Coronavirus spike glycoprotein receptor binding domains and uses thereof

Abstract

The present disclosure relates to polypeptides comprising an antigen fragment of the receptor binding domain (RBD) of coronavirus spike glycoprotein, protein nanostructures thereof, methods of manufacture thereof, and prophylactic and therapeutic uses thereof. In various aspects, the antigen fragment comprises a coronavirus subdomain 1 (SD1).

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 465,978, filed May 12, 2023, and U.S. Provisional Patent Application No. 63 / 598,691, filed November 14, 2023, each of which is incorporated herein by reference in its entirety.

[0003] sequence list This application contains a sequence list that has been submitted by EFS-WEB in .XML format, which is hereby incorporated in its entirety by reference. The .XML copy created on May 8, 2023, is named 061291-509001WO_SeqList_ST26.xml and is 335 kilobytes in size. Background Technology

[0004] The emergence of SARS-CoV-2, the virus that causes COVID-19, has increased focus on coronavirus vaccines, targeting both SARS-CoV-2 and other coronaviruses. There is an unmet need for engineered coronavirus peptides with improved properties and their applications. Summary of the Invention

[0005] In one aspect, this disclosure provides a recombinant polypeptide comprising an antigenic fragment of a coronavirus spike glycoprotein, the antigenic fragment comprising a receptor-binding domain (RBD) and optionally a coronavirus subdomain 1 (SD1). In some embodiments, the antigenic fragment of the polypeptide comprises or is substantially composed of RDB-SD1.

[0006] In some embodiments, SD1 comprises at least one surface-exposed nonpolar amino acid residue replaced by a polar amino acid residue. In some embodiments, the polypeptide comprises one, two, or more amino acids at positions 560, 562, or 569 replaced by polar residues, wherein such replacement is relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the polypeptide comprises an amino acid replacement of one or more, two or more, or three of L560Q, F562T, F562Y, or I569S, relative to the reference sequence according to SEQ ID NO: 1.

[0007] In some embodiments, the polypeptide comprises, relative to the reference sequence according to SEQ ID NO: 1, one, two or more, or three or more amino acid substitutions at positions 544, 546, 560, 562, 564, 569, or 582. In some embodiments, the polypeptide comprises, relative to the reference sequence according to SEQ ID NO: 1, one, two or more, three or more, or four or more amino acid substitutions at positions N544L, N544M, N544Q, L546V, L560Q, F562T, F562Y, Q564C, Q564L, Q564N, Q564W, I569S, or L582S.

[0008] In some embodiments, the polypeptide comprises substitutions of one or more, two or more, three or more, four or more amino acids at positions 338, 358, 363, 365, 392, or 395 relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the polypeptide comprises substitutions of one or more, two or more, three or more, four or more amino acids at positions F338L, I358F, A363L, Y365F, Y365M, Y365W, F392W, or V395I relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the polypeptide comprises amino acid substitutions including F338L / Y365W, F392W, Y365F / V395I, Y365F / F392W / V395I, F338L / A363L / Y365M, and / or I358F / Y365F / V395I.

[0009] In some embodiments, relative to the reference sequence according to SEQ ID NO: 1, the polypeptide comprises one, two or more, three or more, four or more amino acid substitutions at positions 329, 348, 350, 367, 375, 402, 407, 410, 418, 429, 433, 435, 452, 464, 510, 512, 514, 517, 518, 519, 520, 522, 527, or 528. In some embodiments, relative to the reference sequence according to SEQ ID NO: The reference sequence of 1, the polypeptide is contained in positions F329K, F329R, F329Y, A348P, V350L, V367F, F375Y, I402V, V407L, I410F, I418V, F429W, V433I, A435I, A435V, L452R, F464Y, P512Q, V510I, V512F, V512I, S514T, L517A, L517D, L517S, L517T, L518N, L The substitution of one, two or more, three or more, or four or more amino acids at 518Q, L518V, H519D, H519G, H519R, H519S, H519T, A520C, A520D, A520G, A520H, P521A, P521D, P521N, P521Q, P521S, A522G, A522I, P527N, K528I, K528Q, K528T, L518G, or L518S.

[0010] In some embodiments, the polypeptide comprises amino acid substitutions at positions 365, 395, 560, 562, and 569; and one, two, or three or more amino acid substitutions at positions 348, 402, 464, 514, 520, 526, and 527, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the peptide comprises amino acid substitutions, including A348P / Y365F / V395I / L560Q / F562Y / I569S, Y365F / V395I / I402V / L560Q / F562Y / I569S, Y365F / V395I / S514T / L560Q / F562Y / I569S, Y365F / V395I / I402V / F464Y / L560Q / F562Y / I569S and / or Y365F / V395I / G526S / P527N / L560Q / F562Y / I569S.

[0011] In some embodiments, the polypeptide comprises amino acid substitutions at positions 365, 395, 560, 562, and 569; and one, two, or more, or three or more amino acid substitutions at positions 329, 517, 519, 520, and 544, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the polypeptide comprises amino acid substitutions including Y365F / V395I / L560Q / F562Y / I569S, Y365F / V395I / F329Y / L560Q / F562Y / I569S, Y365F / V395I / L517T / H519S / L560Q / F562Y / I569S, Y365F / V395I / L517T / H519S / A520G / L560Q / F562Y / I569S, Y365F / V395I / L517T / H519S / N544L / L560Q / F562Y / I569S and / or Y365F / V395I / F329Y / L517T / H519S / L560Q / F562Y / I569S.

[0012] In some embodiments, the polypeptide comprises amino acid substitutions at positions 365, 392, 395, 560, 562, and 569; and one, two, or more, or three or more amino acid substitutions at positions 329, 517, 519, 520, and 544, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the polypeptide comprises amino acid substitutions including Y365F / F392W / V395I / L560Q / F562Y / I569S, Y365F / F392W / V395I / F329Y / L560Q / F562Y / I569S, Y365F / F392W / V395I / L517T / H519S / L560Q / F562Y / I569S, Y365F / F392W / V395I / L517T / H519S / A520G / L560Q / F562Y / I569S, Y365F / F392W / V395I / L517T / H519S / N544L / L560Q / F562Y / I569S and / or Y365F / F392W / V395I / F329Y / L517T / H519S / L560Q / F562Y / I569S.

[0013] In some embodiments, the polypeptide comprises amino acid substitutions at positions 365, 395, 517, 519, 520, 560, 562, 564, and 569; and one, two, or more, or three or more amino acid substitutions at positions 521, 544, and 546, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the polypeptide comprises amino acid substitutions including Y365F / V395I / L517T / H519G / A520C / Q564C / L560Q / F562Y / I569S and / or Y365F / V395I / L517S / H519R / A520H / P521Q / N544L / L546V / Q564W / F562T / L560Q / F562Y / I569S.

[0014] In some embodiments, the polypeptide includes amino acid substitutions at positions 365, 395, 517, 519, and 520; and one or more amino acid substitutions at positions 518 and 392, which are relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the peptide comprises amino acid substitutions, including Y365F / V395I / L517T / H519S / A520G, Y365F / V395I / L517T / L518G / H519D / A520G, Y365F / V395I / L517T / L518S / H519S / A520G, Y365F / F392W / V395I / L517T / L518G / H519D / A520G and / or Y365F / F392W / V395I / L517T / L518S / H519S / A520G.

[0015] In some embodiments, the polypeptide comprises amino acid substitutions at positions 329, 365, 395, 517, 519, 520, 560, 562, and 56, and one, two, or more, or three or more amino acid substitutions at positions 392, 544, and 564, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the peptide comprises amino acid substitutions, including Y365F / V395I / L517T / H519S / A520G / N544M / L560Q / F562Y / I569S / F329Y, Y365F / V395I / L517T / H519S / A520G / N544Q / L560Q / F562Y / I569S / F329Y, and Y365F / V395I / L517T / H519S / A520G / Q564N / L560Q / F562Y / I569S / F3 29Y, Y365F / F392W / V395I / L517T / H519S / A520G / N544M / L560Q / F562Y / I569S / F329Y, Y365F / F392W / V395I / L517T / H519S / A520G / N544Q / L560Q / F562Y / I569S / F329Y and / or Y365F / F392W / V395I / L517T / H519S / A520G / Q564N / L560Q / F562Y / I569S / F329Y. In some embodiments, the peptide comprises amino acid substitutions, including Y365F / V395I / L517T / H519S / A520G / N544M / L560Q / F562Y / I569S, Y365F / V395I / L517T / H519S / A520G / N544Q / L560Q / F562Y / I569S, and Y365F / V395I / L517T / H519S / A520G / Q564N / L560Q / F562Y / I5 69S, Y365F / F392W / V395I / L517T / H519S / A520G / N544M / L560Q / F562Y / I569S, Y365F / F392W / V395I / L517T / H519S / A520G / N544Q / L560Q / F562Y / I569S and / or Y365F / F392W / V395I / L517T / H519S / A520G / Q564N / L560Q / F562Y / I569S.

[0016] In some embodiments, the polypeptide comprises amino acid substitutions at positions 365, 395, 517, 519, 520, 521, 504, 560, 562, 569, and 329; and one or more amino acid substitutions at position 392, which are relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the polypeptide comprises amino acid substitutions including Y365F / V395I / L517T / H519G / P521A / A520C / Q564C / L560Q / F562Y / I569S / F329Y, Y365F / V395I / L517T / H519G / P521N / A520C / Q564C / L560Q / F562Y / I569S / F329Y, Y365F / F392W / V395I / L517T / H519G / P521A / A520C / Q564C / L560Q / F562Y / I569S / F329Y and / or Y365F / F392W / V395I / L517T / H519G / P521N / A520C / Q564C / L560Q / F562Y / I569S / F329Y.

[0017] In some embodiments, the polypeptide comprises multiple amino acid substitutions at positions 329, 365, 395, 526, 527, 528, 560, 562, and 569. In some embodiments, the polypeptide comprises amino acid substitutions including F329Y / Y365F / V395I / G526S / P527N / K528T / L560Q / F562Y / I569S and / or F329Y / Y365F / V395I / G526S / P527N / K528I / L560Q / F562Y / I569S.

[0018] In some embodiments, the polypeptide comprises a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NO: 11-19. In some embodiments, the polypeptide comprises the same polypeptide sequence as any of SEQ ID NO: 11-19.

[0019] In some embodiments, the peptide is expressed at a higher level than a reference peptide without the amino acid substitution, or optionally a reference peptide according to SEQ ID NO: 2 or 10. In some embodiments, the expressed peptide is more thermostable or more antigenic than SEQ ID NO: 2 or 10.

[0020] In some embodiments, the polypeptide comprises a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 2-10.

[0021] In another aspect, this disclosure provides a self-assembled protein nanostructure comprising a first component and an optional second component, the first component comprising a recombinant coronavirus polypeptide described herein, and the second component comprising a second protein.

[0022] In some embodiments, the protein nanostructure of the recombinant coronavirus polypeptide includes a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 11-19.

[0023] In some embodiments, the protein nanostructure of the recombinant coronavirus polypeptide comprises one or more, two or more, three or more, four or more amino acid substitutions relative to the reference sequence according to SEQ ID NO: 1.

[0024] In some embodiments, the protein nanostructure component peptide and the recombinant coronavirus peptide are non-covalently coupled. In other embodiments, the protein nanostructure component peptide and the recombinant coronavirus peptide are covalently coupled.

[0025] In some embodiments, the protein nanostructure comprises a fusion protein including a recombinant coronavirus peptide, a linker, and protein nanostructure component peptides. In some embodiments, the protein nanostructure comprises a fusion protein that, from its N-terminus to its C-terminus, sequentially includes a recombinant coronavirus peptide, a linker, and protein nanostructure component peptides.

[0026] In some embodiments, the protein nanostructure of the recombinant coronavirus polypeptide includes a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 11-19.

[0027] In some embodiments, the protein nanostructure of the fusion protein includes a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 11-19.

[0028] In some implementations, the first component of the protein nanostructure contains I53-50A.

[0029] In some embodiments, the protein nanostructure is an I3-01 / MI3 protein nanostructure. In some embodiments, the protein nanostructure is an I53-50AB protein nanostructure.

[0030] In some embodiments, the protein nanostructure is a ferritin protein nanostructure. In some embodiments, the protein nanostructure is an encapsulation protein nanostructure. In some embodiments, the protein nanostructure is a CP3 phage capsid protein nanostructure. In some embodiments, the protein nanostructure is a Qβ phage capsid protein nanostructure. In some embodiments, the protein nanostructure is an AP205 phage capsid protein nanostructure.

[0031] In some embodiments, the recombinant polypeptide of the protein nanostructure comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 11-19.

[0032] In some embodiments, the protein nanostructure component peptide comprises a peptide segment having a peptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20-21.

[0033] In some implementations, the protein nanostructure includes a second component, a polypeptide.

[0034] In some implementations, the second component polypeptide is I53-50B.

[0035] In some embodiments, the second component polypeptide comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:22.

[0036] In some embodiments, the linkers of the protein nanostructure contain between 8 and 24 amino acid residues. In some embodiments, the linkers of the protein nanostructure are selected from the group consisting of: GGSGGSGSGGSGGSGS, SGGGSGGSGSGGSGSGGSGS, EPEGGSGGSGSGSGGSGSGSGS, YGGSGGSGGSGSGSGGSGSGS, and GGSGGSGGSGGSGSGGSGSGSGSGSGS.

[0037] In another aspect, this disclosure provides a polynucleotide that encodes the polypeptide or protein nanostructure described herein. In some embodiments, the polynucleotide is mRNA.

[0038] In another aspect, this disclosure provides a pharmaceutical composition comprising the polypeptides, protein nanostructures, or polynucleotides disclosed above.

[0039] In another aspect, this disclosure provides vaccines comprising the polypeptides, protein nanostructures, or polynucleotides disclosed above. In some embodiments, the vaccine further comprises an adjuvant. In some embodiments, the adjuvant comprises squalene. In some embodiments, the vaccine is a bivalent vaccine. In some embodiments, the bivalent vaccine comprises the polypeptide or protein nanostructures described herein.

[0040] In another aspect, this disclosure provides a method for preventing or treating coronavirus-related illness in a subject of need, the method comprising administering to the subject the disclosed peptides, protein nanostructures, polynucleotides, pharmaceutical compositions, or vaccines. In another aspect, this disclosure provides a method for immunizing a subject of need against coronavirus infection by administering to the subject the disclosed vaccines. In yet another aspect, this disclosure provides kits for preparing and administering the peptides, protein nanostructures, polynucleotides, pharmaceutical compositions, or vaccines as described above. Attached Figure Description

[0041] This patent or application document contains at least one color drawing. Upon request and after payment of the necessary fees, the Patent Office will provide a copy of this patent or application publication with a color drawing.

[0042] Figure 1 A- Figure 1 C shows the structure of RBD and the RBD-SD1 antigen fragment (PDB 7UB5). Figure 1 A shows the individual structure of the RBD from residues 328 to 519. The receptor-binding motif (RBM) for ACE2 binding is shown in the box. Figure 1 B shows the structure of RBD-SD1 from residues 319 to 591. SD1 residues are marked in black. Figure 1 C shows the same Figure 1 B has the same structure, wherein regions targeting the designed mutations according to this disclosure are shaded, including regions containing Rpk mutations, RBD-SD1 interface mutations, and RBM neighbor mutations, and are accompanied by a density map. SD1resurf mutations are represented by shaded spheres and outlined with a box.

[0043] Figures 2A-2C Analysis of the supernatant from the initial design is shown, using the original RBD-SD1 fragment fused with the CompA gene. Figure 2A The supernatant shows a protein blot. Figure 2B The first set of ACE2 binding biolayer interferometry (BLI) data collected for selected supernatant samples is shown. Linker differences (in constructs RDBb1-5) are compared using the WT RBD-SD1 antigen (top left), and stabilization mutation differences are compared in constructs with 16-residue linkers. Figure 2CThis shows the second set of BLI data for ACE2 binding collected from selected supernatant samples. The data focus on designs containing mutations in Rpk8 (RBDb016 to 020), Rpk9 (RBDb021 to 025), or SD1resurf (RBDb036 to 040). RBD01 was used as a control for all studies. Figures 2B-2C The maximum offset values ​​are shown in Tables 16 and 17, respectively.

[0044] Figure 3 A- Figure 3 F shows the BLI sensor map of purified CompA derived from the original strain antigen. RBD01 was used as a control, lacking the stabilization mutation and the SD1 domain. Figure 3 A shows ACE2 binding, representing the integrity of a key epitope supporting the neutralizing antibody. Binding levels were similar across all constructs. Figure 3 B shows ACE2 binding, representing the integrity of a key epitope supporting the neutralizing antibody. Binding levels were similar across all constructs. Figure 3 C and Figure 3 D shows CR3022 binding. CR3022 is a non-neutralizing antibody that targets an epitope far from the ACE2 binding site, which is not normally exposed in the spike glycoprotein. Figure 3 E and Figure 3 F shows S309 binding. S309 is a broadly neutralizing antibody against sarbecovirus that binds outside the ACE2 binding site. Figure 3 A- Figure 3 The maximum offset value of F is shown in Table 18.

[0045] Figures 4A-4K show the thermal denaturation curves of the original CompA strain using intrinsic protein fluorescence (ITF) or SYPRO™ orange protein gel staining agent. Figures 4A and 4B show the ITF measurements of the RBD01 control and RBDb001 relative to RBDb002, respectively, to measure the effect of adding the SD1 domain or altering the linker. All show similar melting temperatures. Figures 4C-4F show the ITF measurements of RBD-SD1 CompA containing different Rpk mutations compared to WT RBD-SD1 (RBDb002). The addition of the Rpk9 mutation resulted in the highest melting temperature. Figure 4G is a comparison of the ITF of RBDb002 and RBDb037 (SD1resurf mutation), indicating that the addition of the SD1 resurf mutation does not affect the melting temperature. All plots in Figures 4A through 4G show three replicate measurements (each labeled “rep1”, “rep2”, or “rep3”), and the ITF measurements for RBDb002 shown in each plot from Figures 4A to 4G are identical for cross-plot comparison. The mean melting temperatures of the ITF measurements for RBDb002 and the control sample are represented by gray dashed lines or black dashed lines, respectively, and are also shown in Table 19. Figures 4H through 4K show a comparison of SYPRO for RBD-SD1 CompA with different Rpk mutations compared to WT RBD-SD1 (RBDb002). The Rpk9 mutation (RBDb022) shows a combination of a lower SYPRO baseline (suggesting improved foldability) and a higher melting temperature. All graphs in Figures 4H to 4K show three repeated measurements (each labeled "rep1", "rep2", or "rep3"), and the RBDb002 SYPRO measurements shown in each graph from Figures 4H to 4K are identical for cross-graph comparison. The mean melting temperatures of the RBDb002 and control samples measured by SYPRO are represented by gray dashed lines or black dashed lines, respectively, and are also shown in Table 20.

[0046] Figure 5A and Figure 5B The assembly of the original CompA and complementary CompB molecules is shown, as measured by dynamic light scattering (DLS) (both shown as intensity distributions). Figure 5A The DLS measurements collected after assembly are shown in the presence of a buffer supplemented with 50 mM arginine and 4% sucrose, demonstrating efficient assembly of RBDb022 and RBDb037 (based on the predicted size range of the assembled protein nanostructures), while all other groups show aggregation. Figure 5BThe DLS measurements collected after assembly are shown in the presence of a buffer supplemented with 50 mM arginine, demonstrating efficient assembly of RBDb022 and RBDb037 (based on the predicted size range of the assembled protein nanostructures), while all other groups showed aggregation. The labels “MOPS” and “Tris / glycerol” indicate the buffer background other than the added excipients, referring to either 50 mM MOPS, 0.75% CHAPS, 150 mM NaCl, pH 7.4 or 20 mM Tris, 5% glycerol, 250 mM NaCl, pH 8, respectively. Figure 5A and Figure 5B The DLS results, along with more detailed buffer conditions used for nanostructure assembly and analysis, are summarized in Table 21.

[0047] Figure 6 Assembly analysis of RBDb092 (original strain) and RBDb093 (BA.5) is shown, both containing a combination of Rpk9 and SD1resurf mutations. Analysis was performed using DLS (shown as intensity distribution), with assembly conducted in the presence of buffer supplemented with 100 mM arginine and 5% glycerol. RBDb092 assembled successfully with high efficiency, while RBDb093 showed aggregation. Figure 6 The DLS results are also summarized in Table 22.

[0048] Figure 7 This diagram shows alignments of representative RBD-SD1 sequences (corresponding to residues 319-591 of SARS-CoV-2) from various sabeviruses. The SDI domain is indicated by a horizontal line, and SD1 residues are shown in italics. Regions containing Rpk8 / Rpk9 mutations, RBD-SD1 interface mutations, or SD1resurf mutations are marked with symbols according to the legend. Vacancies relative to other sequences are indicated by dashes. This alignment shows that regions containing Rpk8 / Rpk9 mutations and RBD-SD1 interface mutations are largely conserved, thus validating the widespread presence of these mutations across multiple viral strains. Furthermore, this alignment shows that the SD1resurf mutations L560Q and I569S are present in other sabeviruses and therefore applicable to multiple viral strains. I569V also frequently appears in this alignment.

[0049] Figure 8Western blot analysis of the BA.5 RBD-SD1 CompA design in the supernatant following small-scale expression in HEK293 cells is shown. All two-digit construct numbers refer to the last two digits of the “RBDb0XX” nomenclature, where “XX” is replaced. All designs, except RBDb046, contain both Rpk8 and SD1resurf mutations, with constructs RBDb051-091 containing additional mutations. Constructs RBDb044-050 tested combinations of Rpk8 and SD1resurf mutations in various Omicron strains, while RBDb047 used the BA.5 strain.

[0050] Figure 9 A to Figure 9 C shows a selected, purified, in vitro characterization of BA.5 RBD-SD1 CompA. RBDb046 uses the WT RBD-SD1 antigen, while all other constructs also include stabilization mutations. Figure 9 A shows the melting temperature (T) measured using ITF. m ) and initial temperature (T) 起始 ). Figure 9 B shows the binding rate to ACE2 as measured by BLI. The error bars represent the standard deviation between replicates. Figure 9 C shows the maximum BLI offset for ACE2. The error bars represent the standard deviation between replicates.

[0051] Figure 10 The assembly of selected purified peptides of BA.5 RBD-SD1 CompA is shown, as measured by DLS (shown as intensity distribution). Assembly was performed using a buffer supplemented with 100 mM arginine. All peptides assembled well without aggregation, with the exceptions of RBDb045, RBDb078, and RBDb081.

[0052] Figure 11A and Figure 11B The immunogenicity of selected CompA and protein nanostructures derived from the original strain and BA.5 antigen was demonstrated in mice. Figure 11A The ELISA binding titer against the original strain or BA.5 spike glycoprotein antigen matched to the virus strain is displayed. Figure 11B The data demonstrate pseudovirus neutralization against strain-matched original strains or BA.5 pseudoviruses. These data reveal differences in immunogenicity among various CompA strains, particularly when comparing RBDb046 with other BA.5 CompA strains containing stabilizing mutations.

[0053] Figure 12AThe ACE2 / CompA binding ratios of selected wild-type and stabilized designs from various strains are shown. Figure 12B The total protein yield of selected wild-type and stabilized designs from various strains is shown. Figure 12C The melting temperatures of selected wild-type and stabilized designs from various strains are shown. Detailed Implementation

[0054] definition Where specific features (including method steps) are mentioned herein, it should be understood that the disclosure in this specification includes all possible combinations of such specific features. For example, where a specific feature is disclosed in the context of a particular aspect or embodiment or a particular claim, that feature may also be used, to the extent possible, in combination with other specific aspects and embodiments, and / or in the context of other specific aspects and embodiments.

[0055] When this document refers to a method comprising two or more qualifying steps, the qualifying steps may be performed in any order or simultaneously (unless the context precludes such possibility), and the method may include one or more other steps performed before any of the qualifying steps, between both of the qualifying steps, or after all the qualifying steps (unless the context precludes such possibility).

[0056] Unless otherwise expressly stated, the practice of this technique will employ conventional methods within the scope of the art, including chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology, many of which are described below for illustrative purposes. Such techniques are well explained in the literature. It should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described, as these can vary depending on how those skilled in the art use them.

[0057] All publications and patents mentioned herein are incorporated herein by reference in their entirety, as if each individual publication or patent were specifically and separately indicated to be incorporated by reference. In case of conflict, this application (including any definitions herein) shall prevail. However, any reference to any reference, article, publication, patent, patent publication, or patent application cited herein shall not constitute, nor should be construed as, an admission or implication of any kind that it constitutes valid prior art or is part of common general knowledge in any country of the world.

[0058] The chapter headings used herein are for organizational purposes only and are not intended to limit the subject matter described. Specifically, features described in one chapter may be combined with features from any other chapter of the specification.

[0059] While illustrative embodiments have been described and depicted, it should be understood that various changes may be made to these illustrative embodiments without departing from the spirit and scope of the invention.

[0060] Unless otherwise defined herein, 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 pertains. Various scientific dictionaries including those containing the terms included herein are well known and available to those skilled in the art. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, some preferred methods and materials are described. Therefore, the terms defined below are described more fully by reference to the entire specification.

[0061] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the / that” are also intended to include the plural forms.

[0062] As used herein, the term "about" means a range of values ​​that includes the specified value and that will be reasonably similar to the specified value by one of ordinary skill in the art. In some embodiments, "about" means within the standard deviation range using a measurement method generally accepted in the art. In some embodiments, "about" means a range that reaches + / - 10%, + / - 5%, + / - 3%, or + / - 1% of the specified value.

[0063] In this document, the term "at least" followed by a number indicates the starting point of a range from which to begin (depending on the defined variable, this range may have an upper limit or no upper limit). For example, "at least 1" means 1 or greater than 1.

[0064] In this document, the term "at most" followed by a number indicates the endpoint of a range that ends with that number (depending on the defined variable, it can be a range with a lower limit of 1 or 0, or a range with no lower limit). For example, "at most 4" means 4 or less, and "at most 40%" means 40% or less. In this specification, when a range is given as "(first number) to (second number)" or "(first number) - (second number)", it means a range whose lower limit is the first number and whose upper limit is the second number. For example, 25mm to 100mm means a range whose lower limit is 25mm and whose upper limit is 100mm.

[0065] As used herein, “substantially” or “proximately” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object “substantially” enclosed means that the object is either completely enclosed or nearly completely enclosed. In some cases, the permissible deviation from absolute completeness may depend on the specific circumstances. However, in general, the degree of near-completeness must be sufficient to achieve the same overall result as obtaining absolute and complete completeness. When used in a negative sense, “substantially” is equally applicable to referring to the complete or nearly complete absence of an action, characteristic, property, state, structure, item, or result. For example, a composition “substantially free of” other active agents either completely lacks other active agents or almost completely lacks other active agents, such that its effect is the same as when other active agents are completely absent. In other words, a composition “substantially free of” a certain ingredient or element or another active agent may still contain that item, as long as its measurable effect is absent.

[0066] Throughout this specification, unless the context otherwise requires, the term "comprise / comprises / comprising" shall be understood to imply inclusion of the specified steps or elements or groups of steps or elements, but does not exclude any other steps or elements or groups of steps or elements. For example, a composition "comprising" (or "comprising") components A, B, and C may consist of components A, B, and C (i.e., containing only these components), or may contain not only components A, B, and C, but also one or more other components.

[0067] As used herein, the term "consisting of" means, and is limited to, anything included in the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed element is necessary or mandatory, and no other element may be present. "Substantially consisting of" means any element listed in the phrase, and is limited to other elements that do not interfere with or contribute to the activity or function specified for the listed element in this disclosure. Thus, the phrase "substantially consisting of" indicates that the listed element is necessary or mandatory, but other elements are not optional and may be present or absent depending on whether they affect the activity or function of the listed element.

[0068] Throughout this specification, references to phrases such as "one embodiment," "an embodiment," "another embodiment," "a particular embodiment," "related embodiment," "in some embodiments," "in some embodiments," "another embodiment," or "yet another embodiment," or combinations thereof, indicate that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of this disclosure. Therefore, throughout this specification, the aforementioned phrases appearing in various places do not necessarily refer to the same embodiment. Furthermore, in one or more embodiments, a particular feature, structure, or characteristic may be combined in any suitable manner.

[0069] The following description includes information that may be useful for understanding the invention. This is not an admission that any of the information provided herein is prior art, or related to the currently claimed invention, or that any specific or implied publication is prior art.

[0070] In the case of two or more nucleic acid or peptide sequences, the term "identical" or "percentage of identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when performing a maximum correspondence comparison and alignment. Sequence alignment methods used for comparison are well known in the art. Once alignment is performed, the number of matches is determined by counting the positions in both sequences where identical nucleotide or amino acid residues are present. The percentage of sequence identity is determined by dividing the number of matches in the alignment by the length of the reference sequence and then multiplying the result by 100. For example, when aligned with a test sequence having 1554 amino acids, a peptide sequence with 1166 matches is 75.0% identical to the test sequence (1166 ÷ 1554 * 100 = 75.0). As used herein, vacancies in the alignment do not reduce the percentage of sequence identity.

[0071] Unless otherwise stated, the optimal alignment of sequences used for comparison was performed using the global alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443 (1970), as described by EMBOSS Needle (Website: ebi.ac.uk / Tools / psa / emboss_needle / ) (Madeira et al.) Nucleic Acids Res. This was achieved using 50(W1):W276-W279 (2022)). In the implementation, other alignment methods may be used, including but not limited to those described in: Devereux et al., Nucleic Acids Res. 12:387-95 (1984); Atschul et al. J.Mo. Biol. 215:403-10 (1990) (BLAST); Carrillo and Lipman Siam J. Appl. Math. 48(5)(1988); Computational Molecular Biology (Lesk, AM ed., 1989); BiocomputingInformatics and Genome Projects, (Smith, DW ed., 1993); Computer Analysis of Sequence Data, Part I, (Griffin and Griffin ed., 1994); Sequence Analysis in Molecular Biology (von Heinje, 2012); Sequence Analysis Primer (Gribskov and Devereux, J. ed., 1993). In the implementation scheme, sequence identity calculation was performed using the Needleman-Wunsch algorithm provided by the U.S. National Library of Medicine (Website: blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln).

[0072] For example, sequence identity can be determined using standard methods commonly used to compare the similarity of two polypeptide or two polynucleotide sequences. Computer programs such as the EMBOSS Needle or BLAST are used to align two polypeptide or two polynucleotide sequences to achieve optimal matching of their respective residues (along the full length of one or both sequences, or along predetermined portions of one or both sequences). These programs provide default vacancy opening penalties and default vacancy extension penalties, as well as scoring matrices that can be used in conjunction with computer programs, such as PAM 250 (a standard scoring matrix; see Dayhoff et al., Atlas of Protein Sequence and Structure, Vol. 5, Supplement 3 (1978)).

[0073] The "position" of an amino acid or nucleotide base is represented by a number, which sequentially identifies each amino acid (or nucleotide base) based on its position relative to the N-terminus (or 5' end) in the reference sequence. Because deletions, insertions, truncations, fusions, etc., must be considered when determining the optimal alignment, the amino acid residue numbering in the test sequence determined by simple counting from the N-terminus may not generally be the same as the corresponding position number in the reference sequence. For example, if a variant has a deletion relative to the reference sequence being aligned, the amino acid corresponding to the deletion site in the reference sequence will not be present in the variant. If an insertion exists in the reference sequence being aligned, the insertion will not correspond to the numbered amino acid position in the reference sequence. In the case of truncation or fusion, there may be amino acid segments in the reference or aligned sequences that do not correspond to any amino acid in the corresponding sequence.

[0074] When used in the context of numbering a given sequence, the terms “relative to” or “reference to…number” or “corresponding to” refer to the numbering of residues in a given sequence when compared with a specified reference sequence, such that each position in the numbered sequence is associated with a number that corresponds to the number of the equivalent position in the reference sequence.

[0075] The term "coronavirus" refers to viruses belonging to the subfamily Coronavirinae of the family Coronaviridae in the order Nidovirales. Coronaviruses are enveloped viruses with a positive-sense, single-stranded RNA genome and a helical nucleocapsid. The term "coronavirus" encompasses all strains, genotypes, protective types, and serotypes of infectious bronchitis virus. Examples of avian coronaviruses include infectious bronchitis virus (IBV), guinea fowl coronavirus (GfCo), and turkey coronavirus (TCO, turkey enteritis virus, and blue crown virus).

[0076] The term "SARS-CoV-2" refers to a positive-sense, single-stranded RNA virus belonging to the genus beta-coronavirus, which has become a highly lethal cause of severe acute respiratory infection. The term "SARS-CoV-2" includes its variants, such as, but not limited to: alpha (B.1.1.7 and Q lineages); beta (B.1.351 and progeny lineages); delta (B.1.617.2 and AY lineages); gamma (P.1 and progeny lineages); epsilon (B.1.427 and B.1.429); eta (B.1.525); iota (B.1.526); kappa (B.1.617.1); 1.617.3; mu (B.1.621, B.1.621.1) and zeta (P.2)).

[0077] B.1.1.529, also known as the Omekkaron variant, is a variant of the original SARS-CoV-2. Compared to the original SARS-CoV-2 strain, this variant has a total of 60 mutations, specifically 50 non-synonymous mutations, 8 synonymous mutations, 35 non-coding mutations, and 2 non-coding mutations. Thirty-two mutations affect the spike glycoprotein (see SEQ ID NO: 1, A67V, A69-70, T95I, G142D, A143-145, A211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F), about half of which are located in the receptor-binding domain (319-530).

[0078] The term "spike glycoprotein" refers to a class I fusion glycoprotein, which, in the case of SARS-CoV-2, is initially synthesized as a precursor protein of approximately 1273 amino acids. The S1 subunit is located at the distal end of the viral membrane and contains a receptor-binding domain (RBD), which is believed to mediate the binding of the virus to its host receptor. An exemplary SARS-CoV-2 spike glycoprotein sequence is shown herein as SEQ ID NO: 1 (NCBI reference sequence: YP_009724390.1, the sequence of which is incorporated herein by reference in its entirety). As used herein, the antigen fragment is amino acids 319-591 of the NCBI reference sequence: YP_009724390.1.

[0079] As used herein, the term "receptor-binding domain (RBD)" refers to a portion of the spike glycoprotein. For example, the RBD is amino acids 319-541 in the NCBI reference sequence: YP_009724390.1. As used herein, the RBD is amino acids 328-531 in the NCBI reference sequence: YP_009724390.1. The RBD includes a receptor-binding motif (RBM), which facilitates cell invasion by binding to the ACE2 receptor.

[0080] As used herein, the term “subdomain-1 (SD1)” refers to the conserved portion of the spike glycoprotein adjacent to the receptor-binding domain. For example, SD1 is amino acids 532-591 in the NCBI reference sequence: YP_009724390.1.

[0081] The term "original strain" refers to the antigen or virus directly based on the sequence (Genbank identifier NC_045512.2) of the original SARS-CoV-2 isolate Wuhan-Hu-1.

[0082] The term "recombinant" indicates that a material (e.g., nucleic acid or protein) has undergone artificial or synthetic (non-natural) alteration through human intervention. This alteration can occur while the material is in its natural environment or state, or after it has been isolated from its natural environment or state. Specifically, for example, a coronavirus is recombinant when it is produced through the expression of a recombinant nucleic acid. For example, a "recombinant nucleic acid" is a nucleic acid produced by recombination of nucleic acids, such as in cloning, DNA shuffling, or other procedures, or by chemical mutagenesis or other forms of mutagenesis; a "recombinant polypeptide" or "recombinant protein" is a polypeptide or protein produced by the expression of a recombinant nucleic acid; and a "recombinant virus" (e.g., a recombinant coronavirus) is produced by the expression of a recombinant nucleic acid. The "fusion protein" or "tetramer" described herein is recombinant and does not exist in nature.

[0083] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues in which the polymer may be coupled to portions not composed of amino acids (such as glycans or other post-translational modifications) or non-natural chemical portions. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as both naturally occurring and non-naturally occurring amino acid polymers. A “fusion protein” is a chimeric protein encoding two or more separate protein sequences that are recombined and expressed as a single polymer molecule.

[0084] The term "recombinant polypeptide" refers to a polypeptide that is not naturally occurring, or a polypeptide having a sequence produced by artificially combining two sequence segments that are isolated in their natural state. In some embodiments, the recombinant polypeptide is encoded by a heterologous (e.g., recombinant) nucleic acid that has been introduced into a host cell (such as a bacterium or eukaryotic cell).

[0085] The terms “antigen” or “immunogen” in their common sense refer to a compound or composition that, when administered to or expressed in an immunocompetent subject, induces a cellular or humoral immune response, such as a cytotoxic T-lymphocyte (CTL) response, a B-cell response (e.g., the production of antibodies that specifically bind to an epitope), an NK-cell response, or any combination thereof. Antigens may include polypeptides (including glycoproteins). In all respects, an antigen is a polypeptide or polypeptide complex comprising at least one component designed to elicit an immune response. For example, an antigen may include one or more immunogenic epitopes associated with bacterial pathogens. As used herein, the term “antigen” is not limited to the portion of a polypeptide or polypeptide complex containing an antigenic epitope. The term “epitope” or “antigenic determinant” in its common sense refers to a portion of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells.

[0086] The term "variant" refers to a polypeptide that has one or more insertions, deletions, or amino acid substitutions relative to a reference polypeptide, but retains one or more properties of the reference protein.

[0087] The term "antigen variant" refers to a variant that has one or more of the same epitopes as a reference peptide, and / or a variant that produces the same or similar immune response when administered to a subject as a reference peptide.

[0088] The term "antigen fragment" refers to any fragment of a protein that elicits an immune response (humoral or T-cell response) in vivo. Antigen fragments can be linear epitopes, discontinuous epitopes, or conformational epitopes (e.g., folded domains). Antigen fragments may retain the secondary, tertiary, and / or quaternary structures of the full-length protein.

[0089] The term "functional variant" refers to a variant that exhibits at least some of the activities of a reference polypeptide. For example, a functional variant of an assembly domain is able to promote polymerization and self-assembly to the same extent as the reference assembly domain, and / or is able to polymerize and assemble with the same homologous assembly domain as the reference assembly domain.

[0090] The term "substitution" refers to replacing a single amino acid residue in a sequence with another amino acid residue. The standard abbreviation for amino acid substitutions is used. For example, V94R means that valine (V) in the reference sequence is substituted with arginine (R). The abbreviation Arg94 refers to any sequence in which the 94th residue relative to the reference sequence is arginine (Arg). The term "combination of substitutions" refers to multiple amino acid substitutions included in a single polypeptide. As used herein, the slash " / " character identifies a combination of substitutions at each amino acid position separated by a slash. As an example, "Y365F / V395I" means a combination of a Y-->F substitution at amino acid position 365 and a V-->I substitution at amino acid position 395; "Y365F / F392W / V395I" means a combination of a Y-->F substitution at amino acid position 365, an F-->W substitution at amino acid position 392, and a V-->I substitution at amino acid position 395.

[0091] The following eight groups each contain amino acids that can be conservedly substituted for one another: 1) Alanine (A), glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) (See, for example, Creighton, Proteins (1984)).

[0092] As used herein, "surface-exposed amino acid residues" refers to amino acids located on the exterior of the polypeptide chain that are part of the polypeptide. In some embodiments, surface-exposed amino acid residues can be used to interact with other polypeptides or solvents. In other words, the amino acid residue is not embedded within the polypeptide chain.

[0093] As used herein, a “nonpolar amino acid residue” refers to a hydrophobic amino acid or residue having a side chain that is uncharged at physiological pH, and in which the electron pair shared by two atoms in the bond is usually held equally by each of the two atoms (i.e., the side chain is not polar). Genetically encoded nonpolar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M), and L-Ala (A).

[0094] As used herein, a “polar amino acid residue” refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH, but in at least one of its bonds, an electron pair shared by two atoms is held more tightly by one of the atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S), and L-Thr (T).

[0095] The "position" of an amino acid or nucleotide base is represented by a number, which sequentially identifies each amino acid (or nucleotide base) based on its position relative to the N-terminus (or 5' end) in the reference sequence. Because deletions, insertions, truncations, fusions, etc., must be considered when determining the optimal alignment, the amino acid residue numbering in the test sequence determined by simple counting from the N-terminus may not generally be the same as the corresponding position number in the reference sequence. For example, if a variant has a deletion relative to the reference sequence being aligned, the amino acid corresponding to the deletion site in the reference sequence will not be present in the variant. If an insertion exists in the reference sequence being aligned, the insertion will not correspond to the numbered amino acid position in the reference sequence. In the case of truncation or fusion, there may be amino acid segments in the reference or aligned sequences that do not correspond to any amino acid in the corresponding sequence.

[0096] The term "expression" encompasses any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion. In some respects, expression can be detected using conventional techniques for detecting proteins (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0097] As used herein, a “cell” refers to a membrane-bound biological unit capable of performing metabolic or other functions sufficient to preserve or replicate its genomic DNA. Cells can be identified by methods known in the art, including, for example, the presence of an intact membrane, staining with a specific dye, the ability to produce offspring, or, in the case of gametes, the ability to bind with a second gamete to produce viable offspring. Cells can include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, such as mammals, insects (e.g., spodoptera), and human cells. Cells may be useful when they are naturally non-adhesive or when they are treated (e.g., by trypsin digestion) to prevent them from adhering to surfaces.

[0098] The term "host cell" refers to any cell capable of expressing recombinant polypeptides or polynucleotides. In some embodiments, the host cell is a bacterial cell. In some embodiments, the bacterial cell is *Escherichia coli* (E. coli). E. coli )cell.

[0099] The terms “culture” and “culture medium” refer to standard cell culture and recombinant protein expression techniques.

[0100] The term “manufacturing” refers to the production of recombinant peptides or protein nanostructures or polynucleotides at any scale, including at least 25-mL, 50-mL, 1-L, 1,000-L, 50,000-L or larger.

[0101] The term "purification" refers to the separation of a molecule from other substances present in a composition. Peptides can be purified by affinity (e.g., affinity to an antibody or a tag, such as using a His-tag capture resin), by charge (e.g., ion exchange chromatography), by size (e.g., preparative ultracentrifugation, size exclusion chromatography), or by other means.

[0102] The term "stability" refers to the physical and chemical stability of a peptide formulation. Physical instability in protein formulations can be caused by the aggregation of protein molecules into higher-order polymers or even precipitates. A "stable" formulation is one in which the degree of protein aggregation is acceptablely controlled and does not exhibit an unacceptable increase over time. Physical stability can be assessed using methods known in the art, including the measurement of the apparent light decay (absorbance or optical density) of the sample. This measurement of light decay is related to the turbidity of the formulation. Turbidity arises from the aggregation or precipitation of proteins or complexes in the formulation.

[0103] The term "antigenicity" refers to the ability of an antigen to specifically bind to T-cell receptors or antibodies, and includes the reactivity of the antigen to pre-existing antibodies in the subject's body. "Immunogenicity" refers to the ability of any antigen to induce an immune response, and includes the inherent ability of the antigen to generate antibodies in the subject's body.

[0104] The term "neutralization" (e.g., "neutralizing antibody response") refers to antibodies that prevent infection by pathogens and / or reduce the severity of infection. Neutralizing antibody responses can be measured in vitro (e.g., killing target bacteria in the presence of antibodies) or in vivo (e.g., by administering antibodies to a subject prior to challenge with an infectious dose of pathogen to determine the protective dose of the antibody).

[0105] Antibody “binding” or “specifically binding” or “specifically binding” (which may be used interchangeably herein) to a target (e.g., a bacterial protein) is a well-known term in the art, and methods for determining such specificity or preferential binding are also well-known in the art. A molecule is said to exhibit “specific binding” or “preferential binding” if it reacts or binds to a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with a higher affinity than alternative cells or substances.

[0106] As used herein, the term "protein nanostructure" refers to a symmetrical protein assembly in which subunits self-assemble in aqueous solution without the need for assembly by lipids or macromolecules other than the protein nanostructure. Illustrative protein nanostructures are described in Hsia et al., Nature 35:136-9 (2016) and Bale et al., Science 353:389-394 (2016). In some embodiments, the protein nanostructure is a single-component protein nanostructure in which a single polypeptide type provides the building blocks for self-assembly to form the protein nanostructure. In some embodiments, the protein nanostructure is a two-component protein nanostructure in which two polypeptide types provide the building blocks for self-assembly to form the protein nanostructure. In some embodiments, the polypeptide type includes an assembly domain that causes the polypeptide to form a symmetrical dimer, trimer, tetramer, hexamer component, or another multimer component. In a two-component nanostructure, the two components differ in the choice of assembly domain. In some embodiments, the assembly domain of the first polypeptide type causes the polypeptide to form a trimer; and the assembly domain of the second polypeptide type causes the polypeptide to form a pentamer.

[0107] In some embodiments of single-component nanostructures or related to single-component nanostructures, two or more copies of the component further symmetrically self-assemble to form the nanostructure. In some embodiments of bicomponent nanostructures or related to bicomponent nanostructures, two or more of each of two different components symmetrically self-assemble to form the nanostructure.

[0108] As used herein, the term "assembly domain" refers to a portion of a component's subunit that participates in the formation of protein nanostructures through interactions within the component and with other copies of the same component (in single-component nanostructures) or with other components (e.g., in two-component nanostructures).

[0109] The term "icosahedral particle" refers to a protein nanostructure with an icosahedral core. I53 refers to an icosahedral particle composed of pentamers and trimers. I52 refers to an icosahedral particle composed of pentamers and dimers. T33 refers to a tetrahedral particle composed of two sets of trimers. T32 refers to a tetrahedral particle composed of trimers and dimers. As used herein, the abbreviations I53-50A and I53-50B refer to two components or polypeptide chains (A and B) of a specific I53-type particle (i.e., I53-50 particle).

[0110] Potentially antigenic peptides can be non-covalently or covalently linked to the core of protein nanostructures, including as fusion proteins or in other ways disclosed herein. Multimeric peptides may optionally be arranged along the symmetry axis of the protein nanostructure. Proteins and nucleic acid molecules encoding such proteins, formulations, and methods of use are also provided.

[0111] As used herein, when referring to two parts, the term "linked" means that the two parts are bonded, wherein one or more bonds connecting the two parts may be covalent or non-covalent. In some embodiments, the two parts are covalently bonded to each other (e.g., directly or through a covalently bonded intermediate ("linker")). In some embodiments, the two parts are non-covalently bonded (e.g., through ionic bonds, van der Waals bonds / interactions, hydrogen bonds, polar bonds, or combinations or mixtures thereof). For fusion peptides with N-terminal and C-terminal linkages, the linker is a peptide bond.

[0112] As used herein, the terms “bioconjugate” and “bioconjugate linker” refer to the resulting bond between atoms or molecules of a “bioconjugate reactive group” or a “bioconjugate reactive moiety.” This bond can be direct or indirect. For example, the coupling between a first bioconjugate reactive group (e.g., –NH2, –C(O)OH, –N-hydroxysuccinimide, or –maleimide) and a second bioconjugate reactive group (e.g., thiol, sulfur-containing amino acid, amine, amino acid with amine side chain, or carboxylate) can be direct, for example, through a covalent bond or linker (e.g., the first linker of the second linker), or indirect, for example, through non-covalent bonds (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion forces), ring stacking (π effect), hydrophobic interactions, etc.). In some embodiments, bioconjugation chemistry (i.e., the combination of two bioconjugation reactive groups) is used to form bioconjugates or bioconjugation linkers, including but not limited to nucleophilic substitution (e.g., reactions of amines and alcohols with acyl halides, reactive esters), electrophilic substitution (e.g., enamine reactions), and addition to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reactions, Diels-Alder additions). These and other useful reactions are discussed, for example, in March, ADVANCEDORGANIC CHEMISTRY, 3rd ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982. In some embodiments, a first bioconjugated reactive group (e.g., a maleimide moiety) is covalently linked to a second bioconjugated reactive group (e.g., a thiol group). In some embodiments, a first bioconjugated reactive group (e.g., a haloacetyl moiety) is covalently linked to a second bioconjugated reactive group (e.g., a thiol group). In some embodiments, a first bioconjugated reactive group (e.g., an –N-hydroxysuccinimide moiety) is covalently linked to a second bioconjugated reactive group (e.g., an amine group). In some embodiments, a first bioconjugated reactive group (e.g., a maleimide moiety) is covalently linked to a second bioconjugated reactive group (e.g., a thiol group).In some embodiments, a first biocoupled reactive group (e.g., a –sulfon-N-hydroxysuccinimide moiety) is covalently linked to a second biocoupled reactive group (e.g., an amine).

[0113] Useful bioconjugation reactions used in the bioconjugation chemistry section of this article include, for example: (a) Carboxyl groups and their various derivatives, including but not limited to N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acyl halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b) Hydroxyl group, which can be converted into esters, ethers, aldehydes, etc.

[0114] (c) Haloalkyl, wherein the halogen is subsequently replaced by a nucleophilic group (such as an amine, carboxylate anion, thiol anion, carbocation or alkoxide ion) to achieve covalent bonding of the new group at the site of the halogen atom. (d) Dienephilic groups that can participate in the Diels-Alder reaction, such as maleimide groups or maleimide groups; (e) Aldehyde or ketone group, making subsequent derivatization possible by forming carbonyl derivatives (such as imines, hydrazones, ureas, or oximes) or by mechanisms such as Grignard addition or alkyllithium addition; (f) Sulfonyl halide groups, used for subsequent reactions with amines, for example, to form sulfonamides; (g) Thiol group, which can be converted into a disulfide bond, react with acyl halides, or bond with metals (such as gold), or react with maleimide; (h) Amines or thiol groups (e.g., present in cysteine), which may be, for example, acylated, alkylated or oxidized; (i) Alkenes, which may undergo processes such as cycloaddition, acylation, Michael addition, etc.; (j) Epoxy groups, which can react with, for example, amines and hydroxyl compounds; (k) Phosphite and other standard functional groups that can be used in nucleic acid synthesis; (l) Metal-silicon oxide bonding; and (m) The metal is bonded to a reactive phosphorus group (e.g., phosphine) to form, for example, a phosphate diester bond.

[0115] (n) Azide-alkyne coupling using copper-catalyzed cycloaddition click chemistry.

[0116] (o) Biotin conjugates can react with avidin or streptavidin to form avidin-biotin complex or streptavidin-biotin complex.

[0117] Bioconjugated reactive groups can be selected such that they do not participate in or interfere with the chemical stability of the conjugates described herein. Alternatively, reactive functional groups may be protected from cross-linking reactions by the presence of protecting groups. In some embodiments, the bioconjugates comprise molecular entities derived from reactions of unsaturated bonds (such as maleimide) and thiol groups.

[0118] Another method for covalently linking two peptides is the SpyTag / SpyCatcher system. The peptide SpyTag (13 amino acids) spontaneously reacts with the protein SpyCatcher (12.3 kDa), forming an intermolecular isopeptide bond between the pair. The polynucleotide sequence encoding either the SpyTag or SpyCatcher can be recombined and introduced into the polynucleotide sequence encoding the target peptide, thereby forming a fusion protein. These fusion proteins can be covalently linked via the SpyTag / SpyCatcher system when mixed in the reaction.

[0119] Tag / catcher pairing enables bioconjugation between two recombinant proteins, which may be limited or impossible for traditional direct gene fusion between two proteins. For example, problems concerning protein folding, suboptimal expression hosts, and specialized post-translational modifications can be alleviated by modularizing protein production using tag / catcher systems. Protein nanostructures can be coupled with antigens using a variety of techniques, including but not limited to the SpyCatcher system, described, for example, in Escolano et al. Nature 570:468-473 (2019), He et al. SciAdv. 7(12):eabf1591 (2021), and Tan et al. Nat. Commun. 12(1):542 (2021).

[0120] The term "domain" refers to any part of a polypeptide that adopts a tertiary structure.

[0121] The terms "assembly domain" and "polymerization" refer to the ability of a polypeptide or a domain of a polypeptide to form a tertiary structure with another polypeptide domain. In some embodiments, the assembly domain can form dimers, trimers, tetramers, pentamers, or hexamers and / or heteromers with other assembly domains. In some embodiments, the assembly domain forms protein nanostructures.

[0122] The term "trimerization domain" refers to the assembly domain that forms a trimer.

[0123] The term "fragment" refers to a polypeptide that has one or more N-terminal or C-terminal truncations compared to a reference polypeptide.

[0124] The term "functional fragment" refers to a fragment that retains at least one function of its reference polypeptide.

[0125] As used herein, when referring to two parts, the term "coupling" means that the two parts are bonded, wherein one or more bonds connecting the two parts may be covalent or non-covalent. In some embodiments, the two parts are covalently bonded to each other (e.g., directly or through a covalently bonded intermediate). In some embodiments, the two parts are non-covalently bonded (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals bonds / interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion forces), ring stacking (π effect), hydrophobic interactions, etc.), polar bonds, or combinations or mixtures thereof).

[0126] The term "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombined and expressed as a polypeptide, thereby covalently coupling them. For fusion polypeptides with N-terminal and C-terminal linkages, the linker can be a peptide bond or a peptide of any length.

[0127] The term "linker" refers to a peptide conjugate or polypeptide that chemically links the C-terminus of a first polypeptide segment to the N-terminus of a second polypeptide segment, thereby forming a fusion protein containing two polypeptide segments.

[0128] The term "signal sequence" refers to a polypeptide sequence typically located at the N-terminus of a polypeptide expressed in a host cell, which directs the polypeptide to a specific cellular compartment. A signal sequence can be a secretion signal that causes the host cell to secrete the polypeptide into the culture medium in which the host cell is cultured. A signal sequence can be a "natural" signal sequence, i.e., a signal sequence that exists in nature as part of the polypeptide. A signal sequence can also be a sequence found in nature that does not coexist with the polypeptide in its natural state. Various signal sequences are known, and the selection of an appropriate signal peptide is within the skill of a person skilled in the art.

[0129] The term "secretion" refers to the ability of a host cell to release expressed polypeptides into the culture medium in which they are cultured.

[0130] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment,” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, polymers of nucleotides covalently linked together, which may have various lengths and be deoxyribonucleotides or ribonucleotides, or analogs, derivatives, or modifications thereof. Different polynucleotides may have different three-dimensional structures and may perform a variety of known or unknown functions. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, intergenic DNA (including but not limited to heterochromatin DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched-chain polynucleotides, plasmids, vectors, sequence-isolated DNA, sequence-isolated RNA, nucleic acid probes, and primers. Polynucleotides available in the methods of this disclosure may include natural nucleic acid sequences and their variants, artificial nucleic acid sequences, or combinations of such sequences.

[0131] The term “messenger RNA” or “mRNA” refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA may contain one or more reading frames or regions.

[0132] The terms "pharmaceuticalally acceptable excipient" and "pharmaceuticalally acceptable carrier" refer to substances that facilitate the administration and absorption of an active agent by a subject and can be included in the compositions of this disclosure without causing significant adverse toxicity to the patient. They can also refer to excipients approved by federal or state regulatory agencies or listed in the United States Pharmacopeia or other recognized veterinary and, more particularly, human pharmacopoeias. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline solutions, lactated Ringer's solution, standard sucrose, standard glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose, amylose, or starch), fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and colorants, etc. Such formulations can be sterilized and, if necessary, can be mixed with adjuvants (such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, and / or aromatic substances, etc.) that do not react adversely with the compounds of this disclosure. Those skilled in the art will recognize that other pharmaceutical excipients may also be used in this disclosure.

[0133] The term "vaccine" refers to a composition that provides active acquired immunity against a specific disease (e.g., COVID) or pathogen (e.g., coronavirus). Vaccines typically contain one or more agents that can induce an immune response against a pathogen or disease (i.e., a target pathogen or disease) in a subject. The immunogenic agent stimulates the body's immune system to recognize the agent as a threat or indication of the presence of the target pathogen or disease, thereby inducing immune memory, making it easier for the immune system to recognize and destroy either of the pathogens upon subsequent exposure. Vaccines can be prophylactic (e.g., to prevent or mitigate the effects of future infections caused by any natural or pathogenic factors, or to prevent or mitigate the anticipated occurrence of cancer in a susceptible subject) or therapeutic (e.g., to treat diseases, such as, but not limited to, meningitis in a subject of need). The administration of a vaccine is referred to as vaccination. In some instances, a vaccine composition may deliver an antigen molecule or a nucleic acid (e.g., mRNA) encoding an antigen molecule (e.g., a peptide) to a subject. In the subject, the antigen molecule or nucleic acid delivered via the vaccine composition may be expressed as an antigen molecule, allowing the subject to acquire immunity against that antigen molecule. In the case of vaccination against infectious diseases, the vaccine composition may provide an antigen molecule associated with a specific pathogen or mRNA encoding an antigen molecule, such as one or more peptides known to be expressed in pathogens (e.g., pathogenic bacteria or viruses).

[0134] The term "bivalent vaccine" refers to a vaccine prepared from two different strains of a virus. Such vaccines can be combined with other immunogenic or antigenic components to provide a vaccine composition (e.g., combined with one or more recombinant protein antigens).

[0135] The term "adjuvant" refers to a pharmaceutically acceptable substance that enhances the immune response to an antigen when administered in combination with an antigen or when administered to a subject before, during, or after the administration of the antigen.

[0136] The term "subject" refers to a human or non-human animal to whom the composition may be administered for vaccination, treatment, or other purposes. In some embodiments, a non-human animal is a non-human primate, rabbit, hamster, gerbil, pig, cow, sheep, goat, guinea pig, rat, mouse, squirrel, wolf, fox, horse, zebra, giraffe, hyena, elephant, cat, dog, llama, or ferret.

[0137] The term "effective amount" refers to a specific quantity of a composition that is sufficient to affect such treatment when administered to a patient to treat a condition, symptom, or illness, or sufficient to produce such an immune response when administered to a patient to produce such an immune response. The exact amount will depend on the active ingredient, the condition, symptom, or illness to be treated and its severity, as well as the age, weight, physical condition, and responsiveness of the subject to be treated, and can be determined by a person skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Vols. 1–3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, edited by Gennaro, Lippincott, Williams & Wilkins).

[0138] As used herein, the term “delivery” means providing an entity to a destination. For example, delivering a therapeutic and / or preventative agent to a subject may involve administering a nanoparticle composition comprising the therapeutic and / or preventative agent to the subject (e.g., via intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, or subcutaneous routes). Administering a nanoparticle composition to a mammal or mammalian cells may involve contacting one or more cells with the nanoparticle composition.

[0139] As used in this article, the term "prevention" refers to reducing the occurrence of disease symptoms in patients. Prevention can be complete (without detectable symptoms) or partial, resulting in fewer observed symptoms than would be possible without treatment.

[0140] As used herein (and as is well known in the art), “treating” broadly refers to any method used to obtain a beneficial or desired outcome (including clinical outcomes) in a subject’s condition. Beneficial or desired clinical outcomes may include, but are not limited to: reduction or improvement of one or more symptoms or symptom, attenuation of disease severity, stabilization of disease state (i.e., no worsening), prevention of disease spread or transmission, delay or slowing of disease progression, improvement or relief of disease state, reduction of disease relapse, and remission (whether partial or complete, and whether detectable or undetectable). In other words, as used herein, “treatment” includes any cure, improvement, or prevention of disease. Treatment may prevent the onset of disease, inhibit the spread of disease, alleviate disease symptoms, completely or partially eliminate the root cause of disease, shorten the duration of disease, or accomplish a combination of these.

[0141] The term "administration" refers to the delivery of the composition to a subject in a manner that allows the composition to exert its intended effect. Administration for vaccination or post-exposure prophylaxis can be performed via intramuscular injection, intravenous injection, intraperitoneal injection, or any other suitable route.

[0142] "Combined administration" means that the compositions described herein are administered simultaneously, immediately before, or immediately after the administration of one or more other therapies. The compositions provided herein may be administered to a subject alone or in combination. Combined administration is intended to include the simultaneous or sequential administration of compounds, alone or in combination (more than one compound). Therefore, the formulations may also be combined with other active substances when needed (e.g., to reduce metabolic degradation).

[0143] The terms "immunization" and "immunizing" refer to administering a composition to a subject in an amount sufficient to elicit a desired immune response after one or more administration steps. Immunization may include one to ten or more administrations of the composition (e.g., injection), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more administrations. The first administration may not elicit a detectable immune response, as each subsequent administration typically reinforces the immune response generated by the previous administration. As used herein, the term "immunizing" includes post-exposure prophylaxis.

[0144] The term "protective immune response" refers to an immune response that prevents infection with the pathogen and / or reduces the severity of infection when a subject is reinfected with the pathogen; or it refers to an immune response that produces a level of protection-associated immune response. For example, a vaccination may produce a protective immune response if it results in the production of neutralizing antibodies in the plasma or serum of a subject (e.g., a human, pet, or agricultural animal) that protect the subject against subsequent infection, and / or if it is present in an amount that confers protection as observed in a test subject (e.g., a New Zealand White (NZW) rabbit).

[0145] The term "antibody" refers to a polypeptide encoded by an immunoglobulin gene or a functional segment thereof that specifically binds to and recognizes an antigen. The recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which further define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0146] The term "infection" or "infectious disease" refers to a disease or symptom that can be caused by an organism, such as bacteria, viruses, fungi, or any other pathogenic microorganism. In some embodiments, an infectious disease is caused by pathogenic bacteria. In some embodiments, an infectious disease is an infection with Campylobacter jejuni, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Staphylococcus aureus, Streptococcus pneumoniae, or Vibrio cholerae.

[0147] The terms "viral infection" or "viral disease" refer to illnesses or symptoms caused by viruses. Non-limiting examples of viral infections include: hepatitis virus diseases (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), herpesvirus infections (e.g., HSV-1, HSV-2, shingles), flavivirus infections, Zika virus infections, cytomegalovirus infections, respiratory virus infections (e.g., adenovirus infections, influenza, severe acute respiratory syndrome, coronavirus infections (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV, COVID-19, MERS)), gastrointestinal virus infections (e.g., norovirus infections, rotavirus infections, astrovirus infections), exanthematous virus infections (e.g., measles, shingles, smallpox, rubella), viral hemorrhagic diseases (e.g., Ebola, Lassa fever, dengue fever, yellow fever), neuroviral infections (e.g., West Nile virus infection, poliomyelitis, viral meningitis, viral encephalitis, Japanese encephalitis, rabies), and human papillomavirus infections.

[0148] Recombinant coronavirus peptide In one aspect, this disclosure provides a recombinant polypeptide comprising an antigenic fragment of a coronavirus spike glycoprotein, the antigenic fragment comprising a receptor-binding domain (RBD) and optionally a coronavirus subdomain 1 (SD1). The antigenic fragment comprises RBD-SD1. In some embodiments, the antigenic fragment substantially comprises RBD-SD1. In some embodiments, the antigenic fragment comprises an RBD-SD1 portion isolated from the remainder of the spike glycoprotein. Some flanking sequences around the domain may be used. In some embodiments, the antigenic fragment comprises RBD. Amino acid substitutions may be introduced into coronavirus spike glycoproteins or fragments thereof from a variety of coronavirus strains, including but not limited to MERS, SARS-CoV-1, and SARS-CoV-2.

[0149] An exemplary SARS-CoV-2 spike glycoprotein sequence is shown herein as SEQ ID NO: 1. In some embodiments, the polypeptide comprises a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 1.

[0150]

[0151] Table 1 provides illustrative antigen fragment sequences from various coronavirus strains. In some embodiments, the antigen fragment sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 99%, or 100% identity with any polypeptide sequence listed in Table 1.

[0152] Table 1.

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165] In some embodiments, SD1 comprises at least one surface-exposed nonpolar amino acid residue replaced by a polar amino acid residue. In some embodiments, the polypeptide comprises one, two, or more amino acids at positions 560, 562, or 569 replaced by polar residues, wherein such replacement is relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the polypeptide comprises an amino acid replacement of one or more, two or more, or three of L560Q, F562T, F562Y, or I569S, relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the polypeptide comprises a replacement of one, two, or more amino acids at positions 544, 546, 560, 562, 564, 569, or 582, relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, relative to the reference sequence according to SEQ ID NO: 1, the polypeptide comprises one, two or more, three or more, four or more amino acid substitutions at positions N544L, N544M, N544Q, L546V, L560Q, F562T, F562Y, Q564C, Q564L, Q564N, Q564W, I569S or L582S.

[0166] In some embodiments, the polypeptide comprises substitutions of one or more, two or more, three or more, four or more amino acids at positions 338, 358, 363, 365, 392, or 395, relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the polypeptide comprises substitutions of one or more, two or more, three or more, four or more amino acids at positions F338L, I358F, A363L, Y365F, Y365M, Y365W, F392W, or V395I, relative to the reference sequence according to SEQ ID NO: 1. In some embodiments, the polypeptide comprises amino acid substitutions including those shown in Table 2.

[0167] Table 2.

[0168] In some embodiments, relative to the reference sequence according to SEQ ID NO: 1, the polypeptide comprises one, two or more, three or more, four or more amino acid substitutions at positions 329, 348, 350, 367, 375, 402, 407, 410, 418, 429, 433, 435, 452, 464, 510, 512, 514, 517, 518, 519, 520, 522, 527 or 528.

[0169] In some embodiments, relative to the reference sequence according to SEQ ID NO: 1, the polypeptide is contained at positions F329K, F329R, F329Y, A348P, V350L, V367F, F375Y, I402V, V407L, I410F, I418V, F429W, V433I, A435I, A435V, L452R, F464Y, P512Q, V510I, V512F, V512I, S514T, L517A, L517D, L517S, L517T, L518N, L The substitution of one, two or more, three or more, or four or more amino acids at 518Q, L518V, H519D, H519G, H519R, H519S, H519T, A520C, A520D, A520G, A520H, P521A, P521D, P521N, P521Q, P521S, A522G, A522I, P527N, K528I, K528Q, K528T, L518G, or L518S.

[0170] In some embodiments, the polypeptide comprises amino acid substitutions at positions 365, 395, 560, 562, and 569; and one, two, or three or more amino acid substitutions at positions 348, 402, 464, 514, 520, 526, and 527, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1.

[0171] In some embodiments, the polypeptide includes amino acid substitutions as shown in Table 3.

[0172] Table 3.

[0173] In some embodiments, the polypeptide comprises amino acid substitutions at positions 365, 395, 560, 562, and 569; and one, two, or three or more amino acid substitutions at positions 348, 402, 464, 514, 520, 526, and 527, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1.

[0174] In some embodiments, the polypeptide comprises amino acid substitutions at positions 365, 395, 560, 562, and 569; and one, two, or three or more amino acid substitutions at positions 329, 517, 519, 520, and 544, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1.

[0175] In some embodiments, the polypeptide includes amino acid substitutions as shown in Table 4.

[0176] Table 4.

[0177] In some embodiments, the polypeptide comprises amino acid substitutions at positions 365, 392, 395, 560, 562, and 569; and one, two, or three or more amino acid substitutions at positions 329, 517, 519, 520, and 544, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1.

[0178] In some embodiments, the polypeptide includes amino acid substitutions as shown in Table 5.

[0179] Table 5.

[0180] In some embodiments, the polypeptide comprises amino acid substitutions at positions 365, 395, 517, 519, 520, 560, 562, 564, and 569; and one, two, or more, or three or more amino acid substitutions at positions 521, 544, and 546, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1.

[0181] In some embodiments, the polypeptide includes amino acid substitutions as shown in Table 6.

[0182] Table 6.

[0183] In some embodiments, the polypeptide includes amino acid substitutions at positions 365, 395, 517, 519, and 520; and one or more amino acid substitutions at positions 518 and 392, which are relative to the reference sequence according to SEQ ID NO: 1.

[0184] In some embodiments, the polypeptide includes amino acid substitutions as shown in Table 7.

[0185] Table 7.

[0186] In some embodiments, the polypeptide comprises amino acid substitutions at positions 329, 365, 395, 517, 519, 520, 560, 562, and 569; and one, two, or more, or three or more amino acid substitutions at positions 392, 544, and 564, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1.

[0187] In some embodiments, the polypeptide includes amino acid substitutions as shown in Table 8.

[0188] Table 8.

[0189] In some embodiments, the polypeptide includes amino acid substitutions at positions 365, 395, 517, 519, 520, 521, 504, 560, 562, 569, and 329; and one or more amino acid substitutions at position 392, which are relative to the reference sequence according to SEQ ID NO: 1.

[0190] In some embodiments, the polypeptide includes amino acid substitutions as shown in Table 9.

[0191] Table 9.

[0192] In some embodiments, the polypeptide includes multiple amino acid substitutions at positions 329, 365, 395, 526, 527, 528, 560, 562, and 569.

[0193] In some embodiments, the polypeptide includes amino acid substitutions as shown in Table 10.

[0194] Table 10.

[0195] In some embodiments, the polypeptide comprises a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 2-10.

[0196] In some embodiments, the polypeptide comprises a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NO: 11-19.

[0197] In some embodiments, the polypeptide comprises the same polypeptide sequence as any of SEQ ID NO: 11-19.

[0198] In some embodiments, the polypeptide is expressed at a higher level than a reference polypeptide without the amino acid substitution, or optionally a reference polypeptide according to SEQ ID NO: 2 or 10.

[0199] In some implementations, expression can be detected using conventional techniques for detecting proteins (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0200] In some implementations, the expressed peptide is more thermally stable or more antigenic than SEQ ID NO: 2 or 10.

[0201] In some embodiments, the polypeptide comprises multiple amino acid substitutions at positions 329, 367, 383, 547, 568, 570, and 619. In some embodiments, the polypeptide comprises amino acid substitutions at positions F329Y, F329S, V367F, S383C, T547C, D568N, A570C, and E619Q.

[0202] In some implementations, antigenicity is measured via biological layer interference (BLI) using immobilized ACE2-Fc, S309, and CR3022 antibodies.

[0203] As used herein, the term “thermal stability” or “thermal stability” of a protein is understood as the stability of a protein with respect to denaturation due to temperature changes, and can be determined by the kinetics of protein denaturation during heating. The melting temperature (T0) of the compositions of the invention can be measured using any suitable technique. m ) or starting temperature (T) 起始 Thermal stability is assessed using [a method]. The melting temperature is the temperature at which the first derivative of the melting curve reaches a local maximum. The onset temperature is the temperature at which the linear regression of the baseline intersects with the linear regression of the melting transition.

[0204] In some embodiments, nano-differential scanning fluorescence (nanoDSF) can determine thermal stability by monitoring intrinsic tryptophan fluorescence. In some embodiments, static light scattering (SLS) can be used to reveal the aggregation temperature.

[0205] In some implementations, thermal stability can be assessed by analytical spectroscopy. An exemplary analytical spectroscopic method is circular dichroism (CD) spectroscopy. CD spectroscopy measures the optical activity of a composition as it changes with increasing temperature. Circular dichroism (CD) spectroscopy measures the difference in absorption between left-handed and right-handed polarized light due to structural asymmetry. The CD spectra produced by disordered or unfolded structures are very different from those of ordered or folded structures. CD spectra reflect the sensitivity of proteins to the denaturing effects of elevated temperatures and thus indicate the thermal stability of proteins (see, for example, van Mierlo and Steemsma, J. BiotechnoL, 79(3):281-98, 2000).

[0206] Another exemplary analytical spectroscopic method for measuring thermal stability is fluorescence emission spectroscopy. Fluorescence-based thermal stability assessment methods monitor changes in fluorescence of intrinsic fluorophores (e.g., tryptophan and tyrosine amino acids) or extrinsic fluorophores (e.g., ANS or SYPRO orange) during pyrolysis (see, for example, Niesen F. PL, Berglund H. and Vedadi M.: The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature Protocols 2007, 2:2212-21). Yet another exemplary analytical spectroscopic method for measuring the thermal stability of antibodies, light chains, or heavy chains is nuclear magnetic resonance (NMR) spectroscopy (see, for example, van Mierlo and Steemsma, J. Biotechnol., 79(3):281-98, 2000).

[0207] In some embodiments, the melting temperature (Tm) of the expressed peptide, as described herein, is about 30°C. In some embodiments, the melting temperature (Tm) is about 35°C. In some embodiments, the melting temperature (Tm) is about 40°C. In some embodiments, the melting temperature (Tm) is about 45°C. In some embodiments, the melting temperature (Tm) is about 50°C. In some embodiments, the melting temperature (Tm) is about 55°C. In some embodiments, the melting temperature (Tm) is about 60°C. In some embodiments, the melting temperature (Tm) is about 65°C. In some embodiments, the melting temperature (Tm) is about 70°C. In some embodiments, the melting temperature (Tm) is about 75°C.

[0208] In some embodiments, the melting temperature (Tm) of the expressed peptide, as described herein, is at least about 30°C. In some embodiments, the melting temperature (Tm) is at least about 35°C. In some embodiments, the melting temperature (Tm) is at least about 40°C. In some embodiments, the melting temperature (Tm) is at least about 45°C. In some embodiments, the melting temperature (Tm) is at least about 50°C. In some embodiments, the melting temperature (Tm) is at least about 55°C. In some embodiments, the melting temperature (Tm) is at least about 60°C. In some embodiments, the melting temperature (Tm) is at least about 65°C. In some embodiments, the melting temperature (Tm) is at least about 70°C. In some embodiments, the melting temperature (Tm) is at least about 75°C.

[0209] In some embodiments, the melting temperature (Tm) is 30°C to 75°C. In some embodiments, the melting temperature (Tm) is 35°C to 75°C. In some embodiments, the melting temperature (Tm) is 40°C to 75°C. In some embodiments, the melting temperature (Tm) is 45°C to 75°C. In some embodiments, the melting temperature (Tm) is 50°C to 75°C. In some embodiments, the melting temperature (Tm) is 55°C to 75°C. In some embodiments, the melting temperature (Tm) is 60°C to 75°C. In some embodiments, the melting temperature (Tm) is 65°C to 75°C. In some embodiments, the melting temperature (Tm) is 70°C to 75°C. In some embodiments, the melting temperature (Tm) is 30°C to 70°C. In some embodiments, the melting temperature (Tm) is 35°C to 70°C. In some embodiments, the melting temperature (Tm) is 40°C to 70°C. In some embodiments, the melting temperature (Tm) is 45°C to 70°C. In some embodiments, the melting temperature (Tm) is 50°C to 70°C. In some embodiments, the melting temperature (Tm) is 55°C to 70°C. In some embodiments, the melting temperature (Tm) is 60°C to 70°C. In some embodiments, the melting temperature (Tm) is 65°C to 70°C. In some embodiments, the melting temperature (Tm) is 30°C to 65°C. In some embodiments, the melting temperature (Tm) is 35°C to 65°C. In some embodiments, the melting temperature (Tm) is 35°C to 65°C. In some embodiments, the melting temperature (Tm) is 40°C to 65°C. In some embodiments, the melting temperature (Tm) is 45°C to 65°C. In some embodiments, the melting temperature (Tm) is 50°C to 65°C. In some embodiments, the melting temperature (Tm) is 55°C to 65°C. In some embodiments, the melting temperature (Tm) is 60°C to 65°C. In some embodiments, the melting temperature (Tm) is 35°C to 60°C. In some embodiments, the melting temperature (Tm) is 40°C to 60°C. In some embodiments, the melting temperature (Tm) is 45°C to 60°C. In some embodiments, the melting temperature (Tm) is 50°C to 60°C. In some embodiments, the melting temperature (Tm) is 55°C to 60°C.

[0210] In some embodiments, the melting temperature (Tm) of the expressed peptide as described herein is increased by about 0.1 °C. In some embodiments, the melting temperature (Tm) of the expressed peptide as described herein is increased by about 0.2 °C. In some embodiments, the melting temperature (Tm) of the expressed peptide as described herein is increased by about 0.3 °C. In some embodiments, the melting temperature (Tm) of the expressed peptide as described herein is increased by about 0.4 °C. In some embodiments, the melting temperature (Tm) of the expressed peptide as described herein is increased by about 0.5 °C. In some embodiments, the melting temperature (Tm) of the expressed peptide as described herein is increased by about 0.6 °C. In some embodiments, the melting temperature (Tm) of the expressed peptide as described herein is increased by about 0.7 °C. In some embodiments, the melting temperature (Tm) of the expressed peptide as described herein is increased by about 0.8 °C. In some embodiments, the melting temperature (Tm) of the expressed peptide as described herein is increased by about 0.9 °C. In some embodiments, the melting temperature (Tm) of the expressed peptide as described herein is increased by about 1.0 °C.

[0211] connector In some implementations, recombinant peptides and protein nanostructures can be genetically fused so that they both exist within a single peptide, termed a "fusion protein." The connection between the peptide and the protein nanostructure allows the recombinant peptide to be displayed on the outside of the self-assembled protein nanostructure.

[0212] Various polypeptide sequences can be used to link proteins or their antigenic fragments to protein nanostructures. In some cases, the linker comprises a polypeptide sequence that can be included in the coding polynucleotide sequence. Any suitable linker polypeptide can be used. In some embodiments, the linker maintains a rigid relative orientation between the antigen protein (e.g., a coronavirus polypeptide comprising a receptor-binding domain (RBD) and an RBD-adjacent subdomain 1 (SD1) from the spike glycoprotein) and its antigenic fragments to the protein nanostructure. In some embodiments, the linker flexibly links the antigen protein (e.g., a coronavirus polypeptide comprising a receptor-binding domain (RBD) and an RBD-adjacent subdomain 1 (SD1) from the spike glycoprotein) and its antigenic fragments to the protein nanostructure. In some embodiments, the encoded polypeptide may include a linker located between regions. In some embodiments, the polypeptide is a fusion protein comprising a recombinant coronavirus polypeptide, a linker, and a protein nanostructure component polypeptide. In some embodiments, the polypeptide is a fusion protein comprising, from N-terminus to C-terminus, a recombinant coronavirus polypeptide, a linker, and a protein nanostructure component polypeptide. The linker may be a polypeptide. Various polypeptide sequences are available and are well known in the art. In some embodiments, the linker may comprise a Gly-Ser linker of any suitable length (i.e., a linker composed of glycine and serine residues). In some embodiments, the length of the Gly-Ser linker may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues. Non-limiting examples of Gly-Ser linkers are shown below: Table 11 provides illustrative peptide linkers. In some embodiments, the peptide linker is any sequence in Table 11.

[0213] In some embodiments, the linker contains between 3 and 30 amino acid residues. In some embodiments, the linker contains between 4 and 24 amino acid residues. In some embodiments, the linker contains between 8 and 24 amino acid residues. In some embodiments, the linker contains between 10 and 24 amino acid residues. In some embodiments, the linker contains between 12 and 24 amino acid residues. In some embodiments, the linker contains between 16 and 24 amino acid residues. In some embodiments, the linker contains between 18 and 24 amino acid residues. In some embodiments, the linker contains between 20 and 24 amino acid residues. In some embodiments, the linker contains between 4 and 20 amino acid residues. In some embodiments, the linker contains between 8 and 20 amino acid residues. In some embodiments, the linker contains between 10 and 20 amino acid residues. In some embodiments, the linker contains between 12 and 20 amino acid residues. In some embodiments, the linker contains between 16 and 20 amino acid residues. In some embodiments, the linker contains between 8 and 18 amino acid residues. In some embodiments, the linker contains between 12 and 16 amino acid residues.

[0214] In some embodiments, the linker contains 3 amino acid residues. In some embodiments, the linker contains 4 amino acid residues. In some embodiments, the linker contains 7 amino acid residues. In some embodiments, the linker contains 8 amino acid residues. In some embodiments, the linker contains 10 amino acid residues. In some embodiments, the linker contains 12 amino acid residues. In some embodiments, the linker contains 16 amino acid residues. In some embodiments, the linker contains 18 amino acid residues. In some embodiments, the linker contains 19 amino acid residues. In some embodiments, the linker contains 20 amino acid residues. In some embodiments, the linker contains 24 amino acid residues. In some embodiments, the linker contains 30 amino acid residues.

[0215] In some embodiments, the encoded polypeptide may include a linker located between the regions. In some embodiments, the polypeptide is a fusion protein comprising a recombinant SARS-CoV-2 polypeptide, a linker, an N-terminal extension linker, and a protein nanostructure component polypeptide. In some embodiments, the polypeptide is a fusion protein comprising, from N-terminus to C-terminus, a recombinant SARS-CoV-2 polypeptide, a linker, an N-terminal extension linker, and a protein nanostructure component polypeptide. In some embodiments, the N-terminal extension linker is an I53-50A helical extension. In some embodiments, the polypeptide sequence of the N-terminal extension linker is EKAAKAEEAARK (SEQ ID NO: 237).

[0216] In some implementations, the connectors are selected from the group shown in Table 11.

[0217] Table 11.

[0218] Self-assembled protein nanostructures In another aspect, this disclosure provides a self-assembling protein nanostructure comprising a first component containing a recombinant polypeptide comprising an antigenic fragment of a coronavirus spike glycoprotein as described above. Optionally, the self-assembling protein nanostructure may include a second component containing a second protein. The first component, as a trimer assembly, contains a protein-protein interface that induces regional self-binding of the first component to form a trimer building block. In a protein nanostructure having two or more components, each copy of the first assembly domain further contains a surface-exposed interface that interacts with a complementary surface-exposed interface on the second assembly domain. Similarly, the second component, as a pentamer assembly, is adapted to polymerize with the first component. As described in King et al. (Nature 510, 103-108, 2014), Bale et al. (Science 353, 389-394, 2016), and patent publications WO2014124301 A1 and US20160122392 A1, a complementary protein-protein interface between a first component and a second component drives the assembly of multiple copies of a trimer assembly domain and a second assembly domain into a target protein nanostructure. In some embodiments, each of the trimer assembly domains of the protein nanostructure carries an antigen protein or an antigen fragment thereof (e.g., as a gene fusion) linked thereto; these protein nanostructures exhibit the protein in its full valence state. In other embodiments, the protein nanostructures of this disclosure comprise one or more first assembly domains carrying an antigen protein or an antigen fragment thereof (e.g., as a gene fusion), and one or more first assembly domains not carrying an antigen protein; these protein nanostructures exhibit the protein in a partial valence state. The first assembly domain may be any polypeptide sequence that forms a trimer and interacts with the second assembly domain to drive assembly into a target protein nanostructure. In some embodiments, the protein nanostructure includes a first polypeptide and a second polypeptide selected from those disclosed in US 20130274441 A1, US 20150356240 A1, US 20160122392 A1, and WO2018187325 A1, each of which is incorporated herein by reference in its entirety.

[0219] In some embodiments, a single component self-assembles into a protein nanostructure. In some embodiments, one or more purified samples of the first and second components used to form the protein nanostructure are mixed in an approximately equimolar molar ratio under aqueous conditions (e.g., I53-50A / B icosahedral protein nanostructures). The first and second components interact with each other (through assembly domains) to drive the assembly of the target protein nanostructure. Successful assembly of the target protein nanostructure can be confirmed by analyzing the in vitro assembly reaction using common biochemical or biophysical methods for assessing the physical size of proteins or protein assemblies, including but not limited to size exclusion chromatography, native (non-denaturing) gel electrophoresis, dynamic light scattering, multi-angle light scattering, analytical ultracentrifugation, negative staining electron microscopy, cryo-electron microscopy, or X-ray crystallography. Where necessary, common preparative techniques for separating proteins based on their physical size can be used to purify the assembled protein nanostructure from other substances or molecules present in the in vitro assembly reaction. These preparative techniques include, but are not limited to, size exclusion chromatography, preparative ultracentrifugation, tangential flow filtration, or preparative gel electrophoresis. The presence of antigenic proteins in protein nanostructures can be assessed using common techniques for identifying protein molecules in aqueous solutions, including but not limited to SDS-PAGE, mass spectrometry, protein sequencing, ELISA, surface plasmon resonance, biolayer interferometry, or amino acid analysis. The accessibility of proteins located outside the protein nanostructure, as well as their conformation or antigenicity, can be assessed using common techniques for detecting the presence and conformation of antigens, including but not limited to monoclonal antibody binding, conformation-specific monoclonal antibody binding, surface plasmon resonance, biolayer interferometry, or antigen-specific antiserum binding.

[0220] In various embodiments, the protein nanostructures of this disclosure comprise two or more distinct first polypeptides carrying different antigenic proteins as gene fusions; these protein nanostructures co-exhibit multiple distinct proteins (e.g., different variants of coronavirus polypeptides including a receptor-binding domain (RBD) and an RBD-adjacent subdomain 1 (SD1) from a spike glycoprotein) on the same protein nanostructure. These multi-antigen protein nanostructures are generated by in vitro assembly with a mixture of two or more antigens, each containing an assembly domain. The proportion of each antigen in the mixture determines the average valence state of each antigenic protein in the resulting protein nanostructure. The presence and average valence state of each antigen in a given sample can be assessed quantitatively using the techniques described above for evaluating the presence of antigenic proteins in a full-valence protein nanostructure.

[0221] The protein nanostructures of the present invention may comprise multimeric protein assemblies suitable for displaying coronavirus polypeptides, the coronavirus polypeptides including a receptor-binding domain (RBD) and an RBD-adjacent subdomain 1 (SD1) from a spike glycoprotein, or an antigenic variant or fragment thereof. The protein nanostructures of the present invention comprise at least a first component and optionally a second component. The first component may comprise a substitution of at least one amino acid residue or the addition of one or more amino acid residues at the N-terminus or C-terminus. In some embodiments, the fusion protein comprises a protein sequence determined by computational methods. The first component may form the entire core of the protein nanostructure; or the core of the protein nanostructure may comprise the second component or third, fourth, fifth, etc., components. In some embodiments, the first component is a trimer component, wherein the assembly domains form trimers linked by 3-fold rotational symmetry, and / or the second component is a pentamer component, wherein the assembly domains form pentamers linked by 5-fold rotational symmetry. In some embodiments, a combination of the two components forms an "icosahedral particle" with I53 symmetry. These components may be arranged such that members of each component are linked to each other through symmetry operations. US2015 / 0356240 A1 discloses a general computational method for designing self-assembled protein materials, which involves achieving symmetric docking of protein building blocks in a target-symmetric architecture.

[0222] The term "core" in this document refers to the central portion of a protein nanostructure that links together several copies of a polypeptide comprising an antigenic fragment of the coronavirus spike glycoprotein, as displayed by the protein nanostructure. In some embodiments, the fusion protein comprises a first polypeptide, a linker, and a first assembly domain, the first polypeptide comprising the antigenic fragment of the coronavirus spike glycoprotein. In some embodiments, the antigenic fragment of the coronavirus spike glycoprotein is non-covalently or covalently linked to the assembly domain. For example, an antibody or its antigenic fragment may be fused to the first component and configured to bind a portion of the first component or a chemical tag on the first component. For example, a streptavidin-biotin (or neuroavidin-biotin) linker may be used. Alternatively, various bioconjugated linkers may be used. In some embodiments of this disclosure, the antigen comprises other polypeptide sequences besides the coronavirus polypeptide.

[0223] A non-limiting example of the implementation scheme is an antigenic fragment of the spike glycoprotein of a coronavirus fused with a component gene of a protein nanostructure, which optionally is present in a host cell (e.g., E. coliThe first component is a recombinant protein assembly expressed in the same or different host cells; and the second component is a pentamer protein assembly, optionally recombinantly expressed in the same or different host cells. These two components self-assemble into a protein nanostructure displaying 60 antigen fragment monomers around an icosahedral core. In some embodiments, antigen fragments of the coronavirus spike glycoprotein are mixed with another antigen protein in the same protein nanostructure, such as two different variants of the coronavirus spike glycoprotein. In some embodiments, in addition to one or more antigen fragments of the coronavirus spike glycoprotein (including a receptor-binding domain (RBD) and optionally an RBD-adjacent subdomain 1 (SD1)), the protein nanostructure also contains antigens of other pathogenic organisms and is therefore usable as a combination vaccine. In some embodiments, the protein nanostructure is further linked to a peptide or other agent capable of acting as an adjuvant. In some embodiments, the first and / or second components contain one or more T-cell epitopes, optionally heterologous T-cell epitopes.

[0224] Other potential arrangements of the components disclosed herein include protein nanostructures suitable for displaying up to 12, 24, or 60 monomers. In some embodiments, the components may comprise peptides linked to multiple antigens, such that the protein nanostructure displays different antigens on the same nanostructure. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more different antigens are displayed. Non-limiting illustrative protein nanostructures are provided by Bale et al. Science 353:389-94 (2016); Heinze et al. J. Phys. Chem B. 120:5945-5952 (2016); King et al. Nature 510:103-108 (2014); and King et al. Science 336:1171-71 (2012).

[0225] Connection mode The protein nanostructures disclosed herein display antigen proteins in various ways, including as gene fusions or through other methods disclosed herein. As used herein, “linked / attached” means any method known in the art for binding two peptides. Binding can be direct or indirect, reversible or irreversible, weak or strong, covalent or non-covalent, selective or non-selective.

[0226] In some implementations, the connection is achieved through genetic engineering to generate N-terminal or C-terminal fusions of potential antigenic peptides in protein nanostructures.

[0227] In some embodiments, the linking is achieved through post-translational covalent linkage of one or more antigen proteins. In some embodiments, chemical crosslinking is used to non-specifically link the antigen to the protein nanostructure. In some embodiments, chemical crosslinking is used to specifically link the antigen protein to the protein nanostructure (e.g., to a first polypeptide or a second polypeptide). Various specific and non-specific crosslinking chemistry methods are known in the art, such as click chemistry and other methods. Generally, any crosslinking chemical / bioconjugation used to link two proteins is applicable to the protein nanostructures disclosed in this invention. In particular, chemical methods used to generate immunoconjugates or antibody-drug conjugates can be employed. In some embodiments, cleavable or non-cleavable linkers are used to generate the protein nanostructure. For example, US Patent Publication No. 2008 / 0145373 A1 provides a process and method for conjugating an antigen to a carrier.

[0228] In some embodiments, the connection is achieved through a non-covalent link between the component and the antigen. In some embodiments, the antigen protein is engineered to be negatively charged on at least one surface, and the core polypeptide is engineered to be positively charged on at least one surface, or both positively and negatively charged. This facilitates intermolecular binding between the antigen protein and the component core polypeptide via electrostatic forces. In some embodiments, shape complementarity is employed to generate the connection between the antigen protein and the component core. Shape complementarity may be pre-existing or rationally designed. In some embodiments, the connection is achieved using computational design of protein-protein interfaces. In some embodiments, the antigen is biotinylated, and the polypeptide contains streptavidin, or vice versa. In some embodiments, streptavidin is displayed as a tetramer on the four-fold axis of the component core via gene fusion or otherwise, and the biotinylated antigen is a monomer, dimer, or tetramer, thereby allowing binding to the component core in a conformation suitable for the natural multimerization of the antigen. In some embodiments, a protein-based adaptor is employed to capture the antigen protein. In some embodiments, the polypeptide is fused to a protein capable of binding a complementary protein, which is fused to the antigen protein.

[0229] The immunogenicity of an antigen protein can be controlled by altering its orientation relative to a component core. Depending on how the antigen protein attaches to the component core of the protein nanostructure, it can be displayed in various orientations. In some embodiments, the antigen protein is displayed such that one or more epitopes are oriented distally or toward the distal end of the antigen protein, making these epitopes easily accessible to the immune system. In some embodiments, the orientation will reproduce the orientation of the spike glycoprotein relative to the virus. The choice of orientation can direct the immune system toward one epitope or another.

[0230] In some implementations, epitope preference is controlled in other ways, such as by adding or subtracting N-linked glycan sequence motifs NX-[T / S] at predetermined positions in the amino acid sequence of the component (including in the amino acid sequence of the antigen protein), thereby localizing the glycan onto the protein nanostructure.

[0231] In some embodiments, epitopes found at an intermediate distance from proximal to distal will be preferred over epitopes located further distally, depending on various considerations, including but not limited to: the overall geometry of the protein nanostructure, surface hydrophobicity, surface charge, and competitive binding to endogenously present proteins in the subject or exogenously provided proteins in the vaccine composition. This disclosure covers all known methods of rationally designing protein structures, and the foregoing is not intended to be limiting.

[0232] polypeptide sequence Patent publication US 2015 / 0356240 A1 describes various methods for designing protein assemblies. As described in US Patent Publication US 2016 / 0122392 A1 and International Patent Publication WO 2014 / 124301 A1, the isolated peptides shown in Table 12 are designed to be capable of pairwise self-assembly to form protein nanostructures (such as icosahedral particles). This design involves designing suitable interface residues for each member of the peptide pair, enabling the peptide pair to assemble into a protein nanostructure. The protein nanostructure thus formed comprises a symmetrically repeating, non-natural, non-covalent peptide-peptide interface that orients the first and second assemblies into a protein nanostructure, for example, a structure with icosahedral symmetry. Thus, in one embodiment, the first and second repeating elements of the components are selected from the group consisting of SEQ ID NO: 23-73. In each case, N-terminal methionine residues present in the full-length protein are included but may be removed to produce fusions not included in the sequence. The residues identified in Table 12 are numbered starting from the N-terminal methionine (not shown). In various embodiments, one or more additional residues are deleted from the N-terminus, and / or additional residues are added to the N-terminus (e.g., to form a helical extension).

[0233] Table 12

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246] Table 12 provides the amino acid sequences of the first and second repeating elements of the embodiments of this disclosure. In each case, the sequences are paired together to form an I53 polymer with icosahedral symmetry. The right column in Table 12 identifies the residue numbers (i.e., "identified interface residues") in each exemplary polypeptide that were identified as present at the interface of the resulting assembled protein nanostructure. It can be seen that the number of interface residues in the illustrative polypeptides of SEQ ID NO: 23-56 is in the range of 4-13. In various embodiments, the first and second repeating elements comprise amino acid sequences that, compared to the amino acid sequences of a polypeptide selected from the group consisting of SEQ ID NO: 23-56, have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity in length, and are identical at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 identified interface positions (depending on the number of interface residues in the given polypeptide). SEQ ID NO: 57-84 represents other amino acid sequences from the first and second repeating elements of embodiments of this disclosure. In other embodiments, the first repeating element and / or the second repeating element comprises an amino acid sequence that, compared to the amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NO: 23-84, 209-225, has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity in its length, and is identical at at least 20%, at least 25%, at least 33%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or 100% of the identified interface positions.

[0247] Similar to most proteins, it is expected that some variations in the peptide's designed sequence will not interfere with subsequent assembly into protein nanostructures, especially when such variations involve conserved amino acid substitutions. As used herein, "conserved amino acid substitution" means: a hydrophobic amino acid (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) is substituted for another hydrophobic amino acid; a hydrophobic amino acid with a large side chain (Phe, Tyr, Trp) is substituted for another hydrophobic amino acid with a large side chain; an amino acid with a positively charged side chain (Arg, His, Lys) is substituted for another amino acid with a positively charged side chain; an amino acid with a negatively charged side chain (Asp, Glu) is substituted for another amino acid with a negatively charged side chain; and an amino acid with a polar, uncharged side chain (Ser, Thr, Asn, Gln) is substituted for another amino acid with a polar, uncharged side chain.

[0248] In various embodiments of the protein nanostructures of the present invention, the first and second repeating elements, or vice versa, comprise a polypeptide having an amino acid sequence selected from the following paired amino acid sequences, or a modified form thereof (i.e., permissible modifications as disclosed for the polypeptides of the present invention: a separate polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity in length and / or at least the same position at one identified interface): SEQ ID NO: 23 and SEQ ID NO: 24 (I53-34A and I53-34B); SEQ ID NO: 25 and SEQ ID NO: 26 (I53-40A and I53-40B); SEQ ID NO: 25 and SEQ ID NO: 46 (I53-40A and I53-40B.1); SEQ ID NO: 45 and SEQ ID NO: 26 (I53-40A.1 and I53-40B); SEQ ID NO: 57 and SEQ ID NO: 58 (Class I53-40A and Class I53-40B); SEQ ID NO: 27 and SEQ ID NO: 28 (I53-47A and I53-47B); SEQ ID NO: 27 and SEQ ID NO: 49 (I53-47A and I53-47B.1); SEQ ID NO: 27 and SEQ ID NO: 50 (I53-47A and I53-47B.1NegT2); SEQ ID NO: 47 and SEQ ID NO: 28 (I53-47A.1 and I53-47B); SEQ ID NO: 47 and SEQ ID NO: 49 (I53-47A.1 and I53-47B.1); SEQ ID NO: 47 and SEQ ID NO: 50 (I53-47A.1 and I53-47B.1NegT2); SEQ ID NO: 48 and SEQ ID NO: 28 (I53-47A.1NegT2 and I53-47B); SEQ ID NO: 48 and SEQ ID NO: 49 (I53-47A.1NegT2 and I53-47B.1); SEQ ID NO: 48 and SEQ ID NO: 50 (I53-47A.1NegT2 and I53-47B.1NegT2); SEQ ID NO: 59 and SEQ ID NO: 60 (Class I53-47A and Class I53-47B); SEQ ID NO: 29 and SEQ ID NO: 30 (I53-50A and I53-50B); SEQ ID NO: 29 and SEQ ID NO: 54 (I53-50A and I53-50B.1); SEQ ID NO: 29 and SEQ ID NO: 55 (I53-50A and I53-50B.1NegT2); SEQ ID NO: 29 and SEQ ID NO: 56 (I53-50A and I53-50B.4PosT1); SEQ ID NO: 51 and SEQ ID NO: 30 (I53-50A.1 and I53-50B); SEQ ID NO: 51 and SEQ ID NO: 54 (I53-50A.1 and I53-50B.1); SEQ ID NO: 51 and SEQ ID NO: 55 (I53-50A.1 and I53-50B.1NegT2); SEQ ID NO: 51 and SEQ ID NO: 56 (I53-50A.1 and I53-50B.4PosT1); SEQ ID NO: 52 and SEQ ID NO: 30 (I53-50A.1NegT2 and I53-50B); SEQ ID NO: 52 and SEQ ID NO: 54 (I53-50A.1NegT2 and I53-50B.1); SEQ ID NO: 52 and SEQ ID NO: 55 (I53-50A.1NegT2 and I53-50B.1NegT2); SEQ ID NO: 52 and SEQ ID NO: 56 (I53-50A.1NegT2 and I53-50B.4PosT1); SEQ ID NO: 53 and SEQ ID NO: 30 (I53-50A.1PosT1 and I53-50B); SEQ ID NO: 53 and SEQ ID NO: 54 (I53-50A.1PosT1 and I53-50B.1); SEQ ID NO: 53 and SEQ ID NO: 55 (I53-50A.1PosT1 and I53-50B.1NegT2); SEQ ID NO: 53 and SEQ ID NO: 56 (I53-50A.1PosT1 and I53-50B.4PosT1); SEQ ID NO: 61 and SEQ ID NO: 62 (Class I53-50A and Class I53-50B); SEQ ID NO: 31 and SEQ ID NO: 32 (I53-51A and I53-51B); SEQ ID NO: 33 and SEQ ID NO: 34 (I52-03A and I52-03B); SEQ ID NO: 35 and SEQ ID NO: 36 (I52-32A and I52-32B); SEQ ID NO: 37 and SEQ ID NO: 38 (I52-33A and I52-33B) SEQ ID NO: 39 and SEQ ID NO: 40 (I32-06A and I32-06B); SEQ ID NO: 41 and SEQ ID NO: 42 (I32-19A and I32-19B); SEQ ID NO: 43 and SEQ ID NO: 44 (I32-28A and I32-28B); SEQ ID NO: 45 and SEQ ID NO: 46 (I53-40A.1 and I53-40B.1); SEQ ID NO: 63 and SEQ ID NO: 64 (T32-28A and T32-28B); SEQ ID NO: 65 and SEQ ID NO: 66 (T33-09A and T33-09B); SEQ ID NO: 67 and SEQ ID NO: 68 (T33-15A and T33-15B); SEQ ID NO: 69 and SEQ ID NO: 70 (T33-21A and T33-21B); SEQ ID NO: 71 and SEQ ID NO: 72 (T33-28A and T32-28B); and SEQ ID NO: 73 and SEQ ID NO: 66 (T33-31A and T33-09B (also known as T33-31B)).

[0249] In some embodiments, the assembly domains are I53-50A (trimer, optionally linked to an antigenic fragment of the coronavirus spike glycoprotein as described above) and I53-50B (pentamer). The I53_dn5 nanostructure is described in US2022 / 0072120 A1, the contents of which are incorporated herein by reference. Variants of I53_dn5 may include one or more amino acid substitutions, such as C94A, C119A, W18G, K84R, M88P, E91D, L117I, or L120D (collectively, “I53_dn5A.1”; Ueda et al. eLife 9:e57659 (2020), or A25E, M88A, C119T, L120E, A127E, L131T, I132K, E133A, or deletions at positions 135-137 (“I53_dn5A.2”; Wang et al. bioRxiv 2022.08.04.502842).

[0250] In some embodiments, one or more antigenic fragments of the coronavirus spike glycoprotein or its antigenic fragments are expressed as a fusion protein having a first assembly domain. In some embodiments, the first assembly domain of the coronavirus spike glycoprotein and the antigenic fragments are linked by a linker sequence.

[0251] Non-limiting examples of designed protein complexes that can be used in the protein nanostructures of this disclosure include those disclosed in the following patents: U.S. Patent No. 9,630,994, International Patent Publication No. WO2018187325A1, U.S. Patent Publication No. 2018 / 0137234 A1; and U.S. Patent Publication No. 2019 / 0155988A2, each of which is incorporated herein by reference in its entirety.

[0252] In various embodiments of the protein nanostructures disclosed herein, the assembly domain is a polypeptide having a sequence of amino acid pairs selected from the following, or a modified form thereof (i.e., permissible modifications as disclosed for the polypeptides of the present invention: a separate polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity in length and / or at least at one identified interfacial position compared to the amino acid sequence indicated by the following SEQ ID NO): SEQ ID NO: 75 and SEQ ID NO: 76 (T33_dn2A and T33_dn2B); SEQ ID NO: 77 and SEQ ID NO: 78 (T33_dn5A and T33_dn5B); SEQ ID NO: 79 and SEQ ID NO: 80 (T33_dn10A and T33_dn10B); or SEQ ID NO: 81 and SEQ ID NO: 82 (I53_dn5A and I53_dn5B).

[0253] In another aspect, this disclosure provides a self-assembled protein nanostructure comprising a first component and an optional second component, the first component comprising a recombinant coronavirus polypeptide described herein, and the second component comprising a second protein.

[0254] In some embodiments, the protein nanostructure of the recombinant coronavirus polypeptide includes a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 11-19.

[0255] In some embodiments, the protein nanostructure of the recombinant coronavirus polypeptide comprises one or more, two or more, three or more, four or more amino acid substitutions relative to the reference sequence according to SEQ ID NO: 1.

[0256] In some implementations, the protein nanostructure component peptide and the recombinant coronavirus peptide are non-covalently coupled.

[0257] In some implementations, the protein nanostructure component peptide and the recombinant coronavirus peptide are covalently coupled.

[0258] In some implementations, the protein nanostructure comprises a fusion protein, which includes a recombinant coronavirus peptide, a linker, and protein nanostructure component peptides.

[0259] In some embodiments, the protein nanostructure comprises a fusion protein that includes, from the N-terminus to the C-terminus, a recombinant coronavirus polypeptide, a linker, and a protein nanostructure component polypeptide.

[0260] In some embodiments, the protein nanostructure of the recombinant coronavirus polypeptide includes a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 11-19.

[0261] In some embodiments, the protein nanostructure of the fusion protein includes a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 11-19.

[0262] In some embodiments, the first component of the protein nanostructure is I53-50A. In some embodiments, the first component of the protein nanostructure is I53-34A. In some embodiments, the first component of the protein nanostructure is I53-40B. In some embodiments, the first component of the protein nanostructure is I53-47A. In some embodiments, the first component of the protein nanostructure is I53-51A.

[0263] In some embodiments, the protein nanostructure is an I3-01 / MI3 protein nanostructure, and in other embodiments, the protein nanostructure is an I53-50AB protein nanostructure.

[0264] In some embodiments, the protein nanostructure is a ferritin protein nanostructure. In some embodiments, the protein nanostructure is an encapsulation protein nanostructure. In some embodiments, the protein nanostructure is a CP3 phage capsid protein nanostructure. In some embodiments, the protein nanostructure is a Qβ phage capsid protein nanostructure. In some embodiments, the protein nanostructure is an AP205 phage capsid protein nanostructure. In some embodiments, the protein nanostructure comprises a first polypeptide and a second polypeptide selected from those disclosed in WO 2021252688, which is incorporated herein by reference in its entirety.

[0265] In some embodiments, the recombinant polypeptide of the protein nanostructure comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 11-19.

[0266] In some embodiments, the protein nanostructure component peptide comprises a peptide segment having a peptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 20-21.

[0267] In some implementations, the protein nanostructure includes a second component, a polypeptide.

[0268] In some embodiments, the second component peptide is I53-50B. In some embodiments, the second component peptide is I53-34B. In some embodiments, the second component peptide is I53-40B. In some embodiments, the second component peptide is I53-47B. In some embodiments, the second component peptide is I53-51B. In some embodiments, the second component of the protein nanostructure is I53-50B.4PosT1.

[0269] In some embodiments, the second component polypeptide comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:22.

[0270] Ferritin-based nanostructures In some embodiments, the assembly domain is a ferritin polypeptide. In some embodiments, the assembly domain of the ferritin protein nanostructure comprises a polypeptide sequence having at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the following sequences: MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKVELIGNENHGLYLADQYVKGIAKSRKS. (SEQ ID NO: 204) MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDAQN. (SEQ ID NO: 205) NFHQDCEAGLNRTVNLKFHSSYVYLSMASYFNRDDVALSNFAKFFRERSEEEKEHAEKLIEYQNQRGGRVFLQSVEKPERDDWANGLEALQTALKLQKSVNQALLDLHAVAADKSDPHMTDFLESPYLSESVETIKKLGDHITSLKKLWSSHPGMAEYLFNKHTLG. (SEQ ID NO: 206) QFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS. (SEQ ID NO: 207) SGESQVRQNFKPEMEEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDGS. (SEQ ID NO: 208) Other nanostructures or nanoparticles In some embodiments, the extracellular domains described herein are displayed on any nanostructures or nanoparticles known in the art. Illustrative nanostructures and nanoparticles include, but are not limited to: human papillomavirus (HPV) virus-like particles (VLPs), chikungunya virus VLPs, AP205 capsid protein VLPs, bacteriophage VLPs (e.g., bacterial bacteriophages), encapsulated protein nanostructures, CP3 bacteriophage capsid protein nanostructures, and Qβ bacteriophage capsid protein nanostructures. Display on these and other platforms can be achieved by generating fusion proteins of the extracellular domains with the associated proteins of the system, by bioconjugation chemistry (e.g., SpyCatcher), or by other means known in the art. Protein nanostructures may be lumazine synthase nanoparticles, such as those described, for example, in Geng et al., PLoS Pathog. 17(9):e1009897 (2021). Protein nanostructures can be, for example, ferritin nanoparticles as described in Joyce et al. bioRxiv 2021.05.09.443331 and U.S. Patent Publication No. US 2019 / 0330279 A1.

[0271] Polynucleotides This document provides polynucleotides encoding the polypeptide or protein nanostructures described herein. The polynucleotide sequences may comprise RNA or DNA. Isolated polynucleotides refer to those that have been removed from the normal peripheral nucleic acid sequences in a genome or cDNA sequence. Such polynucleotide sequences may include additional sequences that can be used to facilitate the expression and / or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, output and secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings herein, those skilled in the art will understand which nucleic acid sequences encode the proteins disclosed herein.

[0272] In another aspect, this disclosure provides recombinant expression vectors comprising isolated nucleic acids of any embodiment or combination of embodiments of this disclosure operably linked to suitable control sequences. A “recombinant expression vector” includes a vector operably linked to a nucleic acid coding region or gene to any control sequence capable of influencing the expression of a gene product. The “control sequence” operably linked to a nucleic acid sequence of this disclosure is a nucleic acid sequence capable of influencing the expression of a nucleic acid molecule. Control sequences do not need to be adjacent to the nucleic acid sequence, as long as they function to direct the expression of that nucleic acid sequence. Thus, for example, an intervening untranslated but transcribed sequence may exist between a promoter sequence and a nucleic acid sequence, and the promoter sequence can still be considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including but not limited to plasmid- and virus-based expression vectors. The control sequence used to drive the expression of the disclosed nucleic acid sequence in a mammalian system can be constitutive (driven by any of a variety of promoters, including but not limited to CMV, SV40, RSV, actin, EF) or inducible (driven by any of a variety of inducible promoters, including but not limited to tetracycline, ecdysone, steroid-responsive promoters). The construction of expression vectors for transfecting prokaryotic cells is also well known in the art and can therefore be achieved via standard techniques. (See, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ Murray, The Humana Press Inc., Clifton, NJ) and Ambion 1998 Catalog (Ambion, Austin, TX). The expression vector can replicate in a host organism as an episome or by integration into the host chromosomal DNA. Suitable expression vectors and hosts are well known in the art. In some embodiments, the expression vector comprises a plasmid. However, this disclosure is intended to include other expression vectors that serve an equivalent function, such as viral vectors.

[0273] In another aspect, this disclosure provides host cells transfected or transduced with the recombinant expression vectors disclosed herein, wherein the host cells may be prokaryotic or eukaryotic. Cells may be transiently or stably transfected or transduced. Such transfection or transduction of the expression vector into prokaryotic and eukaryotic cells can be achieved by any technique known in the art, including but not limited to: standard bacterial transformation, calcium phosphate coprecipitation, electroporation, or liposome-mediated, DEAE-glucan-mediated, polycation-mediated, or virus-mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2nd ed. (RI Freshney. 1987. Liss, Inc. New York, NY.))

[0274] In some implementations, the polynucleotide is messenger RNA (mRNA). Methods for generating polynucleotides by chemical synthesis or by in vitro transcription (IVT) (for mRNA) are well known in the art.

[0275] The basic structure of the encoding mRNA can resemble that of a “mature” eukaryotic mRNA and may include some or all of the following features: (i) an open reading frame (ORF) encoding the protein, flanked by (ii) 5′ and 3′ untranslated regions (UTRs), and flanked by (iii) a 7-methylguanosine 5′ cap and (iv) a 3′ poly(A) tail. Non-coding structural features can be individually optimized to modulate mRNA stability, translation efficiency, and immunogenicity. By incorporating modified nucleosides, mRNA transcripts known as “nucleoside-modified mRNAs” can be generated, exhibiting reduced immunostimulatory activity and thus improved safety profiles. Furthermore, modified nucleosides allow for the design of mRNA vaccines with significantly enhanced stability and translational capabilities, as they circumvent the direct antiviral pathway, which is induced by type I interferon and programmed to degrade and inhibit invading mRNA. For example, replacing uridine with pseudouridine reduced the activity of 2′-5′-oligoadenylate synthase, which regulates the cleavage of mRNA by RNase L. Furthermore, a decrease in the activity of protein kinase R, an enzyme involved in inhibiting mRNA translation, was detected.

[0276] The polynucleotides disclosed herein may include one or more modified (e.g., altered or substituted) nucleotides, nucleosides, nucleotides, or combinations thereof. Polynucleotides may include any useful modifications or alterations, such as modifications or alterations to the linkages between nucleotides, sugars, or nucleosides (e.g., to the linking phosphate, to the phosphodiester bond, to the phosphodiester backbone). In some embodiments, the alteration (e.g., one or more alterations) is present in each of the nucleotides, sugars, and nucleosides. The alterations according to this disclosure may be alterations to polynucleotides, such as replacing the 2′-OH of the furanyl ribosome in an RNA loop with a 2′-H, threonine nucleic acid (TNA), glycolic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof.

[0277] Polynucleotides may include a 5′ cap structure. The 5′-cap structure of a polynucleotide is involved in nuclear export and enhances polynucleotide stability, and binds to mRNA cap-binding proteins (CBPs). A 5′-UTR may be provided as a flanking region of the mRNA. In some embodiments, the polynucleotide sequence is codon-optimized. Polynucleotides may include a “polyA sequence” or a “polyadenylation signal,” and these terms are used interchangeably.

[0278] In some implementations, the mRNA contains between 100 bases and 5 kilobases (kb).

[0279] In some implementations, the mRNA contains at least 100 bases, at least 200 bases, at least 400 bases, at least 600 bases, or at least 800 bases.

[0280] In some implementations, the mRNA contains at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, or at least 5 kb.

[0281] In some implementations, the mRNA contains between 100 bases and 5 kb, between 100 bases and 3 kb, between 100 bases and 2 kb, and between 100 bases and 1 kb.

[0282] In some implementations, the mRNA contains bases between 1kb and 5kb, between 1kb and 4kb, or between 1kb and 3kb.

[0283] signal sequence In some embodiments, the encoded polypeptide includes a peptide region that serves as a signal peptide. Signal sequences are well known in the art. The signal peptide may be a native signal sequence or may be replaced by another signal sequence. The function of the signal sequence is to facilitate the transport of proteins within the cell, typically to the cell membrane. The core of a signal sequence typically contains a long segment of hydrophobic amino acids (approximately 5–16 residues long), which tends to form a single α-helix and is also referred to as the “h-region.” Furthermore, many signal sequences begin with a short, positively charged amino acid segment, which may help ensure the correct topology of the polypeptide during transport according to the so-called positive charge-in-the-in rule. Due to its proximity to the N-terminus, this segment is referred to as the “n-region.” At the end of the signal sequence, there is typically an amino acid segment that is recognized and cleaved by a signal peptidase.

[0284] In some implementations, the polypeptide does not include a signal peptide.

[0285] Table 13 provides non-limiting examples of signal peptides. Table 13.

[0286] Trimerization domain In some embodiments, the peptide may include a trimerizing domain, such as FoldOn or GCN4 trimer. In some embodiments, the linker sequence includes FoldOn, wherein the FoldOn sequence is GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 226).

[0287] In some embodiments, the polypeptide may include a trimerizing domain, wherein the trimerizing domain sequence is DKIEEILSKIYHIENEIARIKKLIGE (GEN) (SEQ ID NO: 227). In some embodiments, the polypeptide may include a trimerizing domain, wherein the trimerizing domain sequence is EKFHQIEKEFSEVEGRIQDLEK (HA) (SEQ ID NO: 228).

[0288] In some embodiments, the peptide may include a trimerizing domain, wherein the trimerizing domain sequence is EDKIEEILSKIYHIENEIARIKKLIGEA (a coiled helical isoleucine zipper) (SEQ ID NO: 229).

[0289] In some embodiments, the polypeptide may include a trimerizing domain, wherein the trimerizing domain sequence is GSGYIPEAPRDGQAYVRKDGEWVLLSTFL (phage T4 fibrin) (SEQ ID NO: 230).

[0290] In some embodiments, the trimerizing sequence is RMKQIEDKIEEILSKIYHIENEIARIKKLIGEA (GCN4) (SEQ ID NO: 231). In some embodiments, the trimerizing domain is a GCN4 variant. In some embodiments, the GCN4 variant sequence is RMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGR (SEQ ID NO: 232), RMKQIEDKIEEILSKIYHIENEIARIKKLIGNRTGGR (SEQ ID NO: 233), RMKQIEDKIENITSKIYHIENEIARIKKLIGNRTGGR (SEQ ID NO: 234), RMKQIEDKIEEILSKIYNITNEIARIKKLIGNRTGGR (SEQ ID NO: 235), or RMKQIEDKIENITSKIYNITNEIARIKKLIGNRTGGR (SEQ ID NO: 236).

[0291] Pharmaceutical Composition In another aspect, this disclosure provides a pharmaceutical composition comprising the polypeptides, protein nanostructures, or polynucleotides disclosed above.

[0292] The pharmaceutical compositions described herein may be administered in combination with one or more additional therapeutic agents. These additional therapeutic agents may include, but are not limited to: antibiotics or antibacterial agents, antiemetics, antifungals, anti-inflammatory agents, antiviral agents, immunomodulators, cytokines, antidepressants, hormones, alkylating agents, antimetabolites, antitumor antibiotics, antimitotics, topoisomerase inhibitors, cell quiescent agents, anti-invasive agents, anti-angiogenic agents, growth factor function inhibitors, viral replication inhibitors, viral enzyme inhibitors, anticancer agents, alpha-interferon, beta-interferon, ribavirin, hormones, and other Toll-like receptor modulators, immunoglobulins (Ig), and antibodies that modulate Ig function (such as anti-IgE (omalizumab)).

[0293] The pharmaceutical composition may be sterile and / or pyrogen-free. The pharmaceutical composition may be isotonic with the human body.

[0294] The pharmaceutical composition may include one or more antimicrobial agents, particularly when packaged in multiple doses. Antimicrobial agents include, but are not limited to, thimerosal and 2-phenoxyethanol. In some embodiments, it may be desirable to use mercury-free preservatives or no preservatives at all.

[0295] In some embodiments, the pharmaceutical composition is buffered, including but not limited to Tris buffer, histidine buffer, phosphate buffer, citrate buffer, or acetate buffer. The pharmaceutical composition may also include a lyophilization protectant, such as sucrose, sorbitol, or trehalose. In some embodiments, the composition includes a preservative, such as benzalkonium chloride, benzyl chloride, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a filler, such as glycine. In other embodiments, the pharmaceutical composition includes surfactants, such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, or combinations thereof. The pharmaceutical composition may also include a tonic modifier, such as a compound that makes the formulation substantially isotonic or isotonic with human blood. Exemplary tonic modifiers include sucrose, sorbitol, glycine, methionine, mannitol, dextran, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition further includes a stabilizer in lyophilized or liquid form, such as a molecule that substantially prevents or reduces the chemical and / or physical instability of the protein nanostructure. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0296] The pH of the composition is typically between about 4.5 and about 11, such as between about 5 and about 11, between about 5.5 and about 11, between about 6 and about 11, between about 5 and about 10.5, between about 5.5 and about 10.5, between about 6 and about 10.5, between about 5 and about 10, between about 5.5 and about 10, between about 6 and about 10, between about 5 and about 9.5, between about 5.5 and about 9.5, between about 6 and about pH values ​​can be between 9.5, between about 5 and about 9, between about 5.5 and about 9, between about 6 and about 9, between about 5 and about 8.5, between about 5.5 and about 8.5, between about 6 and about 8.5, between about 5 and about 8, between about 5.5 and about 8, between about 6 and about 8, about 4.5, about 5, about 6.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, etc. A stable pH can be maintained by using a buffer (e.g., Tris buffer, citrate buffer, phosphate buffer, or histidine buffer). Therefore, compositions will typically include a buffer.

[0297] This article provides compositions for use as medicines, for example, for inducing or enhancing an immune response in subjects (such as mammals) in need.

[0298] This article provides compositions for manufacturing a medicine for inducing or enhancing an immune response in a subject (such as a mammal) in need.

[0299] vaccine In another aspect, this disclosure provides vaccines comprising the polypeptides, protein nanostructures, or polynucleotides disclosed above. Such compositions can be used to generate antibodies in mammals (e.g., humans). The vaccine compositions of this disclosure typically include pharmaceutically acceptable vectors, and a detailed discussion of such vectors is available in Remington: The Science and Practice of Pharmacy.

[0300] Alternatively, the antigen can be used in vaccines that do not contain protein nanostructures, which utilize a simpler oligomerization method using a FoldOn tag, wherein the FoldOn tag sequence is EKAAKAEEAARK (SEQ ID NO: 85).

[0301] Vaccine compositions may include immune adjuvants. Exemplary adjuvants include: 1. mineral-containing compositions; 2. oil emulsions; 3. saponin formulations; 4. virions and virus-like particles; 5. bacterial or microbial derivatives; 6. bioadhesives and mucosal adhesives; 7. liposomes; 8. polyoxyethylene ethers and polyoxyethylene ester formulations; 9. polyphosphazene (PCPP); 10. muramyl peptides; 11. imidazoquinone compounds; 12. thiourea compounds; 13. tryptamine compounds; 14. human immunomodulators; 15. lipopeptides; 16. benzonaphthyl; 17. microparticles; 18. immunostimulatory polynucleotides (such as RNA or DNA; for example, oligonucleotides containing cpg).

[0302] For example, the composition may include an aluminum salt adjuvant, an oil-in-water emulsion (e.g., an oil-in-water emulsion containing squalene, such as MF59 or AS03), a TLR7 agonist (such as imidazoquinoline or imiquimod), or a combination thereof. Suitable aluminum salts include hydroxides (e.g., oxyhydroxylates), phosphates (e.g., hydroxyphosphates, orthophosphates) (e.g., see Chapters 8 and 9 of Vaccine Design. (1995) Powell and Newman, ed., ISBN: 030644867X. Plenum), or mixtures thereof. The salt may be in any suitable form (e.g., gel, crystals, amorphous, etc.), one example being an antigen adsorbed onto the salt. In compositions intended for administration to patients, Al +++ The concentration can be below 5 mg / ml, for example, <4 mg / ml, <3 mg / ml, <2 mg / ml, <1 mg / ml, etc. A preferred range is between 0.3 mg / ml and 1 mg / ml. A maximum value of 0.85 mg / dose is preferred. Aluminum hydroxide and aluminum phosphate adjuvants are suitable for use in this disclosure.

[0303] Exemplary adjuvants that may be used in the pharmaceutical compositions provided herein include, but are not limited to: 3M-052, Adju-Phos™, Alhydrogel™, Adjumer™, albumin-heparin microparticles, algal dextran, alginate, aluminum adjuvants, antigen preparations, AS-2 adjuvants, ASO1, ASO3, autologous dendritic cells, autologous PBMCs, Avridine™, B7-2, BAK, BAY R1005, and BECC. TLR-4 agonist, bupivacaine, bupivacaine-HCl, BWZL, calcitriol, calcium phosphate gel, CCR5 peptide, CFA, whole cholera toxin (CT) and cholera toxin B subunit (CTB), cholera toxin A1 subunit-protein AD fragment fusion protein, CpG, CPG-1018, CRL1005, cytokine-containing liposomes, D-Murapalmitine, DDA, DHEA, diphtheria toxoid, DL-PGL, DMPC, DMPG, DOC / aluminum adjuvant complex, fowlpox, Freund's complete adjuvant, γ-inulin, Gerbu adjuvant, GM-CSF, GMDP, hGM-CSF, hIL-12 (N222L), hTNF-α, IFA, IFN-γ in pcDNA3, IL-12 DNA, IL-12 plasmid, IL-12 / GMCSF plasmid (Sykes), IL-2 in pcDNA3, IL-2 / Ig plasmid, IL-2 / Ig protein, IL-4, IL-4 in pcDNA3, Imiquimod™, ImmTher™, immunoliposomes containing antibodies with co-stimulatory molecules, interferon-γ, interleukin-1β, interleukin-12, interleukin-2, interleukin-7, ISCOM™, Iscoprep 7.0.3™, keyhole hemocyanin, lipid-based adjuvants, liposomes, loxoribine, LT (R192G), LT-OA or LT oral adjuvant, LT-R192G, LTK63, LTK72, Matrix-M™ adjuvant, MF59, MONTANIDE ISA 51, MONTANIDE ISA 720, MPL.MPL-SE, MTP-PE, MTP-PE liposomes, Murametide, Murapalmitine, NAGO, Natural Cholera Toxin (nCT), Nonionic Surfactant Vesicles, Non-toxic Mutant of Cholera Toxin E112K (mCT-E112K), Methylparaben, pCIL-10, pCIL12, pCMVmCAT1, pCMVN, Peptide Polymer-NP, Pleuran, PLG, PLGA, PGA and PLA, Pluronic L121, PMMA, PODDS™, Poly rA:Poly rU, Polysorbate 80, Protein Helical Coil, QS-21, Quadri A Saponin, Quil-A, Rehydragel HPA, Rehydragel LV, RIBI, Ribi-like adjuvant systems (MPL, TMD, CWS), S-28463, SAF-1, Sclavo peptide, Sendai virus protein liposomes, Sendai virus-containing lipid matrix, Span 85, Spector, squalane 1, squalene 2, stearoyl tyrosine, SWE, tetanus toxoid (TT), Theramide™, threonyl muramyl dipeptide (TMDP), Ty particles, and Walter Reed liposomes.

[0304] In some embodiments, the adjuvant is aluminum hydroxide gel (e.g., Alhydrogel™). In a preferred embodiment, the adjuvant is SWE. In a preferred embodiment, the adjuvant is MF59. MF59 is an oil-in-water emulsion containing squalene (4.3%) in citrate buffer, with stabilizing nonionic surfactants Tween 80 (0.5%) and Span 85 (0.5%). MF59 has been shown to be well tolerated in humans and is used in vaccines against seasonal influenza (see Ko and Kang, Hum Vaccin Immunother. 2018; 14(12): 3041–3045; U.S. Patent No. 6,299,884).

[0305] For example, the composition may comprise an aluminum salt adjuvant, an oil-in-water emulsion (e.g., an oil-in-water emulsion containing squalene, such as MF59, SWE, or AS03), a TL4 agonist, a TLR9 agonist (such as CpG oligodeoxynucleotides), a TLR7 agonist (such as imidazoquinoline or imiquimod), or a combination thereof. In some embodiments, the adjuvant is a combination of an aluminum salt and CPG1018. Suitable aluminum salts include hydroxides (e.g., oxyhydroxylates), phosphates (e.g., hydroxyphosphates, orthophosphates) (e.g., see Chapters 8 and 9 of Vaccine Design. (1995) Powell and Newman, ed., ISBN: 030644867X. Plenum), or mixtures thereof. The salt may be in any suitable form (e.g., gel, crystals, amorphous, etc.), one example being antigen adsorbed onto the salt. In compositions intended for administration to patients, the concentration of Al+++ may be less than 5 mg / ml, for example, <4 mg / ml, <3 mg / ml, <2 mg / ml, <1 mg / ml, etc. A preferred range is between 0.3 mg / ml and 1 mg / ml. A maximum value of 0.85 mg / dose is preferred. Aluminum hydroxide and aluminum phosphate adjuvants are applicable to this disclosure. In some embodiments, the pharmaceutical compositions provided herein contain aluminum hydroxide as an adjuvant.

[0306] In some embodiments, the adjuvant is a squalene emulsion. In some embodiments, the adjuvant is a TLR4 immunostimulant (e.g., SLA, GLA), such as that described in Van Hoeven et al., PLoS One. 11(2):e0149610 (2016). In some embodiments, the adjuvant is a TLR7 / 8 immunostimulant (e.g., R848, IMQ, 3M-052), such as Dowling D. ImmunoHorizons As described in (6):185-197 (2018). In some embodiments, the adjuvant is a TLR9 immunostimulant (CpG), for example, as Bode et al. Expert Rev Vaccines. As described in 10(4):499–511 (2011). In some embodiments, the adjuvant is a saponin (QS21), for example, as described by Zhu et al. Nat Prod Chem Res. As described in 3(4):e113 (2016).

[0307] In some implementations, the vaccine contains a combination of two or more adjuvants (e.g., squalene emulsion and aluminum adjuvant or TLR4 immunostimulant).

[0308] The vaccine composition contains an immunologically effective amount of its antigen. An “immunologically effective amount” or “effective amount” is the amount that, when administered to a subject, effectively elicits an antibody response against the antigen. This amount can vary depending on factors such as the health and physical condition of the individual to be treated, their age, the individual’s immune system’s ability to synthesize antibodies, the required level of protection, the vaccine formulation, the treating physician’s assessment of the medical condition, and other relevant factors. It is expected that this amount will fall within a relatively broad range that can be determined through routine testing. The antigen content of the compositions disclosed herein is generally expressed as the mass of protein per dose. Doses of 10-500 µg (e.g., 50 µg) per antigen may be useful.

[0309] In some implementations, the vaccine (immunogenic composition) is a stable emulsion.

[0310] In some implementations, the vaccine is a bivalent vaccine. In some implementations, the bivalent vaccine comprises the peptide or protein nanostructures described herein. A bivalent vaccine may comprise peptides, protein nanostructures, or polynucleotides targeting two different strains of the coronavirus in any combination. For example, a bivalent vaccine may contain a mixture of two different immunogens each displaying a separate antigen, or a “mosaic” immunogen co-displaying two antigens. For example, a bivalent vaccine may include a peptide of the original viral strain to provide broad protection against COVID-19, and a component of the Omeprone variant to provide better protection against COVID-19 caused by the Omeprone variant.

[0311] How to use In another aspect, this disclosure provides a method for preventing or treating coronavirus-related illness in a subject in need, the method comprising administering to the subject the disclosed peptides, protein nanostructures, polynucleotides, pharmaceutical components, or vaccines.

[0312] In another aspect, this disclosure provides a method for immunizing subjects in need against coronavirus infection by administering the vaccine disclosed above to the subjects.

[0313] In some embodiments, this method prevents the occurrence of coronavirus-related diseases (e.g., severe illnesses), such as COVID-19. Subjects can be any suitable mammalian subject, including but not limited to human subjects. In some embodiments, the subject is a human infant, e.g., a child less than 12 months of age. In some embodiments, the subject is a human toddler, e.g., a toddler aged approximately 1 to 3 years or approximately 1 to 5 years of age. In some embodiments, the subject is a human adult older than 60 years of age. In some embodiments, the subject is a human adult older than 65 years of age. In certain embodiments, the subject is dependent on others or has serious health problems or risks (e.g., frail elderly). In some embodiments, the subject is a healthy adult aged 18 to 60 years of age. In some embodiments, the subject is a healthy adult aged 18 to 45 years of age. In another embodiment, the subject is a pregnant woman. In some embodiments, the subject is an immunocompromised human adult. In some embodiments, the subject is a human adult with chronic underlying heart and / or lung disease or functional disability. In some embodiments, the subject is at risk of developing a severe viral illness (e.g., LRTI or pneumonia).

[0314] The immunogenic compositions provided herein can be used for fetal vaccination. It is recommended to administer certain inactivated vaccines during pregnancy to induce immunization in the fetus; these vaccines include, for example, tetanus toxoid, attenuated diphtheria toxoid, acellular pertussis (Tdap) vaccine, and influenza vaccine. Therefore, in some embodiments, this document provides a method for generating an immune response in the fetus, comprising administering an effective amount of the immunogenic composition provided herein to the mother of the fetus. The immunogenic composition can be administered at any suitable time during pregnancy, for example, in the last trimester.

[0315] The compositions provided herein can be administered in combination with other treatments, such as other vaccines. Therefore, in some embodiments, one or more seasonal or pandemic vaccines, such as influenza vaccines, may also be administered to subjects treated according to the methods provided herein. In some embodiments, pneumococcal, recombinant herpes zoster (Zoster / Shingles), or Tdap vaccines may also be administered to subjects treated according to the methods provided herein. One, two, or more vaccines may be administered in combination with the immunogenic compositions provided herein. “Combined administration” includes both simultaneous and subsequent administration. For example, one, two, or more vaccines and the immunogenic compositions provided herein may be administered on the same day. In some embodiments, one, two, or more vaccines and the immunogenic compositions provided herein may be administered within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, or 12 hours.

[0316] In another aspect, this document provides a method for treating a subject with a viral infection. As used herein, “treatment” includes, but is not limited to, achieving one or more of the following: (a) reducing the viral titer in the subject; (b) limiting any increase in the viral titer in the subject; (c) reducing the severity of symptoms of viral infection; (d) limiting or preventing the development of symptoms following viral infection; (e) suppressing the worsening of symptoms of viral infection; (f) limiting or preventing the recurrence of symptoms of viral infection in subjects who have previously experienced symptoms of viral infection; and / or (e) improving survival. In some embodiments, the vaccination method reduces the risk of the subject to viral infection. In some embodiments, the vaccination method limits the development of viral infection. In some embodiments, the vaccination method reduces the severity of symptoms of viral infection. Symptoms of coronavirus infection include, but are not limited to: fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, new-onset loss of taste or smell, sore throat, nasal congestion or runny nose, nausea or vomiting, and diarrhea.

[0317] In some implementations, the methods provided herein can be used to prevent viral infections or diseases (e.g., acute respiratory illnesses) in subjects. As used herein, “preventing” includes, but is not limited to, achieving one or more of the following: (a) generating an immune response against a virus in a subject (based on antibodies and / or cells, such as CD4 T cells, memory B cells, and / or CD8 T cells), which is expected to confer protection against lower respiratory tract infections (LRTIs) caused or associated with a virus in the subject; (b) generating neutralizing antibodies against a virus in a subject, which is expected to reduce the severity of LRTIs caused or associated with a coronavirus in the subject; (c) preventing LRTIs caused or associated with a virus in a subject, as detected by an increase in the subject’s viral titer or exacerbation of symptoms of one or more viral infections; (d) preventing severe LRTIs caused or associated with a coronavirus in a subject, as detected by an increase in the subject’s viral titer or exacerbation of symptoms of one or more severe viral infections; (e) reducing the risk of LRTIs caused or associated with a coronavirus or severe LRTIs in a subject population; or (f) inducing a serological response (or seroconversion) in a subject, such as generating neutralizing antibodies against a virus that are at least 4-fold higher than the subject’s baseline antibody level. The preventive effect can be assessed by comparing the immune response, especially protection-related indicators, among the following groups: subjects who received the vaccine with the same subjects before administration (referred to as baseline), subjects who received a placebo, or subjects who received a control vaccine.

[0318] As used herein, “limiting” the development of viral infection means achieving one or more of the following: (a) generating an immune response against the virus in a subject (based on antibodies and / or cells, such as CD4 T cells, memory B cells, and / or CD8 T cells) that is expected to limit the increase in viral titer or the exacerbation of symptoms in the subject; (b) generating neutralizing antibodies against the coronavirus in a subject at levels that are expected to limit the increase in viral titer or the exacerbation of symptoms in the subject; (c) resulting in a decrease in viral titer in a subject after exposure to the coronavirus compared to a subject who has not been given the protein complex; and (d) resulting in a decrease in the incidence or severity of symptoms following viral infection.

[0319] The clinical efficacy of vaccines against coronavirus infection can be assessed using a variety of methods known in the art, including but not limited to placebo-controlled clinical efficacy studies designed to measure viral load or viral disease symptoms in vaccinated subjects compared to control subjects. Protection-related endpoints can also be defined as neutralizing antibody titers (typically expressed as geometric mean titers), fold increases from baseline (typically expressed as geometric increases), and seroresponse rate (the percentage of subjects whose neutralizing antibody titers increase by a fold above a predetermined threshold).

[0320] In some embodiments, the methods described herein generate an immune response in a known virus-free subject, wherein the immune response serves to limit the development of infection and viral infection symptoms. In some embodiments, the immune response includes generating neutralizing antibodies against the virus and / or a cell-based response. In some embodiments, the immune response includes generating a spike glycoprotein protein-specific response having a concentration of at least 1 × 10⁻⁶. 3 At least 1×10 4 At least 1×10 5 At least 1×10 6 At least 1×10 7 At least 1×10 8 Or at least 1×10 9 The average geometric titer. In another embodiment, the immune response includes generating antibodies against multiple antigenic epitopes on the spike glycoprotein protein-specific protein trimer.

[0321] In one aspect, the methods provided herein can lead to an increase in antibody titers in a subject, for example, an increase in virus-specific neutralizing antibodies or virus-specific binding antibodies. Antibody titers can be determined using any suitable assay known in the art or described herein, including but not limited to combined enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot assay (ELISpot), competitive ELISA, immunoprecipitation, immunoblotting, and agglutination assays.

[0322] In some embodiments, a neutralization or micro-neutralization (MN) assay can be used to measure an increase in neutralizing antibodies in a subject following administration of a composition as described herein. Micro-neutralization refers to neutralization performed in a miniaturized form, such as a 96-well plate. A micro-neutralization assay is used to test for the inhibition of a virus by an antibody (e.g., purified antibody, serum, or plasma). This assay measures the level of antibodies present in a sample that are capable of neutralizing the virus in vitro. Generally, micro-neutralization assays for clinical samples are performed using a series of dilutions of serum mixed with a fixed concentration of virus. Methods for performing micro-neutralization assays are well known. Illustrative micro-neutralization assays are described, see, for example, van Baalen et al., Vaccine 35 (2017) 46–52.

[0323] In some implementations, the methods provided herein can lead to an increase in immune cells in a subject (e.g., an increase in virus-specific memory B cells and / or virus-specific T cells). The number of immune cells in a subject can be determined using any suitable assay known in the art or described herein, including but not limited to FACS and flow cytometry.

[0324] In some embodiments, the composition is administered in dose units containing conventional pharmaceutically acceptable carriers, adjuvants, and loads via any suitable route, including intranasal, sublingual, oral, parenteral, inhaled spray, rectal, or topical administration. As used herein, the term “parenteral” includes subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques, or intraperitoneal administration.

[0325] Reagent test kit In another aspect, this disclosure provides kits for the preparation and administration of peptides, protein nanostructures, polynucleotides, pharmaceutical compositions, or vaccines as described above. In some embodiments, the kits provided herein comprise the peptides, protein nanostructures, polynucleotides, pharmaceutical compositions, or vaccines disclosed herein, along with instructions for use with the methods described above. In some embodiments, the kit comprises one or more unit doses as disclosed herein and instructions for use with the methods of this disclosure. In some embodiments, the kit comprises vials containing a single dose of the pharmaceutical composition or vaccine provided herein. In some embodiments, the kit comprises vials containing multiple doses provided herein. In some embodiments, the kit also comprises instructions for use with the pharmaceutical composition or vaccine. In some embodiments, the kit also comprises a diluent for preparing a diluent for use prior to administration of the pharmaceutical composition or vaccine. In some embodiments, the vaccine comprises an adjuvant. In some embodiments, the kit comprises a vaccine and an adjuvant that must be mixed prior to administration.

[0326] Example Materials and methods Structural modeling. PyMol version 2.5.2 or ChimeraX version 1.3 were used to analyze the structural model and generate images. Colabfold (Reference 11) was used with default settings for structure-based predictions of the RDB-SD1 region for each design sequence.

[0327] HEK293 expression. The day before transfection, Expi293F cells (Thermo Fisher Scientific) were counted and divided at 2.5 × 10⁻⁶. 6Cells were seeded at 100 cells / mL and then incubated overnight at 36°C with shaking at 120 rpm. The next day, the cells were counted and diluted to 3 × 10⁻⁶ cells / mL. 6 Cells / mL. Then, transiently transfect the cells with the desired plasmid according to the manufacturer's instructions (Thermo Fisher Scientific), except that the transfection reagent was changed to Transporter 5 (Polysciences), used at a ratio of 3.0 μg Transporter 5 per 1 μg plasmid DNA. The transfected cells were then incubated at 36°C with shaking at 120 rpm for 4 days.

[0328] Western blotting. Western blot analysis was performed on the supernatant of HEK293 cells after small-scale expression using an anti-His antibody conjugated with HRP. The supernatant was heated in a solution containing 5% (v / v) β-... 2x Laemmli loading buffer with mercaptoethanol (Bio- Dilute 2 times in Rad), then heat to Incubate at 95°C for 10 minutes. Load 10 μl and 7 μl of His-labeled protein standard / molecular weight standard (Invitrogen, #LC5606) together onto a NuPAGE 4-12% Bis-Tris protein gel and run at 200V for 30 minutes. Transfer the protein to an Immuno-Blot PDVF membrane (Bio-Rad). After transfer, block the blot with 2% milk powder (w / v) in 1xPBS, 0.05% Tween 20, and agitate at room temperature for 1 hour. Dilute the HRP-conjugated anti-His antibody (R&D systems MAB050H) 1:7000 in 2% milk powder (w / v) in 1xPBS, 0.05% Tween 20, and incubate with the membrane at room temperature for 1 hour with agitation. The membrane was then washed three times with 1x PBS and 0.05% Tween 20 for five minutes each time, and His-tagged proteins were detected using (BioFx TMB enhanced single-component HRP membrane substrate, Surmodics), with the signal captured on the BioRad ChemiDoc MP imaging system.

[0329] IMAC purification. NA protein samples were purified directly from HEK Expi293F cell supernatant using immobilized metal affinity chromatography (IMAC). Ni was washed with 5 column volumes (CV) of water. 2+ - Agarose resin (Cube Biotech, Indigo), followed by washing with 5 CV of equilibration buffer (20 mM Tris pH 8.0, 250 mM NaCl, 5% glycerol, 30 mM imidazole), and then resuspending in 1 CV of equilibration buffer. The supernatant was subjected to 4,000 × 10⁻⁶ ppm. gCentrifuge to clarify, then filter using a 0.2 mm vacuum filter unit. Add the resin suspension to each supernatant at a ratio of 4 μL resin per mL of supernatant. Incubate the supernatant-resin slurry with gentle shaking at 4°C for approximately 16–18 hours, then collect the resin by applying it to an empty gravity column (Biorad, 7321010). Wash the resin bed with 20 CV equilibration buffer, and elute the protein three times with 6 CV elution buffer (20 mM Tris pH 8.0, 250 mM NaCl, 5% glycerol, 300 mM imidazole), incubating for 30 minutes between each application of elution buffer. After IMAC purification, the protein sample was further purified by size exclusion chromatography (SEC), or dialyzed in “RBD Buffer 1” (20 mM Tris pH 8.0, 150 mM NaCl), “RBD Buffer 2” (5% glycerol, 20 mM Tris pH 8.0, 250 mM NaCl, 5% glycerol), or “RBD Buffer 3” (50 mM MOPS, 150 mM NaCl, pH 7.4).

[0330] SEC purification. IMAC eluents from each antigen-CompA sample were injected onto a Superdex 200 Increase 10 / 300 GL column (Cytiva, equilibrated in RBD buffer 1, RBD buffer 2, or RBD buffer 3) and eluted with 1.2 column volumes of the corresponding run buffer to further purify the antigen by size exclusion chromatography (SEC). For assembled protein nanostructure formulations, samples were loaded onto a Superose 6 Increase 10 / 300 GL column (Cytiva, equilibrated in 20 mM Tris Ph 8.0, 150 mM NaCl, 100 mM L-arginine, and 5% glycerol) and eluted with 1.2 column volumes of the same buffer. After analysis, the purified samples were flash-frozen in liquid nitrogen and stored at -80°C.

[0331] Biolayer Interference Method. In BLI assay buffer (PBS, 0.5% BSA, 0.05% Tween 20, pH 7.4), the concentrations of ACE2-Fc, CR3022 (Reference 12), or S309 (Reference 16) were normalized to 10 μg / mL, with a volume sufficient to add 200 μL to each well of a black 96-well microplate (Greiner, 655209). In short, on an Octet Red96e instrument, the pre-hydrated protein G biosensor (Sartorius, 18-5022) was immersed in the assay buffer for 60 seconds to acquire a baseline. Next, the biosensor was immersed in ACE2-Fc, CR3022, or S309 for 60 seconds or 120 seconds, respectively, to complete immobilization, followed by another baseline step. Bind the immobilized antibody to 200 μL of purified antigen-CompA fusion (10 μg / mL in BLI assay buffer) or cell supernatant for 120 to 700 seconds, and then transfer the biosensor back to the assay buffer for 120 to 700 seconds to observe any possible dissociation. Unless otherwise specified, the maximum offset during the binding phase is reported.

[0332] The intrinsic protein fluorescence (ITF) was measured using a nanoDSF instrument (UNchained Laboratories, UNcle). Protein thermal stability and fluorescence baseline measurements were performed on a nanoDSF instrument (UNchained Laboratories, UNcle) using intrinsic protein fluorescence (ITF). Antigen concentrations were normalized to 0.5 mg / mL–1 mg / mL. Samples were loaded into a quartz capillary sample holder (UNchained Laboratories, UNi), equilibrated at 15°C for 5 minutes, and then ITF measurements were performed at a temperature gradient (15°C–95°C, 0.5°C / min–1°C / min). The melting temperature of a representative single sample was determined using the default settings or a customized Python script in Uncle Analysis V6.0 software, calculated from the centroid mean. This Python script calculated the melting temperature using the second derivative of the temperature-dependent centroid mean. The onset temperature was calculated from the intersection of the linear fit between the pre-transition region baseline and the melting transition region.

[0333] SYPRO nanoDSF was used. Thermal melting profiles of the NA constructs were determined by normalizing sample concentrations to 0.5 mg / mL–1 mg / mL. Then, 3.5 μL of SYPRO™ Orange Protein Gel Stain (Invitrogen S6651) was diluted 1:20 in each sample buffer and added to 31.5 μL of the normalized NA construct. Samples were loaded into a quartz capillary sample holder (UNchained Laboratories, UNi), equilibrated at 15 °C for 5 min, and then ITF measurements were performed at a temperature gradient (15 °C–95 °C, 0.5 °C / min–1 °C / min). Melting temperatures were determined using fluorescence area measurements from representative single samples, using the default settings of Uncle Analysis V6.0 software. Baseline fluorescence near 30 °C was measured by integrating fluorescence intensity based on three replicates, averaging the points acquired at the temperature closest to 30 °C for each replicate.

[0334] Assembly of antigen-CompA samples with CompB. For any protein nanostructure assembly process, purified CompB (I53-50B.4PosT1) was mixed with the antigen-CompA sample by pipetting at a molar ratio of 1.1 parts antigen-CompA to 1 part CompB. For small-scale assembly reactions, the assembly process was sometimes performed with the addition of additional concentrated buffer solutions to add excipients to the final solution, including 50 mM–100 mM L-arginine, 0.75% CHAPS, 4% sucrose, or 5% glycerol to a final concentration. The excipients used in each case are detailed in the following tables. For scale-up assembly prior to SEC, the assembly process was performed against a background of 20 mM Tris Ph 8.0, 150 mM NaCl, 100 mM L-arginine, and 5% glycerol. All assembly processes were allowed to stand at room temperature for at least 30 minutes before SEC or dynamic light scattering (DLS) analysis using the default protocol of a DLS instrument (UNchained Laboratories, UNcle).

[0335] Animal immunization. Female BALB / c mice were intramuscularly immunized on day 0 or day 21 with 0.027-1.0 μg of purified antigen-CompA or antigen-VLP protein mixed 1:1 with Addavax™ adjuvant. Serum samples were collected on day 0 (before the first immunization), day 21 (before the second immunization), and day 35. Antigen binding and neutralization tests were performed on the corresponding antigens and pseudoviruses of the original strain and BA.5 strain in Nexelis.

[0336] Example 1: Design of SD1resurf mutations and their functional enhancement in nanostructure assembly In the first round of design, initial characterization was tested using RBD antigens from the original SARS-CoV-2 strain. All tested antigens were fused with the I53-50A “CompA” trimer gene, a protein-based component that, upon addition of a complementary “CompB” pentamer (in this case, I53-50B.4PosT1), could be assembled in vitro into protein nanostructures (Reference 2). In the case of this RBD-SD1 antigen, several previously described stabilization mutations (Rpk2, Rpk4, Rpk8, Rpk9, Rpk11, and Rpk15) were tested (Reference 5) (Table 14). A new set of mutations (named “SD1resurf”) targeting exposed hydrophobic groups on the SD1 domain were designed, making these groups more polar to reduce aggregation (L560Q, F562Y, I569S). L560Q and I569S were designed based on other sabevir SD1 sequences containing these polar residues at these positions, while F562Y was designed to make these residues more polar. All RBD-SD1 designs were tested individually using one of five different flexible linkers (Table 15) between the RBD-SD1 antigen and CompA, with linker lengths ranging from 16 to 24 amino acids. For all designs with an added secretion signal sequence, codon-optimized DNA sequences were generated and then cloned into vectors for expression in HEK293 cells using transient transfection.

[0337] Table 14. Mutations tested in the first round of design.

[0338]

[0339] Table 15. Connectors for the test located between the RBD-SD1 antigen and CompA

[0340] First, a small-scale evaluation design was performed using the supernatant, which was used to assess expression using Western blotting. Figure 2A It is also used to assess antigenicity after screening using immobilized ACE2-Fc receptors via biolayer interference (BLI). Figure 2B and Figure 2C(Tables 16 and 17). Surprisingly, most tested samples showed higher ACE2 binding signals compared to the “RBD01” control, which was characterized by the fusion of the RBD fragment with CompA (Reference 14). These data suggest improved expression and / or antigenicity. The higher levels of binding were observed to be independently influenced by the addition of stabilizing mutations (including multiple Rpk mutations and SD1resurf mutations) and by the use of linkers with 18 or 20 residues. Based on these data, various designs containing 18-residue linkers were scaled up by HEK293 transfection and purified by immobilized metal ion affinity chromatography (IMAC) for more detailed characterization and comparison with wild-type RBD antigens without the SD1 domain (residues 328-531) fused to the CompA gene. BLI was performed on immobilized ACE2-Fc or S309 neutralizing monoclonal antibodies (Reference 12) or CR3022 non-neutralizing monoclonal antibodies (Reference 16), with ACE2-Fc and S309 results used for screening because they bind to the epitopes targeted by the neutralizing antibodies. BLI for ACE2-Fc and S309 showed broadly similar results. Figure 3 (Table 18), among which some constructs retaining the native isoleucine (I) at position 358 showed superior performance as measured by S309. Nano-differential scanning fluorescence (NanoDSF) based on intrinsic tryptophan fluorescence showed that designs containing the Rpk4, Rpk8, Rpk9, and Rpk15 stabilization mutations had increased melting temperatures relative to wild-type antigens with and without the SD1 domain (Figs. 4A to 4G, Table 19). Melting temperatures were similar for wild-type RBD antigens with and without the SD1 domain (Figs. 4A to 4B, Table 19) and for designs containing the SD1resurf mutation (Fig. 4G, Table 19). Similar results were observed for NanoDSF monitoring using SYPRO dye, but the Rpk4 and Rpk9 designs showed significantly lower baselines compared to other designs, suggesting that improved foldability reduced the hydrophobicity of these designs (Figs. 4H to 4K, Table 20). Finally, by adding complementary CompB pentamer components and monitoring particle size using dynamic light scattering (DLS) on the original strain, the assemblies of different designs were tested under various excipient conditions. Figure 5A and Figure 5B (Table 21). When using a milder excipient (arginine) Figure 5B (Table 21) or arginine and sucrose ( Figure 5A When assembled in the buffer of Table 21, significant aggregation was observed in all designs, with the exception of those designs containing Rpk9 stabilization mutations and SD1resurf mutations, which provided DLS signals (35nm-45nm) consistent with successful protein nanostructure assembly.

[0341] In summary, this first-round design demonstrates that the previously described Rpk9 mutations successfully improve the thermostability of RBD-SD1 antigens from the original strain and further enhance their ability to assemble into I53-50 protein nanostructures with minimal aggregation in a simpler buffer formulation. Unexpectedly, a novel SD1resurf mutation was identified that assists in the assembly of I53-50 protein nanostructures with minimal aggregation. Both sets of mutations at least demonstrate antigenicity of key epitopes targeted by the neutralizing antibodies.

[0342] Table 16 shows the first round of BLI using supernatant designed from a small-scale expression of the original strain using immobilized ACE2-Fc.

[0343] Table 16

[0344] Table 17 shows the second round of BLI performed on the supernatant of the original strain designed for small-scale expression using immobilized ACE2-Fc.

[0345] Table 17:

[0346] Table 18 shows the BLI targeting the purified antigen-CompA protein using immobilized ACE2-Fc, CR3022, or S309.

[0347] Table 18:

[0348] Table 19. NanoDSF analysis of purified, original-strain-based antigen-CompA samples using intrinsic tryptophan fluorescence. Measurements are reported as the average of three replicates.

[0349] Table 19:

[0350] Table 20. NanoDSF analysis of purified, original-strain-based antigen-CompA samples using SYPRO dye. Measurements are reported as the average of three replicates.

[0351] Table 20:

[0352] Table 21 shows the DLS results of the purified antigen-CompA sample assembled with CompB.

[0353] Table 21:

[0354]

[0355]

[0356] Example 2: Design of additional stabilization mutations based on structural analysis of highly conserved regions of the RBD-SD1 antigen Constructs containing the RBD-SD1 antigen fragment, Rpk9 mutation, SD1resurf mutation, and an 18-residue linker were generated, and these combinations were tested with the original strain and BA.5 (an Omeprón-derived variant). Assembly of the purified protein with CompB was tested, and the ancestral antigen assembled successfully as expected. Figure 6 Table 22) (References 3, 13).

[0357] Based on the structural location of the added stabilizing mutation and the close proximity of F392W to the RBD-SD1 interface, a design based on a combination of the Rpk8 mutation (identical to Rpk9 but lacking F392W) and the SD1resurf mutation was created, with additional stabilizing mutations added to improve stability and assembly. RBD sequences with poor thermal stability, such as the Omeprone variant, form the basis for other designs. This reduced stability presents a disadvantage for the reliable fabrication of RBD antigens and derived protein nanostructure-based vaccines, but allows for more rigorous testing of novel stabilizing mutations. Due to the global relevance of the Omeprone-derived BA.5 variant and its representativeness in bivalent mRNA vaccines in 2022, BA.5 was selected for testing novel stabilizing mutations in the RBD-SD1 antigen.

[0358] To optimally select stabilizing mutations of the RBD-SD1 antigen from various strains of SARS-CoV-2 or other sabanaviridae viruses, structural data were analyzed to identify highly conserved but seemingly lacking local stability regions of the RBD-SD1 antigen, such as structural cavities or other non-ideal features. The interface between RBD and SD1 was highlighted as a region exhibiting local instability. Figure 1 C). Multiple cavities and poorly formed polar groups were observed between the two domains, and the loop region from residues 517-522 on the RBD appeared to form antagonistic tension with the SD1 domain and also contained exposed hydrophobic groups. Structural analysis of the spike glycoprotein structures from various SARS-CoV-2 variants and SARS-CoV-1 showed that this interface region (PDB 6VXX, 7UB5, 5XLR) was conserved at both the structural and residue levels. To further confirm this conservation, sequences from several representative SARS-CoV-2 variants and Sabie virus were compared and analyzed. The comparison showed strong conservation of residues at this interface ( Figure 7Furthermore, no potent neutralizing antibodies characterized to target any residues in this region were identified, making this region more suitable for mutation without affecting key antigenicity. Based on modeling in PyMol, Rosetta (Reference 10), and Colabfold (Reference 11), multiple sets of mutations were designed to enhance the stability of the RBD-SD1 interface. Some mutant sets aimed to strengthen the interaction between the RBD and SD1 domains by improving stacking and / or hydrogen bonding or adding disulfide bonds. Other sets mutated the 517-522 ring to make the hydrophobic group more polar and / or eliminate ring strain, such as by adding glycine mutations or removing native proline. Selected combinations of different design strategies were also evaluated. Similarly, many designs were also tested in other parts of the RBD adjacent to the RBM. Figure 1 C) The aim was to improve stability by enhancing other non-covalent interactions. DNA sequences were generated for all designs, and these sequences were fused with CompA using 18-residue linkers. As mentioned earlier, the Rpk8 mutation was added to all designs to further support stability, and the SD1resurf mutation was also added to all designs to assist in nanostructure assembly.

[0359] Table 22 shows the DLS results of the purified antigen-CompA sample assembled with CompB.

[0360] Table 22: Example 3: Evaluation of stabilizing mutations and their functional enhancements to antigenicity and thermal stability First, through protein blotting ( Figure 8 Designs were screened from small-scale transfections in HEK293 cells, and some designs with RBM-adjacent mutations were removed based on poor secretion. For designs with good expression, further screening was conducted based on BLI signals against ACE2-Fc from the supernatant (Table 23) to select designs with high expression and / or improved antigenicity. Several designs with high BLI signals were selected for scale-up and purification and compared with wild-type antigen sequences lacking Rpk8 and SD1resurf mutations. NanoDSF of designs using intrinsic tryptophan fluorescence showed varying degrees of improvement in thermal stability. Figure 9A (Table 24) shows that the melting and onset temperatures were increased by up to 6-7 °C compared to RBDb046 (wild-type design). The wild-type design showed a melting temperature of approximately 37 °C and an onset temperature of 40.5 °C, respectively. This low thermal stability poses a manufacturing risk due to the increased temperature and associated stress, and the stabilization mutation directly provides a way to mitigate such risks. Surprisingly, BLI targeting the immobilized ACE2-Fc receptor showed improved binding signaling from the stabilization design compared to the wild-type design. Figure 9 B and Figure 9 (C, Table 24), particularly regarding binding rate. All added stabilizing mutations were structurally distant from the ACE2 binding site on the RBD, suggesting an improvement in antigenicity by enhancing the foldability of the RBD-SD1 antigen. Finally, these designed subgroups were found to reduce aggregation during the assembly of the I53-50 protein nanostructures in the presence of small amounts of excipients after the addition of CompB. Figure 10 (Table 24), while the wild-type design showed DLS signals inconsistent with the correct protein nanostructure assembly.

[0362] Table 23 shows the BLI binding shift in the supernatant screening of ACE2 binding in the BA.5 design.

[0363] Table 23:

[0364] Table 24 provides a summary of the in vitro analyses of the purified BA.5-based antigen-CompA samples. The onset and melting temperatures are averages from three replicates, calculated based on the custom analytical method described in "Materials and Methods". "NC" indicates "Not Collected" because subsequently labeled constructs exhibited other poorer properties. The desired Z-mean d values ​​ranged from 30 nm to 45 nm, with larger values ​​indicating aggregation during nanostructure assembly.

[0365] Table 24:

[0366] Example 4: Immunogenicity of selected stabilization mutations Next, the immunogenicity of a small group of assembled protein nanostructures and a large group of trimer CompA was tested. For constructs based on the original RBD strain, the protein nanostructures and CompA of RBD01 (WT, without SD1) and RBDb022 (containing a stabilizing mutation, with SD1) were tested. For constructs based on BA.5 RBD, only RBDb047 was tested as a protein nanostructure, while RBDb046 (WT, with SD1), RBDb047, RBDb075, and RBDb078 (all three containing stabilizing mutations, with SD1) were tested as CompA. Mice were immunized on days 0 and 21 using a two-dose regimen, with protein nanostructures ranging from 0.027 μg to 0.030 μg (matched to the antigenic molar equivalents between individual protein nanostructures) and CompA ranging from 0.85 μg to 1.0 μg (matched to the antigenic molar equivalents between individual CompA units). All groups were adjuvanted using Addavax™. Binding titers against strain-matched spike glycoprotein antigens against the original strain and the BA.5 strain were measured. Figure 11A Furthermore, the neutralizing titers of the original strain and the BA.5 pseudovirus were measured against the strain-matched pseudovirus. Figure 11BBlood samples were collected on day 21 (after primary immunization) and day 35 (after booster immunization) for serological measurements. In the original CompA strain, RBDb022 maintained a slightly higher binding titer than RBD01 on both day 21 and day 35. Although no neutralizing titer was detected on day 21, RBDb022 CompA also showed a higher neutralizing titer on day 35 compared to RBD01 CompA. This was unexpected, as previous comparisons between immunogens similar to RBD01 CompA and different stabilized RBDs lacking the SD1 domain in published results did not show differences (Reference 5). Binding and neutralizing titers were generally similar between the RBD01 and RBDb022 protein nanostructure groups. Unexpectedly, significant differences were observed between CompA strains of the BA.5 antigen. Among the CompA strains, only RBDb075 showed a clear signal above baseline on day 21. On day 35, CompA with the stabilized antigen exhibited higher binding titers compared to RBDb046, with RBDb075 showing the highest binding titer. While neutralizing activity of BA.5 CompA was not detected on day 21, a significant difference was observed on day 35, with RBDb046 showing a near-baseline response, while all CompA groups with stabilizing mutations showed higher responses. Most surprisingly, RBDb075 showed a higher neutralizing titer than both RBDb047 and RBDb078. These data suggest that, in addition to improvements in thermal stability, antigenicity, and nanostructure assembly, the added specific stabilizing mutant groups provide unique assistance to immunogenicity in a combination that cannot be predicted using existing methods based on the antigen sequence. Of all measurements, the protein nanostructure of RBDb047 was more immunogenic than CompA.

[0367] Example 5: Antigenicity, expression, and melting of selected stable mutations of various variant RBD-SD1 antigens Solution temperature To understand how the stabilizing mutation used in RBDb075 functions in RBDs from other variants, equivalent designs for other variant RBDs were generated using the same stabilizing mutation, antigen fragments (origin and endpoint), and CompA linkers. Variants selected for analysis included the original strain variants BA.1, BA.5, XBB, and SARS-CoV-1. As controls, these stabilization designs were compared to equivalent designs lacking the stabilizing mutation, with RBDb002 and RBDb046 used for the original strain and BA.5 variant, respectively.

[0368] First, five stable designs and five equivalent wild-type designs were secreted on a small scale by HEK293 cells, and antigenicity against ACE2, S309, and 16A8 (anti-CompA) was assessed by BLI. The binding signal ratios against ACE2 and 16A8 were compared for each sample. For BA.1, BA.5, and XBB, the modified constructs showed a significantly increased ACE2 / 16A8 binding ratio compared to the wild-type constructs, indicating that the stabilization mutations improved the folding of the antigen for better ACE2 binding. For constructs derived from the original strain variant or SARS-CoV-1, this ratio was generally unchanged, indicating that these RBD variants were correctly folded in their wild-type form. Figure 12A (See Table 25). Similar results were observed for the ratio of S309 to 16A8.

[0369] Next, all ten designs were scaled up and purified using IMAC, and the total protein yield was compared between the stable and wild-type designs from each variant. For BA.1, BA.5, and XBB, the yield was significantly higher than the stable design, with particularly large increases observed in BA.1 and BA.5. The yields of the wild-type and stable designs from the original strain and SARS-CoV-1 variants were generally similar. Figure 12B (and Table 25).

[0370] Finally, the melting temperatures of the purified proteins were measured by intrinsic tryptophan fluorescence and compared between RBDs from each variant. The wild-type BA.1 construct was not analyzed due to its low yield. For each of the remaining four variant RBD pairs, the melting temperatures of all stabilized antigens were increased. The increase ranged from 4.7°C for SARS-CoV-1 to 6.8°C for BA.5. Figure 12C (and Table 25).

[0371] In summary, the stabilization design elements established using RBDb075 were successfully extended to various RBD variants without adverse effects on any of them, and showed improvements in antigenicity, yield, and / or thermal stability.

[0372] Table 25 shows the designs used in RBDb002 (“wild type”) and RBDb075 extended to multiple strain sequences from SARS-CoV-2 and SARS-CoV-1. The mean normalized ACE2 binding measurement was based on BLI and calculated by dividing two repeated measurements of the ACE2 binding signal by the binding signal with anti-CompA mAb. Yields were measured from equal volumes of transfected cell cultures after IMAC purification. Melting temperatures were measured using intrinsic tryptophan fluorescence and based on values ​​calculated by Uncle software. “NC” indicates “Not collected”.

[0373] Table 25:

[0374] Table 26 shows the constructs used in the experiment.

[0375]

[0376]

[0377]

[0378]

[0379]

[0380] In some embodiments, the signal sequence is cleaved post-translation when the protein is expressed. In some embodiments, the underscore / italic signal and the polyhis tag sequence are optionally included in the sequence.

[0381] Table 27 provides the sequences of the constructs used in the experiment (signal sequences and labels with underlined / italic text).

[0382] Table 27:

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401] References

[0402]

[0403] Implementation Plan This disclosure also provides the following listed implementation schemes: Implementation Scheme 1. A recombinant polypeptide comprising an antigenic fragment of a coronavirus spike glycoprotein, said antigenic fragment comprising a receptor-binding domain (RBD) and optionally a coronavirus subdomain 1 (SD1).

[0404] Implementation Scheme 2. The polypeptide as described in Implementation Scheme 1, wherein the antigen fragment comprises RBD-SD1.

[0405] Implementation Scheme 3. The polypeptide as described in Implementation Scheme 1 or Implementation Scheme 2, wherein the SD1 comprises at least one surface-exposed nonpolar amino acid residue replaced by a polar amino acid residue.

[0406] Implementation Scheme 4. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises one, two or more, or three amino acids at positions 560, 562, or 569 being replaced by polar residues, wherein the replacement is relative to the reference sequence according to SEQ ID NO: 1.

[0407] Implementation Scheme 5. The polypeptide as described in any of the preceding embodiments, wherein, relative to the reference sequence according to SEQ ID NO:1, the polypeptide comprises one or more, two or more, or three of the amino acid substitutions L560Q, F562T, F562Y, or I569S.

[0408] Implementation Scheme 6. The polypeptide as described in any of the preceding embodiments, wherein, relative to the reference sequence according to SEQ ID NO:1, the polypeptide comprises one, two or more, or three or more amino acid substitutions at positions 544, 546, 560, 562, 564, 569, or 582.

[0409] Implementation Scheme 7. The polypeptide as described in any of the preceding embodiments, wherein, relative to the reference sequence according to SEQ ID NO:1, the polypeptide comprises one, two or more, three or more, four or more amino acid substitutions at positions N544L, N544M, N544Q, L546V, L560Q, F562T, F562Y, Q564C, Q564L, Q564N, Q564W, I569S or L582S.

[0410] Implementation Scheme 8. The polypeptide as described in any of the preceding embodiments, wherein, relative to the reference sequence according to SEQ ID NO:1, the polypeptide comprises one or more, two or more, three or more, four or more amino acid substitutions at positions 338, 358, 363, 365, 392 or 395.

[0411] Implementation Scheme 9. The polypeptide as described in any of the preceding embodiments, wherein, relative to the reference sequence according to SEQ ID NO:1, the polypeptide comprises one or more, two or more, three or more, four or more amino acid substitutions at positions F338L, I358F, A363L, Y365F, Y365M, Y365W, F392W or V395I.

[0412] Implementation Scheme 10. The polypeptide as described in any of the preceding implementation schemes, wherein the polypeptide comprises amino acid substitutions, the amino acid substitutions including F338L / Y365W, F392W, Y365F / V395I, Y365F / F392W / V395I, F338L / A363L / Y365M and / or I358F / Y365F / V395I.

[0413] Implementation Scheme 11. The polypeptide as described in any of the preceding embodiments, wherein, relative to the reference sequence according to SEQ ID NO:1, the polypeptide comprises one, two or more, three or more, four or more amino acid substitutions at positions 329, 348, 350, 367, 375, 402, 407, 410, 418, 429, 433, 435, 452, 464, 510, 512, 514, 517, 518, 519, 520, 522, 527 or 528.

[0414] Implementation Scheme 12. The polypeptide as described in any of the preceding embodiments, wherein, relative to the reference sequence according to SEQ ID NO:1, the polypeptide comprises positions F329K, F329R, F329Y, A348P, V350L, V367F, F375Y, I402V, V407L, I410F, I418V, F429W, V433I, A435I, A435V, L452R, F464Y, P512Q, V510I, V512F, V512I, S514T, L517A, L517D, L517S, L517T, L518 Substitution of one, two or more, three or more, or four or more amino acids at N, L518Q, L518V, H519D, H519G, H519R, H519S, H519T, A520C, A520D, A520G, A520H, P521A, P521D, P521N, P521Q, P521S, A522G, A522I, P527N, K528I, K528Q, K528T, L518G, or L518S.

[0415] Implementation Scheme 13. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises amino acid substitutions at positions 365, 395, 560, 562 and 569; and one, two or more or three or more amino acid substitutions at positions 348, 402, 464, 514, 520, 526 and 527, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1.

[0416] Implementation Scheme 14. The polypeptide as described in any of the preceding implementation schemes, wherein the polypeptide comprises amino acid substitutions, the amino acid substitutions comprising A348P / Y365F / V395I / L560Q / F562Y / I569S, Y365F / V395I / I402V / L560Q / F562Y / I569S, Y365F / V395I / S514T / L560Q / F562Y / I569S, Y365F / V395I / I402V / F464Y / L560Q / F562Y / I569S and / or Y365F / V395I / G526S / P527N / L560Q / F562Y / I569S.

[0417] Implementation Scheme 15. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises amino acid substitutions at positions 365, 395, 560, 562 and 569; and one or more, two or more, or three or more amino acid substitutions at positions 329, 517, 519, 520 and 544, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO:1.

[0418] Implementation Scheme 16. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises amino acid substitutions, the amino acid substitutions comprising: Y365F / V395I / L560Q / F562Y / I569S; Y365F / V395I / F329Y / L560Q / F562Y / I569S; Y365F / V395I / L517T / H519S / L560Q / F562Y / I569S ;Y365F / V395I / L517T / H519S / A520G / L560Q / F562Y / I569S;Y365F / V395I / L517T / H519S / N544L / L560Q / F562Y / I569S;and / or Y365F / V395I / F329Y / L517T / H519S / L560Q / F562Y / I569S;

[0419] Implementation Scheme 17. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises amino acid substitutions at positions 365, 392, 395, 560, 562 and 569; and one or more, two or more or three or more amino acid substitutions at positions 329, 517, 519, 520 and 544, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1.

[0420] Implementation Scheme 18. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises amino acid substitutions, said amino acid substitutions comprising: Y365F / F392W / V395I / L560Q / F562Y / I569S; Y365F / F392W / V395I / F329Y / L560Q / F562Y / I569S; Y365F / F392W / V395I / L517T / H519S / L560Q / F562Y / I569S ;Y365F / F392W / V395I / L517T / H519S / A520G / L560Q / F562Y / I569S;Y365F / F392W / V395I / L517T / H519S / N544L / L560Q / F562Y / I569S;and / or Y365F / F392W / V395I / F329Y / L517T / H519S / L560Q / F562Y / I569S;

[0421] Implementation Scheme 19. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises amino acid substitutions at positions 365, 395, 517, 519, 520, 560, 562, 564, and 569; and one, two, or more, or three or more amino acid substitutions at positions 521, 544, and 546, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1.

[0422] Implementation Scheme 20. The polypeptide as described in any of the preceding implementation schemes, wherein the polypeptide comprises an amino acid substitution, the amino acid substitution comprising Y365F / V395I / L517T / H519G / A520C / Q564C / L560Q / F562Y / I569S and / or Y365F / V395I / L517S / H519R / A520H / P521Q / N544L / L546V / Q564W / F562T / L560Q / F562Y / I569S.

[0423] Implementation Scheme 21. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises amino acid substitutions at positions 365, 395, 517, 519 and 520; and one or more amino acid substitutions at positions 518 and 392, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1.

[0424] Implementation Scheme 22. The polypeptide as described in any of the preceding implementation schemes, wherein the polypeptide comprises an amino acid substitution, the amino acid substitution comprising Y365F / V395I / L517T / H519S / A520G, Y365F / V395I / L517T / L518G / H519D / A520G, Y365F / V395I / L517T / L518S / H519S / A520G, Y365F / F392W / V395I / L517T / L518G / H519D / A520G and / or Y365F / F392W / V395I / L517T / L518S / H519S / A520G.

[0425] Implementation Scheme 23. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises amino acid substitutions at positions 329, 365, 395, 517, 519, 520, 560, 562, and 569; and one, two, or more, or three or more amino acid substitutions at positions 392, 544, and 564, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1.

[0426] Implementation Scheme 24. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises amino acid substitutions, the amino acid substitutions comprising: Y365F / V395I / L517T / H519S / A520G / N544M / L560Q / F562Y / I569S / F329Y; Y365F / V395I / L517T / H519S / A520G / N544Q / L560Q / F562Y / I569S / F329Y; Y365F / V395I / L517T / H519S / A520G / Q564N / L560Q / F562Y / I5 69S / F329Y; Y365F / F392W / V395I / L517T / H519S / A520G / N544M / L560Q / F562Y / I569S / F329Y; Y365F / F392W / V395I / L517T / H519S / A520G / N544Q / L560Q / F562Y / I569S / F329Y; and / or Y365F / F392W / V395I / L517T / H519S / A520G / Q564N / L560Q / F562Y / I569S / F329Y.

[0427] Implementation Scheme 25. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises amino acid substitutions, the amino acid substitutions comprising: Y365F / V395I / L517T / H519S / A520G / N544M / L560Q / F562Y / I569S; Y365F / V395I / L517T / H519S / A520G / N544Q / L560Q / F562Y / I569S; Y365F / V395I / L517T / H519S / A520G / Q564N / L560Q / F5 62Y / I569S; Y365F / F392W / V395I / L517T / H519S / A520G / N544M / L560Q / F562Y / I569S; Y365F / F392W / V395I / L517T / H519S / A520G / N544Q / L560Q / F562Y / I569S; and / or Y365F / F392W / V395I / L517T / H519S / A520G / Q564N / L560Q / F562Y / I569S.

[0428] Implementation Scheme 26. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises amino acid substitutions at positions 365, 395, 517, 519, 520, 521, 504, 560, 562, 569 and 329; and one or more amino acid substitutions at position 392, the amino acid substitutions being relative to the reference sequence according to SEQ ID NO: 1.

[0429] Implementation Scheme 27. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises amino acid substitutions, the amino acid substitutions comprising: Y365F / V395I / L517T / H519G / P521A / A520C / Q564C / L560Q / F562Y / I569S / F329Y; Y365F / V395I / L517T / H519G / P521N / A520C / Q564C / L560Q / F562Y / I569S / F329Y; Y365F / F392W / V395I / L517T / H519G / P521A / A520C / Q564C / L560Q / F562Y / I569S / F329Y; and / or Y365F / F392W / V395I / L517T / H519G / P521N / A520C / Q564C / L560Q / F562Y / I569S / F329Y.

[0430] Implementation Scheme 28. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises a plurality of amino acid substitutions at positions 329, 365, 395, 526, 527, 528, 560, 562 and 569.

[0431] Implementation Scheme 29. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises an amino acid substitution, the amino acid substitution comprising: F329Y / Y365F / V395I / G526S / P527N / K528T / L560Q / F562Y / I569S; and / or F329Y / Y365F / V395I / G526S / P527N / K528I / L560Q / F562Y / I569S.

[0432] Implementation Scheme 30. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of SEQ ID NO: 11-19.

[0433] Implementation Scheme 31. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises the same polypeptide sequence as any of SEQ ID NO: 11-19.

[0434] Implementation Scheme 32. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide is expressed at a higher level than a reference polypeptide without the amino acid substitution, or optionally a reference polypeptide according to SEQ ID NO: 2 or 10.

[0435] Implementation Scheme 33. The polypeptide as described in any of the preceding embodiments, wherein the expressed polypeptide is more thermally stable or more antigenic than SEQ ID NO: 2 or 10.

[0436] Implementation Scheme 34. The polypeptide as described in any of the preceding embodiments, wherein the polypeptide comprises a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2-10.

[0437] Implementation Scheme 35. A self-assembled protein nanostructure comprising: a first component comprising a recombinant coronavirus polypeptide according to any one of Implementation Schemes 1 to 34; and an optional second component comprising a second protein.

[0438] Implementation Scheme 36. The protein nanostructure of Implementation Scheme 35, wherein the recombinant coronavirus polypeptide comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11-19.

[0439] Implementation Scheme 37. The protein nanostructure as described in Implementation Scheme 35 or Implementation Scheme 36, wherein the recombinant coronavirus polypeptide comprises one or more, two or more, three or more, four or more amino acid substitutions relative to the reference sequence according to SEQ ID NO: 1.

[0440] Implementation Scheme 38. The protein nanostructure as described in any one of Implementation Schemes 35 to 37, wherein the protein nanostructure component polypeptide and the recombinant coronavirus polypeptide are non-covalently coupled.

[0441] Implementation Scheme 39. The protein nanostructure as described in any one of Implementation Schemes 35 to 38, wherein the protein nanostructure component peptide and the recombinant coronavirus peptide are covalently coupled.

[0442] Implementation Scheme 40. A protein nanostructure as described in any one of Implementation Schemes 35 to 39, wherein the protein nanostructure comprises a fusion protein, the fusion protein comprising the recombinant coronavirus polypeptide, a linker, and a protein nanostructure component polypeptide.

[0443] Implementation Scheme 41. A protein nanostructure as described in any one of Implementation Schemes 35 to 40, wherein the protein nanostructure comprises a fusion protein, the fusion protein comprising, from the N-terminus to the C-terminus, the recombinant coronavirus polypeptide, a linker, and the protein nanostructure component polypeptide.

[0444] Implementation Scheme 42. The protein nanostructure of any one of Implementation Schemes 35 to 41, wherein the recombinant coronavirus polypeptide comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11-19.

[0445] Implementation Scheme 43. The protein nanostructure of any one of Implementation Schemes 35 to 42, wherein the fusion protein comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11-19.

[0446] Implementation Scheme 44. The protein nanostructure as described in any one of Implementation Schemes 35 to 43, wherein the first component comprises I53-50A.

[0447] Implementation Scheme 45. The protein nanostructure as described in any one of Implementation Schemes 35 to 44, wherein the protein nanostructure is an I53-50AB protein nanostructure; or an I3-01 / MI3 protein nanostructure.

[0448] Implementation Scheme 46. The protein nanostructure as described in any one of Implementation Schemes 35 to 45, wherein the protein nanostructure is a ferritin protein nanostructure, an encapsulating protein nanostructure, a CP3 phage capsid protein nanostructure, a Qβ phage capsid protein nanostructure, or an AP205 phage capsid protein nanostructure.

[0449] Implementation Scheme 47. The protein nanostructure of any one of Implementation Schemes 35 to 46, wherein the recombinant polypeptide comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the polypeptide sequence of SEQ ID NO: 11-19.

[0450] Implementation Scheme 48. A protein nanostructure as described in any one of Implementation Schemes 35 to 47, wherein the protein nanostructure component polypeptide comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the polypeptide sequence of SEQ ID NO: 20-21.

[0451] Implementation Scheme 49. The protein nanostructure as described in any one of Implementation Schemes 35 to 48, wherein the protein nanostructure comprises a second component polypeptide.

[0452] Implementation Scheme 50. The protein nanostructure as described in any one of Implementation Schemes 35-49, wherein the second component polypeptide is I53-50B.

[0453] Implementation Scheme 51. The protein nanostructure of any one of Implementation Schemes 35 to 50, wherein the second component polypeptide comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 22.

[0454] Implementation Scheme 52. The protein nanostructure of any one of Implementation Schemes 35 to 51, wherein the linker comprises between 8 and 24 amino acid residues.

[0455] 53. The protein nanostructure of any one of embodiments 35 to 52, wherein the linker is selected from the group consisting of: GGSGGSGSGGSGGSGS; SGGGSGGSGSGGSGGSGS; EPEGGSGGSGSGGSGGSGSGS; YGGSGGSGGSGSGGSGSGS; and GGSGGSGGSGGSGGSGSGGSGSGS.

[0456] Implementation Scheme 54. A protein nanostructure as described in any one of Implementation Schemes 35 to 53, wherein the recombinant polypeptide comprises a polypeptide segment having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the polypeptide sequences shown in Table 27.

[0457] Implementation Scheme 55. A polynucleotide encoding a polypeptide as described in any one of Implementation Schemes 1 to 34 or a protein nanostructure as described in any one of Implementation Schemes 35 to 54.

[0458] Implementation Scheme 56. The polynucleotide as described in Implementation Scheme 55, wherein the polynucleotide is mRNA.

[0459] Implementation Scheme 57. A pharmaceutical composition comprising a polypeptide as described in any one of Implementation Schemes 1 to 34, a protein nanostructure as described in any one of Implementation Schemes 35 to 54, or a polynucleotide as described in any one of Implementation Schemes 55 to 56.

[0460] Implementation Scheme 58. A vaccine comprising a polypeptide as described in any one of Implementation Schemes 1 to 34, a protein nanostructure as described in any one of Implementation Schemes 35 to 54, or a polynucleotide as described in any one of Implementation Schemes 55 to 56.

[0461] Implementation Scheme 59. The vaccine as described in Implementation Scheme 58 further comprises an adjuvant.

[0462] Implementation Scheme 60. The vaccine as described in Implementation Scheme 58 or Implementation Scheme 59, wherein the adjuvant comprises squalene.

[0463] Implementation Scheme 61. The vaccine as described in any one of Implementation Schemes 58 to 60, wherein the vaccine is a bivalent vaccine.

[0464] Implementation Scheme 62. The vaccine as described in any one of Implementation Schemes 58 to 61, wherein the bivalent vaccine comprises a polypeptide as described in any one of Implementation Schemes 1 to 34 or a protein nanostructure as described in any one of Implementation Schemes 35 to 54.

[0465] Implementation Scheme 63. A method for preventing coronavirus disease in a subject of need, the method comprising administering to the subject a polypeptide as described in any one of Implementation Schemes 1 to 34, a protein nanostructure as described in any one of Implementation Schemes 35 to 54, a polynucleotide as described in any one of Implementation Schemes 55 to 56, a pharmaceutical component as described in Implementation Scheme 57, or a vaccine as described in any one of Implementation Schemes 58 to 62.

[0466] Implementation Scheme 64. A method for immunizing a subject in need against coronavirus infection, the method comprising administering to the subject a vaccine as described in any one of Implementation Schemes 58 to 62.

[0467] Implementation Scheme 65. A kit comprising a polypeptide as described in any one of Implementation Schemes 1 to 34, a protein nanostructure as described in any one of Implementation Schemes 35 to 54, a polynucleotide as described in any one of Implementation Schemes 55 to 56, a pharmaceutical component as described in any one of Implementation Schemes 57, a vaccine as described in any one of Implementation Schemes 58 to 62, and instructions for use thereof.

[0468] **** Although the invention has been described in conjunction with the specific embodiments presented, it should be understood that the invention is capable of other modifications, and this application is intended to cover any variations, uses, or adaptations of the invention that generally follow the principles described herein, as well as such deviations that may occur in known or customary practice within the field to which this invention pertains.

Claims

1. A recombinant polypeptide comprising an antigenic fragment of a coronavirus spike glycoprotein, said antigenic fragment comprising a receptor-binding domain (RBD) and optionally a coronavirus subdomain 1 (SD1).

2. The polypeptide of claim 1, wherein the antigen fragment comprises RBD-SD1.

3. The polypeptide of claim 1, wherein the SD1 comprises at least one surface-exposed nonpolar amino acid residue replaced by a polar amino acid residue.

4. The polypeptide of claim 3, wherein the polypeptide comprises one, two or more, or three amino acids at positions 560, 562, or 569 being replaced by polar residues, wherein the replacement is relative to the reference sequence according to SEQ ID NO:

1.

5. The polypeptide of claim 3, wherein, relative to the reference sequence according to SEQ ID NO: 1, the polypeptide comprises one or more, two or more, or three of the amino acid substitutions L560Q, F562T, F562Y, or I569S.

6. The polypeptide of claim 3, wherein, relative to the reference sequence according to SEQ ID NO: 1, the polypeptide comprises one, two or more, or three or more amino acid substitutions at positions 544, 546, 560, 562, 564, 569, or 582.

7. The polypeptide of claim 3, wherein, relative to the reference sequence according to SEQ ID NO: 1, the polypeptide comprises one, two or more, three or more, four or more amino acid substitutions at positions N544L, N544M, N544Q, L546V, L560Q, F562T, F562Y, Q564C, Q564L, Q564N, Q564W, I569S or L582S.

8. The polypeptide of claim 1, wherein, relative to the reference sequence according to SEQ ID NO: 1, the polypeptide comprises one, two or more, three or more, four or more amino acid substitutions at positions 338, 358, 363, 365, 392 or 395.

9. The polypeptide of claim 1, wherein, relative to the reference sequence according to SEQ ID NO: 1, the polypeptide comprises one, two or more, three or more, four or more amino acid substitutions at positions F338L, I358F, A363L, Y365F, Y365M, Y365W, F392W or V395I.

10. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid substitution, the amino acid substitution comprising: F338L / Y365W; F392W; Y365F / V395I; Y365F / F392W / V395I; F338L / A363L / Y365M; and / or I358F / Y365F / V395I.

11. The polypeptide of claim 1, wherein, relative to the reference sequence according to SEQ ID NO: 1, the polypeptide comprises one, two or more, three or more, four or more amino acid substitutions at positions 329, 348, 350, 367, 375, 402, 407, 410, 418, 429, 433, 435, 452, 464, 510, 512, 514, 517, 518, 519, 520, 522, 527 or 528.

12. The polypeptide of claim 1, wherein, relative to the reference sequence according to SEQ ID NO: 1, the polypeptide comprises positions F329K, F329R, F329Y, A348P, V350L, V367F, F375Y, I402V, V407L, I410F, I418V, F429W, V433I, A435I, A435V, L452R, F464Y, P512Q, V510I, V512F, V512I, S514T, L517A, L517D, L517S, L517T, L518N, Substitution of one, two or more, three or more, or four or more amino acids at L518Q, L518V, H519D, H519G, H519R, H519S, H519T, A520C, A520D, A520G, A520H, P521A, P521D, P521N, P521Q, P521S, A522G, A522I, P527N, K528I, K528Q, K528T, L518G, or L518S.

13. The polypeptide of claim 1, wherein the polypeptide comprises Amino acid substitutions at positions 365, 395, 560, 562, and 569; and One or more, two or more, or three or more amino acid substitutions at positions 348, 402, 464, 514, 520, 526, and 527 The amino acid substitution is relative to the reference sequence according to SEQ ID NO:

1.

14. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid substitution, the amino acid substitution comprising: A348P / Y365F / V395I / L560Q / F562Y / I569S; Y365F / V395I / I402V / L560Q / F562Y / I569S; Y365F / V395I / S514T / L560Q / F562Y / I569S; Y365F / V395I / I402V / F464Y / L560Q / F562Y / I569S; and / or Y365F / V395I / G526S / P527N / L560Q / F562Y / I569S.

15. The polypeptide of claim 1, wherein the polypeptide comprises Amino acid substitutions at positions 365, 395, 560, 562, and 569; and One or more, two or more, or three or more amino acid substitutions at positions 329, 517, 519, 520, and 544. The amino acid substitution is relative to the reference sequence according to SEQ ID NO:

1.

16. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid substitution, the amino acid substitution comprising: Y365F / V395I / L560Q / F562Y / I569S; Y365F / V395I / F329Y / L560Q / F562Y / I569S; Y365F / V395I / L517T / H519S / L560Q / F562Y / I569S; Y365F / V395I / L517T / H519S / A520G / L560Q / F562Y / I569S; Y365F / V395I / L517T / H519S / N544L / L560Q / F562Y / I569S; and / or Y365F / V395I / F329Y / L517T / H519S / L560Q / F562Y / I569S.

17. The polypeptide of claim 1, wherein the polypeptide comprises Amino acid substitutions at positions 365, 392, 395, 560, 562, and 569; and One or more, two or more, or three or more amino acid substitutions at positions 329, 517, 519, 520, and 544. The amino acid substitution is relative to the reference sequence according to SEQ ID NO:

1.

18. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid substitution, the amino acid substitution comprising: Y365F / F392W / V395I / L560Q / F562Y / I569S; Y365F / F392W / V395I / F329Y / L560Q / F562Y / I569S; Y365F / F392W / V395I / L517T / H519S / L560Q / F562Y / I569S; Y365F / F392W / V395I / L517T / H519S / A520G / L560Q / F562Y / I569S; Y365F / F392W / V395I / L517T / H519S / N544L / L560Q / F562Y / I569S; and / or Y365F / F392W / V395I / F329Y / L517T / H519S / L560Q / F562Y / I569S.

19. The polypeptide of claim 1, wherein the polypeptide comprises Amino acid substitutions at positions 365, 395, 517, 519, 520, 560, 562, 564, and 569; and One or more, two or more, or three or more amino acid substitutions at positions 521, 544, and 546. The amino acid substitution is relative to the reference sequence according to SEQ ID NO:

1.

20. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid substitution, the amino acid substitution comprising: Y365F / V395I / L517T / H519G / A520C / Q564C / L560Q / F562Y / I569S; and / or Y365F / V395I / L517S / H519R / A520H / P521Q / N544L / L546V / Q564W / F562T / L560Q / F562Y / I569S.

21. The polypeptide of claim 1, wherein the polypeptide comprises Amino acid substitutions at positions 365, 395, 517, 519, and 520; and One or more amino acid substitutions at positions 518 and 392 The amino acid substitution is relative to the reference sequence according to SEQ ID NO:

1.

22. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid substitution, the amino acid substitution comprising: Y365F / V395I / L517T / H519S / A520G; Y365F / V395I / L517T / L518G / H519D / A520G; Y365F / V395I / L517T / L518S / H519S / A520G; Y365F / F392W / V395I / L517T / H519S / A520G; Y365F / F392W / V395I / L517T / L518G / H519D / A520G; and / or Y365F / F392W / V395I / L517T / L518S / H519S / A520G.

23. The polypeptide of claim 1, wherein the polypeptide comprises Amino acid substitutions at positions 329, 365, 395, 517, 519, 520, 560, 562, and 569; and One or more, two or more, or three or more amino acid substitutions at positions 392, 544, and 564. The amino acid substitution is relative to the reference sequence according to SEQ ID NO:

1.

24. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid substitution, the amino acid substitution comprising: Y365F / V395I / L517T / H519S / A520G / N544M / L560Q / F562Y / I569S / F329Y; Y365F / V395I / L517T / H519S / A520G / N544Q / L560Q / F562Y / I569S / F329Y; Y365F / V395I / L517T / H519S / A520G / Q564N / L560Q / F562Y / I569S / F329Y; Y365F / F392W / V395I / L517T / H519S / A520G / N544M / L560Q / F562Y / I569S / F329Y; Y365F / F392W / V395I / L517T / H519S / A520G / N544Q / L560Q / F562Y / I569S / F329Y; and / or Y365F / F392W / V395I / L517T / H519S / A520G / Q564N / L560Q / F562Y / I569S / F329Y.

25. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid substitution, the amino acid substitution comprising: Y365F / V395I / L517T / H519S / A520G / N544M / L560Q / F562Y / I569S; Y365F / V395I / L517T / H519S / A520G / N544Q / L560Q / F562Y / I569S; Y365F / V395I / L517T / H519S / A520G / Q564N / L560Q / F562Y / I569S; Y365F / F392W / V395I / L517T / H519S / A520G / N544M / L560Q / F562Y / I569S; Y365F / F392W / V395I / L517T / H519S / A520G / N544Q / L560Q / F562Y / I569S; and / or Y365F / F392W / V395I / L517T / H519S / A520G / Q564N / L560Q / F562Y / I569S.

26. The polypeptide of claim 1, wherein the polypeptide comprises Amino acid substitutions at positions 365, 395, 517, 519, 520, 521, 504, 560, 562, 569, and 329; and One or more amino acid substitutions at position 392 The amino acid substitution is relative to the reference sequence according to SEQ ID NO:

1.

27. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid substitution, the amino acid substitution comprising: Y365F / V395I / L517T / H519G / P521A / A520C / Q564C / L560Q / F562Y / I569S / F329Y; Y365F / V395I / L517T / H519G / P521N / A520C / Q564C / L560Q / F562Y / I569S / F329Y; Y365F / F392W / V395I / L517T / H519G / P521A / A520C / Q564C / L560Q / F562Y / I569S / F329Y; and / or Y365F / F392W / V395I / L517T / H519G / P521N / A520C / Q564C / L560Q / F562Y / I569S / F329Y.

28. The polypeptide of claim 1, wherein the polypeptide comprises a plurality of amino acid substitutions at positions 329, 365, 395, 526, 527, 528, 560, 562 and 569.

29. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid substitution, the amino acid substitution comprising: F329Y / Y365F / V395I / G526S / P527N / K528T / L560Q / F562Y / I569S; and / or F329Y / Y365F / V395I / G526S / P527N / K528I / L560Q / F562Y / I569S.

30. The polypeptide of claim 1, wherein the polypeptide comprises a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of SEQ ID NO: 11-19.

31. The polypeptide of claim 1, wherein the polypeptide comprises the same polypeptide sequence as any of SEQ ID NO: 11-19.

32. The polypeptide of claim 1, wherein the polypeptide is expressed at a higher level than a reference polypeptide without the amino acid substitution, or optionally a reference polypeptide according to SEQ ID NO: 2 or 10.

33. The polypeptide of claim 1, wherein the expressed polypeptide is more thermally stable or more antigenic than SEQ ID NO: 2 or 10.

34. The polypeptide of claim 1, wherein the polypeptide comprises a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2-10.

35. A self-assembled protein nanostructure comprising: A first component comprising a recombinant coronavirus polypeptide according to any one of claims 1 to 34; and An optional second component, which contains a second protein.

36. The protein nanostructure of claim 35, wherein the recombinant coronavirus polypeptide comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11-19.

37. The protein nanostructure of claim 35, wherein the recombinant coronavirus polypeptide comprises one or more, two or more, three or more, four or more amino acid substitutions relative to the reference sequence according to SEQ ID NO:

1.

38. The protein nanostructure of claim 35, wherein the protein nanostructure component polypeptide and the recombinant coronavirus polypeptide are non-covalently coupled.

39. The protein nanostructure of claim 35, wherein the protein nanostructure component polypeptide and the recombinant coronavirus polypeptide are covalently coupled.

40. The protein nanostructure of claim 39, wherein the protein nanostructure comprises a fusion protein, the fusion protein comprising the recombinant coronavirus polypeptide, a linker, and a protein nanostructure component polypeptide.

41. The protein nanostructure of claim 39, wherein the protein nanostructure comprises a fusion protein, the fusion protein comprising, from the N-terminus to the C-terminus, the recombinant coronavirus polypeptide, a linker, and the protein nanostructure component polypeptide.

42. The protein nanostructure of claim 39, wherein the recombinant coronavirus polypeptide comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the polypeptide sequence of SEQ ID NO: 11-19.

43. The protein nanostructure of claim 40, wherein the fusion protein comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the polypeptide sequence of SEQ ID NO: 11-19.

44. The protein nanostructure of claim 39, wherein the first component comprises I53-50A.

45. The protein nanostructure of claim 35, wherein the protein nanostructure is I3-01 / MI3 protein nanostructure; or I53-50AB protein nanostructure.

46. ​​The protein nanostructure of claim 35, wherein the protein nanostructure is Ferritin protein nanostructure; Encapsulated protein nanostructures; CP3 phage capsid protein nanostructure; Qβ phage capsid protein nanostructure; or AP205 phage capsid protein nanostructure.

47. The protein nanostructure of claim 45, wherein the recombinant polypeptide comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the polypeptide sequence of SEQ ID NO: 11-19.

48. The protein nanostructure of claim 35, wherein the protein nanostructure component polypeptide comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the polypeptide sequence of SEQ ID NO: 20-21.

49. The protein nanostructure of claim 35, wherein the protein nanostructure comprises a second component, a polypeptide.

50. The nanoparticles of claim 48, wherein the second component polypeptide is I53-50B.

51. The protein nanostructure of claim 49, wherein the second component polypeptide comprises a polypeptide segment having a polypeptide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the polypeptide sequence of SEQ ID NO:

22.

52. The protein nanostructure of claim 35, wherein the linker comprises between 8 and 24 amino acid residues.

53. The protein nanostructure of claim 35, wherein the linker is selected from the group consisting of: GGSGGSGSGGSGGSGS; SGGGSGGSGSGGSGGSGS; EPEGGSGGSGSGGSGGSGS; YGGSGGSGGSGSGGSGGSGS; and GSGGSGGSGGSGGSGGSGGSGGSGS.

54. The protein nanostructure of claim 35, wherein the recombinant polypeptide comprises a polypeptide segment having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the polypeptide sequences shown in Table 18.

55. A polynucleotide encoding a polypeptide as described in any one of claims 1 to 34 or a protein nanostructure as described in any one of claims 35 to 54.

56. The polynucleotide of claim 55, wherein the polynucleotide is mRNA.

57. A pharmaceutical composition comprising a polypeptide as described in any one of claims 1 to 34, a protein nanostructure as described in any one of claims 35 to 54, or a polynucleotide as described in any one of claims 55 to 56.

58. A vaccine comprising a polypeptide as claimed in any one of claims 1 to 34, a protein nanostructure as claimed in any one of claims 35 to 54, or a polynucleotide as claimed in any one of claims 55 to 56.

59. The vaccine of claim 58, further comprising an adjuvant.

60. The vaccine of claim 59, wherein the adjuvant comprises squalene.

61. The vaccine as claimed in any one of claims 58 to 60, wherein the vaccine is a bivalent vaccine.

62. The vaccine of claim 61, wherein the bivalent vaccine comprises a polypeptide as described in any one of claims 1 to 34 or a protein nanostructure as described in any one of claims 35 to 54.

63. A method for preventing coronavirus disease in a subject of need, comprising administering to the subject a polypeptide as described in any one of claims 1 to 34, a protein nanostructure as described in any one of claims 35 to 54, a polynucleotide as described in any one of claims 55 to 56, a pharmaceutical component as described in claim 57, or a vaccine as described in any one of claims 58 to 62.

64. A method for immunizing a subject in need against coronavirus infection, the method comprising administering to the subject the vaccine as described in any one of claims 58 to 62.

65. A kit comprising a polypeptide as claimed in any one of claims 1 to 34, a protein nanostructure as claimed in any one of claims 35 to 54, a polynucleotide as claimed in any one of claims 55 to 56, a pharmaceutical component as claimed in any one of claims 57, a vaccine as claimed in any one of claims 58 to 62, and instructions for use thereof.

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