHLGI
VEVLLKNGQI DRLITGRLQSLQTGHTPLHLAAYTGHLEIVE
VLLKNGMWLSYFIASFRLFARTRSMWGTAPLHLAAMWGH
LEIVEVLLKNGKWPVVYIWLGFIAGLITPKDLARDNGNEWIR
ELLEKAERKLKDLDRKLLEAARAGHRDEVEDLIKNGKRSFI
EDLLFNKVTLGFTPLHLAAWEGHLGIVEVLLKNGRFLYIIKL
I FLWLLWPGHTPLHLAAYTGHLEIVEVLLKNGMAYRFNGIG
VTQNVLGTAPLHLAAMWGHLEIVEVLLKNGTRGCSGLTSI
TFPNSLTGKTPKDLARDNGNEWIRELLEKAERKLKDLDRK
LLEAARAGHRDEVEDLIKNGGESAFKGCSGLKSITGFTPL
HLAAWEGHLGIVEVLLKNGAMQTMLFTMLRKLDNGHTPL
HLAAYTGHLEIVEVLLKNGALSGCTGLTSITIPNAPLHLAAM
WGHLEIVEVLLKNGALSGCTGLTSITIPNTPFDLAIDNGN E
DIAEVLQKGSAAAGGSGG DI SGI NASVVN IQKEIDRLN EVA
KNLNESLIDLQELGNSDYKDDDDKDYKDDDDKDYKDDDD
K
Construct #11 MDVVTWILFLVAAATRVHSGNITNLCPFGEVFNATRFASVY 76 (Combined AWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLC
Components 1 FTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG
and 2) CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEI
YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV
VVLSFELLHAPGGGSGGGSGGAGSRMKQIEDKIEEILSKIY
HI EN EIARIKKLIGERTSGGSGGTGGSGGTGGSADLGKKL
LEAARAGQDDEVTQNVLYENQKLIANQPLHLAAWEGHLGI
VEVLLKNGQI DRLITGRLQSLQTGHTPLHLAAYTGHLEIVE
VLLKNGMWLSYFIASFRLFARTRSMWGTAPLHLAAMWGH
LEIVEVLLKNGKWPVVYIWLGFIAGLITPKDLARDNGNEWIR
ELLEKAERKLKDLDRKLLEAARAGHRDEVEDLIKNGKRSFI
EDLLFNKVTLGFTPLHLAAWEGHLGIVEVLLKNGRFLYIIKL
I FLWLLWPGHTPLHLAAYTGHLEIVEVLLKNGMAYRFNGIG
VTQNVLGTAPLHLAAMWGHLEIVEVLLKNGTRAQVINTNS
LSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAI
ANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQR
VRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQ
FNGVKVLAQDNTLTIQVGAN DGETI D I D LKQI N SQTLG LDT
LNVQQKYKVSDTAVKMSYTDNNGKTIDGGLAVKVGDDYY
SATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEV
VSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKID
AALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSR
I EDSDYATEVSNMSRAQI LQQAGTSVLAQANQVPQNVLSL
LRAAAGGSGGD ISG I NASVVN IQKEIDRLNEVAKN LN ES LI
DLQELGNSDYKDDDDKDYKDDDDKDYKDDDDK

Date Recue/Date Received 2022-04-29 Trimeric RBD M NTQI LVFALIAI I PTNADKI GSGARVQPTESIVRF PN ITN LC 77 PFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFST
FKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG
KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF
RKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYG
FQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKGSAAAG
PGPGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGGGSYQT
QTNSPRRARSVASQSIIAGSGARVQPTESIVRFPNITNLCP
FGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTF
KCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGK
IADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFR
KSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGF
QPTNGVGYQPYRVVVLSFELLHAPATVCGPKKGSAAAGP
GPGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGGGSYQTQ
TNSPRRARSVASQSIIAGSGARVQPTESIVRFPNITNLCPF
GEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFK
CYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKI
ADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFR
KSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGF
QPTNGVGYQPYRVVVLSFELLHAPATVCGPKKGSAAAGP
GPGYIPEAPRDGQAYVRKDGEVVVLLSTFL
NC2 hetero- GKEASEQRIRELCGGMISEQIAQLAAHLRKPLAPG 78 trimerization domain 1 Human NC2 hetero- G RDATDQH IVDVALKM LQEQLAEVAVSAKREALG 79 trimerization domain 2 Human NC2 hetero- GRAPTDQHIKQVCMRVIQEHFAEMAASLKRPDSG 80 trimerization domain 3 Human furin cleavage YQTQTNSPRRARSVASQSIIA 81 site Human C1r-like VCGRPVTPIAQNQTTLGSSRAKLGNFPWQ 82 protease cleavage site Human Construct #19 MDVVTWILFLVAAATRVHSGDLGKKLLEAARAGQDDEVTQ 83 NVLYENQKLIANQPLHLAAWEGHLGIVEVLLKNGQI DRLIT
GRLQSLQTGHTPLHLAAYTGHLEIVEVLLKNGMWLSYFIA
SFRLFARTRSMWGTAPLHLAAMWGHLEIVEVLLKNGKWP
VVYIWLGFIAGLITPKDLARDNGNEWIRELLEKAERKLKDLD
RKLLEAARAGHRDEVEDLIKNGKRSFIEDLLFNKVTLGFTP
LHLAAWEGHLGIVEVLLKNGRFLYIIKLIFLWLLWPGHTPLH
LAAYTGH LEIVEVLLKNG MAYRFNG I GVTQNVLGTAPLH LA
AMWGHLEIVEVLLKNGTRGCSGLTSITFPNSLTGKTPKDL
ARDNGNEWIRELLEKAERKLKDLDRKLLEAARAGHRDEV
EDLIKNGGESAFKGCSGLKSITGFTPLHLAAWEGHLGIVEV

Date Recue/Date Received 2022-04-29 LLKNGAMQTMLFTMLRKLDNGHTPLH LAAYTGH LE IVEVL
LKNGALSGCTGLTSITIPNAPLHLAAMWGHLEIVEVLLKNG
ALSGCTGLTSITI PNTPFDLAI DNG N ED IAEVLQKGSAAAG
NSGSGDYKDDDDKDYKDDDDKDYKDDDDK
Construct #20 MDVVTWILFLVAAATRVHSGTQDCSFQHSPISSDFAVKIRE 84 LSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERL
KTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLP
PPWSPRPLEATAPTAPQPGAANGASGGGGSGGGGTPLG
DTTHTSGGELEELLKHLKELLKGPRKGELEELLKHLKELLK
GLSGALDLGKKLLEAARAGQDDEVTQNVLYENQKLIANQP
LHLAAWEGHLGIVEVLLKNGQIDRLITGRLQSLQTGHTPLH
LAAYTGHLEIVEVLLKNGMWLSYFIASFRLFARTRSMWGT
APLHLAAMWGHLEIVEVLLKNGKWPWYIWLGFIAGLITPK
DLARDNGNEWIRELLEKAERKLKDLDRKLLEAARAGHRD
EVEDLIKNGKRSFIEDLLFNKVTLGFTPLHLAAWEGHLGIV
EVLLKNGRFLYIIKLIFLWLLWPGHTPLHLAAYTGHLEIVEV
LLKNGMAYRFNGIGVTQNVLGTAPLHLAAMWGHLEIVEVL
LKNGTRGCSGLTSITFPNSLTGKTPKDLARDNGNEWIREL
LEKAERKLKDLDRKLLEAARAGHRDEVEDLIKNGGESAFK
GCSGLKSITGFTPLHLAAWEGHLGIVEVLLKNGAMQTMLF
TMLRKLDNGHTPLHLAAYTGHLEIVEVLLKNGALSGCTGL
TSITIPNAPLHLAAMWGHLEIVEVLLKNGALSGCTGLTSITI
PNTPFDLAIDNGNEDIAEVLQKGSAAAGNSGSGDYKDDD
DKDYKDDDDKDYKDDDDK
Construct #26 MDLGKKLLEAARAGQDDEVTQNVLYENQKLIANQPLHLAA 85 WEGHLGIVEVLLKNGQIDRLITGRLQSLQTGHTPLHLAAYT
GHLEIVEVLLKNGMWLSYFIASFRLFARTRSMWGTAPLHL
AAMWGHLEIVEVLLKNGKWPWYIWLGFIAGLITPKDLARD
NGNEWIRELLEKAERKLKDLDRKLLEAARAGHRDEVEDLI
KNGKRSFIEDLLFNKVTLGFTPLHLAAWEGHLGIVEVLLKN
GRFLYIIKLIFLWLLWPGHTPLHLAAYTGHLEIVEVLLKNGM
AYRFNGIGVTQNVLGTAPLHLAAMWGHLEIVEVLLKNGNS
DYKDDDDKDYKDDDDKDYKDDDDK
Construct #24 MNTQILVFALIAIIPTNADKIGSGADLGKKLLEAARAGQDDE 86 VTQNVLYENQKLIANQPLHLAAWEGHLGIVEVLLKNGQID
RLITGRLQSLQTGHTPLHLAAYTGHLEIVEVLLKNGMWLS
YFIASFRLFARTRSMWGTAPLHLAAMWGHLEIVEVLLKNG
KWPWYIWLGFIAGLITPKDLARDNGNEWIRELLEKAERKL
KDLDRKLLEAARAGHRDEVEDLIKNGKRSFIEDLLFNKVTL
GFTPLHLAAWEGHLGIVEVLLKNGRFLYIIKLIFLWLLWPG
HTPLHLAAYTGHLEIVEVLLKNGMAYRFNGIGVTQNVLGT
APLHLAAMWGHLEIVEVLLKNGNSDYKDDDDKDYKDDDD
KDYKDDDDK
Construct #28 MDVVTWILFLVAAATRVHSGTQDCSFQHSPISSDFAVKIRE 87 LSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERL
KTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLP
PPWSPRPLEATAPTAPQPGAANGASGGGGSGGGGTPLG
DTTHTSGGELEELLKHLKELLKGPRKGELEELLKHLKELLK
GLSGALDLGKKLLEAARAGQDDEVTQNVLYENQKLIANQP

Date Recue/Date Received 2022-04-29 LHLAAWEGHLGIVEVLLKNGQIDRLITGRLQSLQTGHTPLH
LAAYTGHLEIVEVLLKNGMWLSYFIASFRLFARTRSMWGT
APLHLAAMWGHLEIVEVLLKNGKWPWYIWLGFIAGLITPK
DLARDNGN EWIRELLEKAERKLKDLDRKLLEAARAGH RD
EVEDLIKNGKRSFIEDLLFNKVTLGFTPLHLAAWEGHLGIV
EVLLKNGRFLYIIKLIFLWLLWPGHTPLHLAAYTGHLEIVEV
LLKNGMAYRFNGIGVTQNVLGTAPLHLAAMWGHLEIVEVL
LKNGNSGSGDYKDDDDKDYKDDDDKDYKDDDDK
Construct #50 MDVVTWILFLVAAATRVHSGTQDCSFQHSPISSDFAVKIRE 88 LSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERL
KTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLP
PPWSPRPLEATAPTAPQPGAANGASGGGGSGGGGTPLG
DTTHTSGGELEELLKHLKELLKGPRKGELEELLKHLKELLK
GLSGALDLGKKLLEAARAGQDDEVTQNVLYENQKLIANQP
LHLAAWEGHLGIVEVLLKNGQIDRLITGRLQSLQTGHTPLH
LAAYTGHLEIVEVLLKNGMWLSYFIASFRLFARTRSMWGT
APLHLAAMWGHLEIVEVLLKNGKWPWYIWLGFIAGLITPK
DLARDNGN EWIRELLEKAERKLKDLDRKLLEAARAGH RD
EVEDLIKNGKRSFIEDLLFNKVTLGFTPLHLAAWEGHLGIV
EVLLKNGRFLYIIKLIFLWLLWPGHTPLHLAAYTGHLEIVEV
LLKNGMAYRFNGIGVTQNVLGTAPLHLAAMWGHLEIVEVL
LKNGTRSGSSALSGCTGLTSITIPNTGSSGESAFKGCSGL
KSITAAAGNSGSGDYKDDDDKDYKDDDDKDYKDDDDK
Construct #22 MNTQILVFALIAIIPTNADKIGSGADLGKKLLEAARAGQDDE 89 VTQNVLYENQKLIANQPLHLAAWEGHLGIVEVLLKNGQID
RLITGRLQSLQTGHTPLHLAAYTGHLEIVEVLLKNGMWLS
YFIASFRLFARTRSMWGTAPLHLAAMWGHLEIVEVLLKNG
KWPWYIWLGFIAGLITPKDLARDNGNEWIRELLEKAERKL
KDLDRKLLEAARAGHRDEVEDLIKNGKRSFIEDLLFNKVTL
GFTPLHLAAWEGHLGIVEVLLKNGRFLYIIKLIFLWLLWPG
HTPLHLAAYTGHLEIVEVLLKNGMAYRFNGIGVTQNVLGT
APLHLAAMWGHLEIVEVLLKNGAAAGPGPMSAPASTTQA
TGSTTSTTTKTAGATPATASGLFTIPDGDFFSTARAIVASN
AVATNEDLSKIEAIWKDMKVPTDTMAQAAWDLVRHCADV
GSSAQTEMIDTGPYSNGISRARLAAAIKEVCTLRQFCMKY
APVVWNWMLTNNSPPANWQAQGFKPEHKFAAFDFFNGV
TNPAAIMPKEGLIRPPSEAEMNAAQTAAFVKITKARAQSN
DFASLDAAVTRGRITGTTTAEAVVTLPPPGNSDYKDDDDK
DYKDDDDKDYKDDDDK
Construct #27 MDLGKKLLEAARAGQDDEVRILLANGTPLHLAAWEGHLGI 90 VEVLLKNGPYRVVVLSFELLHAPGHTPLHLAAYTGHLEIVE
VLLKNGGDAVVYRGTTTYKLNGTAPLHLAAMWGHLEIVEV
LLKNGKPFERDISTEIYQAGTPKDLARDNGNVLNDILSRLD
PPEAEVSFKELLVYAADPAMHAASYVTQQLIRAAEIRASA
NLAATKMSECVGHRDEVEDLIKNGSQLGGLHLLIGLAKRF
KGFTPLHLAAWEGHLGIVEVLLKNGVIDLLLDDFVEIIKSGH
TPLHLAAYTGHLEIVEVLLKNGTLEQYVFCTVNALPEGTAP
LHLAAMWGHLEIVEVLLKNGFGLFCLLNRYFRLTLGVYDY
GKTPKDLARDNGNTERLKLFAAETLKATEETFKLSYGIATV
RGHRDEVEDLIKNGMIERFVSLAIDAYPLGFTPLHLAAWE
GHLGIVEVLLKNGFYAYLRKHFSMMILSGHTPLHLAAYTG
HLEIVEVLLKNGTQMNLKYAISAKNRARTVAGAPLHLAAM

Date Recue/Date Received 2022-04-29 WGH LEIVEVLLKNGVRKI FVDGVPFVVSTTPKDLARD NG N
SDYKDDDDKDYKDDDDKDYKDDDDK
Construct #25 MNTQILVFALIAIIPTNADKIGSGADLGKKLLEAARAGQDDE 91 VRILLANGTPLHLAAWEGHLGIVEVLLKNGPYRVVVLSFEL
LHAPGHTPLHLAAYTGHLEIVEVLLKNGGDAVVYRGTTTY
KLNGTAPLHLAAMWGHLEIVEVLLKNGKPFERDISTEIYQA
GTPKDLARDNGNVLNDILSRLDPPEAEVSFKELLVYAADP
AMHAASYVTQQLI RAAEI RASAN LAATKMS ECVGH RD EVE
DLIKNGSQLGGLHLLIGLAKRFKGFTPLHLAAWEGHLGIVE
VLLKNGVIDLLLDDFVEIIKSGHTPLHLAAYTGHLEIVEVLLK
NGTLEQYVFCTVNALPEGTAPLHLAAMWGHLEIVEVLLKN
GFGLFCLLNRYFRLTLGVYDYGKTPKDLARDNGNTERLKL
FAAETLKATEETFKLSYGIATVRGHRDEVEDLIKNGMIERF
VSLAIDAYPLGFTPLHLAAWEGHLGIVEVLLKNGFYAYLRK
HFSMMILSGHTPLHLAAYTGHLEIVEVLLKNGTQMNLKYAI
SAKNRARTVAGAPLHLAAMWGHLEIVEVLLKNGVRKIFVD
GVPFVVSTTPKDLARDNGNSDYKDDDDKDYKDDDDKDY
KDDDDK
Construct #29 M DVVTWI LFLVAAATRVHSGTQDCSFQHS PI SSDFAVKI RE 92 LSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERL
KTVAGSKMQGLLERVNTE I H FVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLP
PPWSPRPLEATAPTAPQPGAANGASGGGGSGGGGTPLG
DTTHTSGGELEELLKHLKELLKGPRKGELEELLKHLKELLK
GLSGALDLGKKLLEAARAGQDDEVRILLANGTPLHLAAWE
GHLGIVEVLLKNGPYRVVVLSFELLHAPGHTPLHLAAYTG
HLEIVEVLLKNGGDAVVYRGTTTYKLNGTAPLHLAAMWG
HLEIVEVLLKNGKPFERDISTEIYQAGTPKDLARDNGNVLN
DI LSRLDPPEAEVSFKE LLVYAADPAM HAASYVTQQLI RAA
El RASANLAATKMSECVGHRDEVEDLIKNGSQLGGLHLLI
GLAKRFKGFTPLHLAAWEGHLGIVEVLLKNGVIDLLLDDFV
El IKSGHTPLHLAAYTGHLEIVEVLLKNGTLEQYVFCTVNAL
PEGTAPLHLAAMWGHLEIVEVLLKNGFGLFCLLNRYFRLT
LGVYDYGKTPKDLARDNGNTERLKLFAAETLKATEETFKL
SYGIATVRGHRDEVEDLIKNGMIERFVSLAIDAYPLGFTPL
HLAAWEGHLGIVEVLLKNGFYAYLRKHFSMMILSGHTPLH
LAAYTGHLEIVEVLLKNGTQMNLKYAISAKNRARTVAGAPL
HLAAMWGHLEIVEVLLKNGVRKIFVDGVPFVVSTTPKDLA
RDNGNSDYKDDDDKDYKDDDDKDYKDDDDK
Construct #9 MDVVTWI LFLVAAATRVHSGTQDCSFQHSPISSDFAVKI RE 93 LSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERL
KTVAGSKMQGLLERVNTE I H FVTKCAFQPPPSCLRFVQTN
ISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLP
PPWSPRPLEATAPTAPQPGAANGASGGGGSGGGGTPLG
DTTHTSGGELEELLKHLKELLKGPRKGELEELLKHLKELLK
GLSGALDLGKKLLEAARAGQDDEVRILLANGTPLHLAAWE
GHLGIVEVLLKNGPYRVVVLSFELLHAPGHTPLHLAAYTG
HLEIVEVLLKNGGDAVVYRGTTTYKLNGTAPLHLAAMWG
HLEIVEVLLKNGKPFERDISTEIYQAGTPKDLARDNGNVLN
DI LSRLDPPEAEVSFKE LLVYAADPAM HAASYVTQQLI RAA
El RASANLAATKMSECVGHRDEVEDLIKNGSQLGGLHLLI
GLAKRFKGFTPLHLAAWEGHLGIVEVLLKNGVIDLLLDDFV
El IKSGHTPLHLAAYTGHLEIVEVLLKNGTLEQYVFCTVNAL

Date Recue/Date Received 2022-04-29 PEGTAPLHLAAMWGHLEIVEVLLKNGFGLFCLLNRYFRLT
LGVYDYGKTPKDLARDNGNTERLKLFAAETLKATEETFKL
SYGIATVRGHRDEVEDLIKNGMIERFVSLAIDAYPLGFTPL
HLAAWEGHLGIVEVLLKNGFYAYLRKHFSMMILSGHTPLH
LAAYTGHLEIVEVLLKNGTQMNLKYAISAKNRARTVAGAPL
HLAAMWGHLEIVEVLLKNGVRKIFVDGVPFVVSTTPKDLA
RDNTRSGSSALSGCTGLTSITIPNTGSSGESAFKGCSGLK
SITAAAGNSGSGDYKDDDDKDYKDDDDKDYKDDDDK
Construct #23 MNTQILVFALIAII PTNADKIGSGADLGKKLLEAARAGQDDE 94 VRILLANGTPLHLAAWEGHLGIVEVLLKNGPYRVVVLSFEL
LHAPGHTPLHLAAYTGHLEIVEVLLKNGGDAVVYRGTTTY
KLNGTAPLHLAAMWGHLEIVEVLLKNGKPFERDISTEIYQA
GTPKDLARDNGNVLNDILSRLDPPEAEVSFKELLVYAADP
AMHAASYVTQQLIRAAEIRASANLAATKMSECVGHRDEVE
DLIKNGSQLGGLHLLIGLAKRFKGFTPLHLAAWEGHLGIVE
VLLKNGVIDLLLDDFVEIIKSGHTPLHLAAYTGHLEIVEVLLK
NGTLEQYVFCTVNALPEGTAPLHLAAMWGHLEIVEVLLKN
GFGLFCLLNRYFRLTLGVYDYGKTPKDLARDNGNTERLKL
FAAETLKATEETFKLSYGIATVRGHRDEVEDLIKNGMIERF
VSLAIDAYPLGFTPLHLAAWEGHLGIVEVLLKNGFYAYLRK
HFSMMILSGHTPLHLAAYTGHLEIVEVLLKNGTQMNLKYAI
SAKNRARTVAGAPLHLAAMWGHLEIVEVLLKNGVRKIFVD
GVPFVVSTTPKDLARDNAAAGPGPMSAPASTTQATGSTT
STTTKTAGATPATASGLFTIPDGDFFSTARAIVASNAVATN
EDLSKIEAIWKDMKVPTDTMAQAAWDLVRHCADVGSSAQ
TEMIDTGPYSNGISRARLAAAIKEVCTLRQFCMKYAPVVW
NWMLTNNSPPANWQAQGFKPEHKFAAFDFFNGVTNPAAI
MPKEGLIRPPSEAEMNAAQTAAFVKITKARAQSNDFASLD
AAVTRGRITGTTTAEAVVTLPPPGNSDYKDDDDKDYKDD
DDKDYKDDDDK
COV-X1 Formula I 95,109 COV-X2 Formula II 96,116 COV-X3 Formula III 97,129 COV-X4 Formula IV 98,137 COV-X5 Formula V 99, COV-X6 Formula VI 100, COV-X7 Formula VII 101, Linker EAAAK 102 Date Recue/Date Received 2022-04-29 Linker RVRR 103 Linker GGGS 104 Linker GPGPG 105 Linker HEYGAEALERAG 106 TLR2 agonist ALSGCTGLTSITIPNTGSSGESAFKGCSGLKSIT 107 peptides construct Construct #21 MDLGKKLLEAARAGQDDEVRILLANGTPLHLAAWEGHLGI 108 VEVLLKNGPYRVVVLSFELLHAPGHTPLHLAAYTGHLEIVE
VLLKNGGDAVVYRGTTTYKLNGTAPLHLAAMWGHLEIVEV
LLKNGKPFERDISTEIYQAGTPKDLARDNGNVLNDILSRLD
PPEAEVSFKELLVYAADPAMHAASYVTQQLIRAAEIRASA
NLAATKMSECVGHRDEVEDLIKNGSQLGGLHLLIGLAKRF
KGFTPLHLAAWEGHLGIVEVLLKNGVIDLLLDDFVEIIKSGH
TPLHLAAYTGHLEIVEVLLKNGTLEQYVFCTVNALPEGTAP
LHLAAMWGHLEIVEVLLKNGFGLFCLLNRYFRLTLGVYDY
GKTPKDLARDNGNTERLKLFAAETLKATEETFKLSYGIATV
RGHRDEVEDLIKNGMIERFVSLAIDAYPLGFTPLHLAAWE
GHLGIVEVLLKNGFYAYLRKHFSMMILSGHTPLHLAAYTG
HLEIVEVLLKNGTQMNLKYAISAKNRARTVAGAPLHLAAM
WGHLEIVEVLLKNGVRKIFVDGVPFVVSTTPKDLARDNAA
AGPGPMSAPASTTQATGSTTSTTTKTAGATPATASGLFTI
PDGDFFSTARAIVASNAVATNEDLSKIEAIWKDMKVPTDT
MAQAAWDLVRHCADVGSSAQTEMIDTGPYSNGISRARLA
AAIKEVCTLRQFCMKYAPVVWNWMLTNNSPPANWQAQG
FKPEHKFAAFDFFNGVTNPAAIMPKEGLIRPPSEAEMNAA
QTAAFVKITKARAQSNDFASLDAAVTRGRITGTTTAEAVVT
LPPPGNSDYKDDDDKDYKDDDDKDYKDDDDK
TLR2 agonist ALSGCTGLTSITIPN 168 peptide ((Tannerella forsythia virulence factor BspA) TLR2 agonist GESAFKGCSGLKSIT 169 peptide (Tannerella forsythia virulence factor BspA) Short tail KDLARDN 170 Long tail FDLAIDNGNEDIAEVLQK 171 *H_ is helicase (nsp13) Wuhan-Hu-1 Date Recue/Date Received 2022-04-29 *USE_ is Uridylate-specific endoribonuclease (nsp15) Wuhan-Hu-1 *S_ is SARS-2 spike protein

[00260] While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[00261] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers and/or biomarker sequences (e.g.
protein and/or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely.

[00262] The scope of the claims should not be limited by the preferred embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole.

Date Recue/Date Received 2022-04-29

Claims (90)

CLAIMS:

1. A nucleic acid molecule comprising a polynucleotide, the polynucleotide encoding a polypeptide comprising:
a receptor-binding domain (RBD) of a spike (S) protein of a coronavirus, and one or more of:
a heptad repeat 2 (HR2) domain of the S protein;
a potato virus X capsid protein (PVXCP); and a trimerization domain.

2. The nucleic acid molecule of claim 1, wherein the coronavirus is a human beta coronavirus, optionally SARS-CoV-2 or a variant or mutant thereof.

3. The nucleic acid molecule of claim 2, wherein the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of any one of SEQ ID NOs: 2 to 14, or is or comprises the sequence of any one of SEQ ID NOs: 2 to 14.

4. The nucleic acid molecule of claim 2, wherein the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 2, or has or comprises the sequence of SEQ ID NOs: 2.

5. The nucleic acid molecule of any one of claims 1 to 4, wherein the polypeptide comprises the HR2 domain, the HR2 domain optionally having an amino acid sequence having the sequence of SEQ ID NO: 15 or at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID
NO: 15, or has or comprises the sequence of SEQ ID NO:15.

6. The nucleic acid molecule of any one of claims 1 to 5, wherein the polypeptide comprises the PVXCP, the PVXCP optionally having a sequence of SEQ ID NO: 16 or at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity to the sequence of SEQ ID NO: 16, or has or comprises the sequence of SEQ ID NO:16.

7. The nucleic acid molecule of any one of claims 1 to 6, wherein the polypeptide comprises the trimerization domain, optionally a GCN4 leucine zipper or a T4 foldon domain.

8. The nucleic acid of claim 7, wherein the GCN4 leucine zipper has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 17, or has or comprises the sequence of SEQ ID NO: 17.

9. The nucleic acid of claim 7, wherein the T4 foldon domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 18, or has or comprises the sequence of SEQ ID NO: 18.

10. The nucleic acid molecule of any one of claims 1 to 9, further comprising a truncated Salmonella typhimurium flagellin (tFliC), optionally having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 19, or having or comprising the sequence of SEQ ID NO:
19.

11. The nucleic acid molecule of any one of claims 1 to, further comprising a leader peptide.

12. The nucleic acid molecule of claim 11, wherein the leader peptide is an influenza virus hemagglutinin (HA) leader peptide, optionally having at least 80% sequence identity to the sequence of SEQ ID NO: 21, or having or comprising the sequence of SEQ ID
NO:
21.

13. The nucleic acid molecule of claim 11, wherein the leader peptide is a leader peptide of the human immunoglobulin E protein (IgE), optionally having at least 80%
sequence identity to the sequence of SEQ ID NO: 22, or having or comprising the sequence of SEQ ID NO: 22.

14. The nucleic acid molecule of any one of claims 1 to 13, wherein the polypeptide comprises one or more linkers, optionally wherein the one or more linker is or comprises AAY, GSGA (SEQ ID NO: 20), EAAAK (SEQ ID NO: 102), RVRR (SEQ ID
NO: 103), GGGS (SEQ ID NO: 104), GPGPG (SEQ ID NO: 105), HEYGAEALERAG
(SEQ ID NO: 106).

15. The nucleic acid molecule of any one of claims 11 to 13, wherein the polypeptide comprises a linker between the leader peptide and the RBD.

16. The nucleic acid molecule of any one of claims 1 to 15, wherein the polypeptide comprises the leader peptide, the RBD, the GCN4 leucine zipper and the HR2 domain (from N- to C- terminus).

17. The nucleic acid molecule of any one of claims 1 to 12, wherein the polypeptide has or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ
ID
NOs: 23 to 28.

18. The nucleic acid molecule of any one of claims 1 to 15, wherein the polypeptide comprises one or more additional RDBs and/or one or more additional trimerization domains.

19. The nucleic acid molecule of claim 18, wherein the polypeptide comprises three RBDs and three trimerization domains.

20. The nucleic acid molecule of claims 18 or 19, wherein the polypeptide comprises one or more cleavage sites, optionally wherein cleavage site is a furin cleavage site or a C1r-like protease cleavage site, and/or one or more linkers.

21. A nucleic acid molecule comprising a polynucleotide, the polynucleotide encoding a polypeptide comprising a plurality of antigenic coronavirus peptides interspersed in an ankyrin repeat scaffold, the ankyrin repeat scaffold comprising a plurality of ankyrin repeat motifs, optionally wherein the ankyrin repeat scaffold is a designed ankyrin repeat protein (DARPin).

22. The nucleic acid molecule of claim 21, wherein the polypeptide further comprises a leader peptide.

23. The nucleic acid molecule of claim 22, wherein the leader peptide is an influenza virus hemagglutinin (HA) leader peptide, optionally having at least 80% sequence identity to the sequence of SEQ ID NO: 21, or having or comprising the sequence of SEQ ID
NO:
21.

24. The nucleic acid molecule of claim 22, wherein the leader peptide is a leader peptide of the human immunoglobulin E protein (IgE), optionally having at least 80%
sequence identity to the sequence of SEQ ID NO: 22, or having or comprising the sequence of SEQ ID NO: 22.

25. The nucleic acid molecule of any one of claims 21 to 24, the polypeptide further comprising a Toll-Like receptor 2 (TLR2) agonist peptide, optionally having at least 80% sequence identity to the sequence of SEQ ID NO: 107, or having or comprising the sequence of SEQ ID NO: 107.

26. The nucleic acid molecule of any one of claims 21 to 25, the polypeptide further comprising a FMS-like tyrosine kinase 3 ligand (FLT3L), optionally having at least 80%
sequence identity to the sequence of SEQ ID NO: 29, or having or comprising the sequence of SEQ ID NO: 29.

27. The nucleic acid molecule of any one of claims 21 to 26, the polypeptide further comprising an a-helix dimerization domain (dHLX) or a CH3 dimerization domain, the dHLX optionally having at least 80% sequence identity to the sequence of SEQ
ID NO:
30, or having or comprising the sequence of SEQ ID NO: 30.

28. The nucleic acid molecule of any one of claims 21 to 26, the polypeptide further comprising a PVXCP, the PVXCP optionally having a sequence of SEQ ID NO: 16 or at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity to the sequence of SEQ ID NO: 16, or has or comprises the sequence of SEQ ID NO:16.

29. The nucleic acid molecule of any one of claims 21 to 28, wherein the polypeptide comprises a plurality of linkers flanking one or more of the antigenic coronavirus peptides, optionally wherein the linker is or comprises AAY, GSGA (SEQ ID NO:
20), EAAAK (SEQ ID NO: 102), RVRR (SEQ ID NO: 103), GGGS (SEQ ID NO: 104), GPGPG (SEQ ID NO: 105), HEYGAEALERAG (SEQ ID NO: 106).

30. The nucleic acid molecule of any one of claims 21 to 29, wherein the polypeptide comprises non-structural antigenic coronavirus peptides.

31. The nucleic acid molecule of claim 30, wherein each of the non-structural coronavirus peptides has or comprises an amino acid sequence selected from Table 1 or 2 or has at least 80% sequence identity to the amino acid sequence selected from Table 1 or 2.

32. The nucleic acid molecule of any one of claims 21 to 31, wherein the polypeptide comprises structural antigenic coronavirus peptides.

33. The nucleic acid molecule of claim 32, wherein each of the structural coronavirus peptides has or comprises amino acid sequence selected from Table 1 or 3 or at least 80% sequence identity to the amino acid sequence selected from Table 1 or 3.

34. The nucleic acid molecule of any one of claims 21 to 30, wherein the antigenic coronavirus peptides are epitopes of replicative proteins or capsid proteins.

35. The nucleic acid molecule of any one of claims 21 to 29, wherein the polypeptide has or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ
ID
NOs: 83 to 94, 108 or Formulas I-VII, including any one or more of SEQ ID NOs:

to101 and 109 to 167, 170 and/or 171.

36. The nucleic acid molecule of any one of claims 1 to 35, wherein the nucleic acid molecule is associated with a lipid, optionally wherein the nucleic acid molecule or vector is encapsulated in a liposome, interspersed within a liposome lipid bilayer or attached to a liposome.

37. A vector comprising the nucleic acid molecule of any one of claims 1 to 20.

38. A vector comprising the nucleic acid molecule of any one of claims 21 to 36.

39. The vector of claim 37 or 38, wherein the vector is a plasmid vector.

40. The vector of any one of claims 37 to 39, wherein the vector is associated with a lipid, optionally wherein the nucleic acid molecule or vector is encapsulated in a liposome, interspersed within a liposome lipid bilayer or attached to a liposome.

41. A polypeptide comprising:
a receptor-binding domain (RBD) of a spike (S) protein of a coronavirus, and one or more of:
a heptad repeat 2 (HR2) domain of the S protein;

a potato virus X capsid protein (PVXCP); and a trimerization domain.

42. The polypeptide of claim 41, wherein the coronavirus is a human beta coronavirus, optionally SARS-CoV-2 or a variant or mutant thereof.

43. The polypeptide of claim 42, wherein the RBD has an amino acid sequence having at least has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%
or at least 99% sequence identity to the sequence of any one of SEQ ID NOs: 2 to 14, or is or comprises the sequence of any one of SEQ ID NOs: 2 to 14.

44. The polypeptide of claim 42, wherein the RBD has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 2, or has or comprises the sequence of SEQ ID NOs: 2.

45. The polypeptide of any one of claims 41 to 44, wherein the polypeptide comprises the HR2 domain, the HR2 domain optionally having an amino acid sequence having the sequence of SEQ ID NO: 15 or at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO:
15, or has or comprises the sequence of SEQ ID NO:15.

46. The polypeptide of any one of claims 41 to 45, wherein the polypeptide comprises the PVXCP, the PVXCP optionally having a sequence of SEQ ID NO: 16 or at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity to the sequence of SEQ ID NO: 16, or has or comprises the sequence of SEQ ID
NO:16.

47. The polypeptide of any one of claims 41 to 46, wherein the polypeptide comprises the trimerization domain, optionally a GCN4 leucine zipper or a T4 foldon domain.

48. The polypeptide acid of claim 47, wherein the GCN4 leucine zipper has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity to the sequence of SEQ ID NO: 17, or has or comprises the sequence of SEQ ID
NO:
17.

49. The polypeptide acid of claim 47, wherein the T4 foldon domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 18, or has or comprises the sequence of SEQ ID
NO:
18.

50. The polypeptide of any one of claims 41 to 49, further comprising a truncated Salmonella typhimurium flagellin (tFliC), optionally having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO: 19, or having or comprising the sequence of SEQ ID NO:
19.

51. The polypeptide of any one of claims 41 to 50, further comprising a leader peptide.

52. The polypeptide of claim 51, wherein the leader peptide is an influenza virus hemagglutinin (HA) leader peptide, optionally having at least 80% sequence identity to the sequence of SEQ ID NO: 21, or having or comprising the sequence of SEQ ID
NO:
21.

53. The polypeptide of claim 51, wherein the leader peptide is a leader peptide of the human immunoglobulin E protein (IgE), optionally having at least 80% sequence identity to the sequence of SEQ ID NO: 22, or having or comprising the sequence of SEQ ID NO: 22.

54. The polypeptide of any one of claims 41 to 53, wherein the polypeptide comprises one or more linkers, optionally wherein the one or more linker is or comprises AAY, GSGA
(SEQ ID NO: 20), EAAAK (SEQ ID NO: 102), RVRR (SEQ ID NO: 103), GGGS (SEQ
ID NO: 104), GPGPG (SEQ ID NO: 105), HEYGAEALERAG (SEQ ID NO: 106).

55. The polypeptide of any one of claims 51 to 53, wherein the polypeptide comprises a linker between the leader peptide and the RBD.

56. The polypeptide of any one of claims 41 to 55, wherein the polypeptide comprises the leader peptide, the RBD, the GCN4 leucine zipper and the HR2 domain (from N-to C-terminus).

57. The polypeptide of any one of claims 41 to 55, wherein the polypeptide has or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ
ID
NOs: 23 to 28.

58. The polypeptide of any one of claims 41 to 55, wherein the polypeptide comprises one or more additional RDBs and/or one or more additional trimerization domains.

59. The polypeptide of claim 58, wherein the polypeptide comprises three RBDs and three trimerization domains.

60. The polypeptide of claims 58 or 59, wherein the polypeptide comprises one or more cleavage sites, optionally wherein cleavage site is a furin cleavage site or a C1r-like protease cleavage site, and/or one or more linkers

61. A polypeptide comprising a polypeptide comprising a plurality of antigenic coronavirus peptides interspersed in an ankyrin repeat scaffold, the ankyrin repeat scaffold comprising a plurality of ankyrin repeat motifs.

62. The polypeptide of claim 61, wherein the polypeptide further comprises a leader peptide.

63. The polypeptide of claim 62, wherein the leader peptide is an influenza virus hemagglutinin (HA) leader peptide, optionally having at least 80% sequence identity to the sequence of SEQ ID NO: 21, or having or comprising the sequence of SEQ ID
NO:
21.

64. The polypeptide of claim 62, wherein the leader peptide is a leader peptide of the human immunoglobulin E protein (IgE), optionally having at least 80% sequence identity to the sequence of SEQ ID NO: 22, or having or comprising the sequence of SEQ ID NO: 22.

65. The polypeptide of any one of claims 61 to 64, the polypeptide further comprising a Toll-Like receptor 2 (TLR2) agonist peptide, optionally having at least 80%
sequence identity to the sequence of SEQ ID NO: 107, or having or comprising the sequence of SEQ ID NO: 107.

66. The polypeptide of any one of claims 61 to 65, the polypeptide further comprising a FMS-like tyrosine kinase 3 ligand (FLT3L), optionally having at least 80%
sequence identity to the sequence of SEQ ID NO: 29, or having or comprising the sequence of SEQ ID NO: 29.

67. The polypeptide of any one of claims 61 to 66, the polypeptide further comprising an a-helix dimerization domain (dHLX) or a CH3 dimerization domain, the dHLX
optionally having at least 80% sequence identity to the sequence of SEQ ID NO: 30, or having or comprising the sequence of SEQ ID NO: 30.

68. The polypeptide of any one of claims 61 to 66, the polypeptide further comprising a PVXCP, the PVXCP optionally having a sequence of SEQ ID NO: 16 or at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%
sequence identity to the sequence of SEQ ID NO: 16, or has or comprises the sequence of SEQ ID
NO:16.

69. The polypeptide of any one of claims 61 to 68, wherein the polypeptide comprises a plurality of linkers flanking one or more of the antigenic coronavirus peptides, optionally wherein the linker is or comprises AAY, GSGA (SEQ ID NO: 20), EAAAK (SEQ ID
NO:
102), RVRR (SEQ ID NO: 103), GGGS (SEQ ID NO: 104), GPGPG (SEQ ID NO: 105), HEYGAEALERAG (SEQ ID NO: 106).

70. The polypeptide of any one of claims 61 to 69, wherein the polypeptide comprises non-structural antigenic coronavirus peptides.

71. The polypeptide of claim 70, wherein each of the non-structural coronavirus peptides has or comprises an amino acid sequence selected from Table 1 or 2 or has at least 80% sequence identity to the amino acid sequence selected from Table 1 or 2.

72. The polypeptide of any one of claims 61 to 71, wherein the polypeptide comprises structural antigenic coronavirus peptides.

73. The polypeptide of claim 72, wherein each of the structural coronavirus peptides has or comprises amino acid sequence selected from Table 1 or 3 or at least 80%
sequence identity to the amino acid sequence selected from Table 1 or 3.

74. The polypeptide of any one of claims 61 to 70, wherein the antigenic coronavirus peptides are epitopes of replicative proteins or capsid proteins.

75. The polypeptide of any one of claims 61 to 69, wherein the polypeptide has or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ ID
NOs:
83 to 94. 108, formulas I-VII including for example SEQ ID Nos: 95-101 and 109-167, 170 and/or171.

76. The polypeptide of any one of claims 41 to 75, wherein the polypeptide molecule is associated with a lipid, optionally wherein the polypeptide molecule is encapsulated in a liposome, interspersed within a liposome lipid bilayer or attached to a liposome.

77. The polypeptide of any one of claims 41 to 75, wherein the polypeptide is associated with a lipid, optionally wherein the polypeptide molecule is encapsulated in a liposome, interspersed within a liposome lipid bilayer or attached to a liposome.

78. A composition comprising the nucleic acid molecule of any one of claims 1 to 20 and/or the nucleic acid molecule of any one of claims 21 to 36.

79. The composition of claim 78, wherein the nucleic acid is comprised in a vector, optionally the vector of claim 37 and/or the vector of claim 38 or 39 or 40.

80. A composition comprising the polypeptide of any one of claims 41 to 60 and/or the polypeptide of any one of claims 61 to 77.

81. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1 to 20 and/or the nucleic acid molecule of any one of claims 21 to 36, and optionally a pharmaceutically acceptable carrier, buffer, diluent or adjuvant.

82. The pharmaceutical composition of claim 81, comprising the vector of claim 37 and/or the vector of claim 38, and optionally a pharmaceutically acceptable carrier, buffer, diluent or adjuvant.

83. A pharmaceutical composition comprising the polypeptide of any one of claims 41 to 60 and/or the polypeptide of any one of claims 61 to 77, and optionally a pharmaceutically acceptable carrier, buffer, diluent or adjuvant.

84. A method of eliciting an immune response in a subject, comprising administering an effective amount of the nucleic acid molecule of any one of claims 1 to 36, the vector of any one of claims 37 to 40, the polypeptide of any one of claims 41 to 77, the composition of any one of claims 78 to 80 or a pharmaceutical composition of any one of claims 81 to 83.

85. The method of claim 84, wherein the nucleic acid molecule, the vector, the composition or the pharmaceutical composition is administered by electroporation, injection, optionally needleless injection, using a virus or using living bacteria.

86. Use of the nucleic acid molecule of any one of claims 1 to 36, the vector of any one of claims 37 to 40, the polypeptide of any one of claims 41 to 77, the composition of any one of claims 78 to 80 or the pharmaceutical composition of any one of claims 81 to 83 for eliciting an immune response in a subject.

87. The method of claim 84 or 85 or the use of claim 86, wherein the immune response is against a coronavirus, optionally a human beta coronavirus.

88. The method of claim 84 or 85 or the use of claim 86, wherein the subject is a human.

89. Use of the nucleic acid molecule of any one of claims 1 to 36, the vector of any one of claims 37 to 40, the polypeptide of any one of claims 41 to 77, the composition of any one of claims 78 to 80 or a pharmaceutical composition of any one of claims 81 to 83 in the manufacture of a medicament.

90. A method of producing a polypeptide in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1 to 36, the vector of any one of claims 37 to 40, the polypeptide of any one of claims 41 to 77, the composition of any one of claims 78 to 80 or a pharmaceutical composition of any one of claims 81 to 83.