Vaccine antigens

Abstract

The field of this specification relates generally to coronavirus vaccine (CoV) antigens and methods of using and making CoV antigens. The invention also relates to vector vaccines, kits, devices, and test strips comprising the coronavirus vaccine antigens. The invention also relates generally to ribonucleic acids encoding S protein monomers of coronavirus vaccine (CoV) antigens and methods of using and making the ribonucleic acids. The invention also relates to vectors, lipid nanoparticles, RNA vaccines, kits, devices, and test strips comprising the ribonucleic acids.

Description

Technical Field

[0001] This specification generally relates to coronavirus vaccine (CoV) antigens and methods for using and preparing CoV antigens. The invention also relates to vaccines, kits, devices, and test strips comprising CoV antigens. Furthermore, the invention relates to ribonucleic acid encoding a monomeric S protein of a coronavirus vaccine (CoV) antigen and methods for using and preparing said ribonucleic acid. The invention also relates to carriers, lipid nanoparticles, RNA vaccines, kits, devices, and test strips comprising said ribonucleic acid. Background Technology

[0002] Since December 2019, the COVID-19 pandemic has resulted in approximately 770 million infections and about 7 million deaths. SARS-CoV-2, the coronavirus that causes COVID-19, has mutated its surface spike (S) glycoprotein over time, which can enhance transmissibility and allow it to evade protective antibodies induced by previous infection and / or vaccination. Since the start of the pandemic in late 2019, viral variants have emerged intermittently, eventually dominating the pandemic by replacing the previous dominant variant (called variant of concern (VOC)). In addition to the ancestral lineage, four consecutive variant lineages dominated the pandemic for approximately six months before being replaced by subsequent variants: α (December 20 to July 21), δ (April 20 to February 22), Omicron BA.1 (November 21 to May 22), and Omicron BA.2 (January to July 22). The most recent VOCs are sublineages of Omicron BA.2: BA.4 / BA.5 (May 22 to February 23), BQ.1 (September 22 to March 23), and the XBB sublineage (October 22 to 23). Currently, the main BA.2.86-derived strains include JN.1, KP.1, KP.2, and KP.3 (October 23 to present). S is the sole target of protective neutralizing antibodies (NAb), thus forming the basis of currently licensed SARS-CoV-2 vaccines, delivered either as mRNA (Pfizer-BioNTech and Moderna) or as the full-length S of a recombinant protein reconstructed in adjuvanted nanoparticles (Novavax). COVID-19 vaccines have saved over 20 million lives since their introduction. To address the emergence of VOCs and variants of interest (VOIs), COVID-19 vaccines are being regularly updated with newly emerging VOC sequences (such as BA.1 in 2022 and BA.4 and XBB in 2023). However, by the time a vaccine update is rolled out, a new VOC has usually already appeared.

[0003] S is a trimeric, membrane-integrated protein with a transmembrane sequence at its C-terminus. The spike contains the ACE2 receptor-binding subunit S1 and S2, which mediates virus-cell membrane fusion. S1 and S2 are derived from the precursor trimeric S, which is cleaved by furin in the producer cell. The heterodimeric S1-S2 trimer forms an ordered head domain, with three receptor-binding domains (RBDs) at the apex and the S2 trimer facing the base (Ke et al., 2020). A 64-amino acid-long stem provides a flexible connection between the head domain and the transmembrane sequence, providing orientational flexibility to the head when on the virion (Turonova et al., 2020).

[0004] The RBD contains the ACE2 receptor-binding motif (RBM) and is located at the top of the trimer, away from the viral envelope. On the virion, the RBD can be observed in either an "upward" RBM exposed conformation or a "downward" RBM closed conformation. The RBD is an immunodominant target of NAbs induced by infection and / or vaccination, but it is also a major site for mutation accumulation in omicron lineage variants (e.g., 15, 16, and 22 mutations, respectively, in the BA.1, BA.4 / BA.5, and XBB.1.5 RBDs). First-generation therapeutic NAbs targeting epitopes of overlapping RBMs have lost their effectiveness against omicron BA.2 lineage subvariants due to mutations in the RBM that also improve ACE2 binding affinity (e.g., K417N, E484K / A, N501Y). Furthermore, the accumulation of mutations in XBB subvariants is associated with increased resistance to serum derived from vaccinated individuals receiving the ancestral S sequence, as well as in cases of breakthrough infection in the early omicron subvariant (Malato et al., 2023 and Uraki et al., 2023). The N-terminal domain (NTD) of S1 contains a NAb “supersite,” but its immunogenicity is lower than that of the RBD. Point mutations, deletions, and insertions occurring at the supersite can also lead to escape from NAbs.

[0005] The S2 subunit is a class I fusion glycoprotein comprising a fusion peptide, a hydrophobic heptapeptide repeat 1 (HR1), a central coiled helix (CH), a second heptapeptide repeat (HR2) within the stem, and a transmembrane sequence. Following receptor attachment, S2 is cleaved on the cell surface by the TMPRSS2 protease or by cathepsin L after endocytosis to release the fusion peptide and achieve complete fusion activation. The S glycoprotein mediates membrane fusion via a class I mechanism, where activation triggers (via ACE2 binding of S1 and TMPRSS2 cleavage of S2) the metastable pre-fusion trimer of the S2 subunit refolds into a stable hairpin trimer, linking the N-terminal fusion peptide and the C-terminal transmembrane sequence together for their associated membrane fusion. S2 contains highly conserved NAb epitopes in both the fusion peptide and the stem, which can elicit antibodies with broad pan-coronavirus neutralizing activity.

[0006] Currently licensed SARS-CoV-2 vaccines are delivered as mRNA (Pfizer-BioNTech, Moderna) or as the full-length S-terminus of a recombinant protein reconstituted in adjuvanted nanoparticles (Novavax). The emergence of a simple method for generating stable, soluble S-terminus trimers would fill a significant gap in the current COVID-19 vaccine library, allowing for storage and distribution without the need for ultra-cold chains. Stabilization of the trimer quaternary assembly has been achieved by replacing the transmembrane sequence with a trimerizing clamp derived from an exogenous, typically immunogenic, protein. Unfortunately, off-target antibody responses to the trimerizing clamp in a Phase I clinical trial halted further development of this vaccine modality.

[0007] Previous observations have shown that the S2 glycoprotein subunit contains a central coiled helix with an unusual 3 to 4 inward-facing position repeats, predominantly occupied by polar residues that mediate minimal interhelical contact in the pre-fusion trimer. The insertion of bulky hydrophobic residues (Val and Ile, respectively) to fill cavities adjacent to Ala1016 and Ala1020 in the 3 to 4 repeats was found to be associated with increased thermal stability of the pre-fusion stable S trimer derived from the omicron BA4 / 5 isolates (PCT / AU2022 / 050429 and PCT / AU2022 / 050880). Despite the lack of external trimerizing clamps, this A1016V / A1020I(VI) glycoprotein variant (S2P.BA45.VI-1208), which contains the entire head domain and stem (residues 16 to 1208), remained trimerized after freeze-thaw cycles, but was obtained in low yields (Poumbourios et al., 2023).

[0008] Therefore, there is a need for improved antigens to elicit an immune response against coronaviruses. Specifically, vaccine antigens with improved stability, melting temperature, immunogenicity, antigenicity, and production yield are required. Summary of the Invention

[0009] In one aspect, the present invention provides a coronavirus (CoV) vaccine antigen comprising a CoV S protein trimer having at least one non-endogenous protomer disulfide bond.

[0010] In one embodiment, the non-endogenous protopolymer disulfide bond is formed between cysteines selected from: i) cysteine ​​at positions corresponding to amino acid numbers 914 and 1123 of SEQ ID NO: 1 or SEQ ID NO: 2 (L23); ii) cysteine ​​at positions corresponding to amino acid numbers 571 and 967 of SEQ ID NO: 1 or SEQ ID NO: 2 (D17); and iii) cysteine ​​at positions corresponding to amino acid numbers 570 and 967 of SEQ ID NO: 1 or SEQ ID NO: 2 (I1).

[0011] In one aspect, the present invention provides a coronavirus (CoV) vaccine antigen comprising a CoV S protein trimer having a C-terminus truncated in the stem region.

[0012] In one aspect, the present invention provides protein nanoparticles comprising coronavirus (CoV) vaccine antigens as described herein.

[0013] In one aspect, the present invention provides virus-like particles comprising coronavirus (CoV) vaccine antigens as described herein.

[0014] In one aspect, the present invention provides deoxyribonucleic acid encoding a coronavirus vaccine antigen as described herein.

[0015] In one aspect, the present invention provides a carrier comprising deoxyribonucleic acid as described herein.

[0016] In one aspect, the present invention provides a host cell comprising deoxyribonucleic acid as described herein or a vector as described herein.

[0017] In one aspect, the present invention provides a method for generating coronavirus (CoV) vaccine antigens as described herein, comprising culturing host cells as described herein in a culture medium to generate the vaccine antigens.

[0018] In one aspect, the present invention provides a vaccine comprising a coronavirus (CoV) vaccine antigen as described herein, or a protein nanoparticle as described herein, or a virus-like particle as described herein, or a deoxyribonucleic acid as described herein, or a vector as described herein.

[0019] In one aspect, the present invention provides a vaccine comprising a coronavirus (CoV) vaccine antigen as described herein, or a protein nanoparticle as described herein, or a virus-like particle as described herein.

[0020] In one aspect, the present invention provides a method for inducing an immune response against a coronavirus (CoV) in a subject, the method comprising delivering to the subject a vaccine antigen as described herein or a vaccine as described herein.

[0021] In one aspect, the present invention provides a method for enhancing an immune response against a coronavirus (CoV) in an object, the method comprising delivering to the object a vaccine antigen as described herein or a vaccine as described herein.

[0022] In one aspect, the present invention provides a method for preventing or reducing the possibility of coronavirus (CoV) infection in a subject, the method comprising delivering to the subject a vaccine antigen as described herein or a vaccine as described herein.

[0023] In one aspect, the present invention provides a method for preventing symptoms of coronavirus (CoV) infection or reducing the likelihood or severity of symptoms of coronavirus (CoV) infection in a subject, the method comprising delivering to the subject a vaccine antigen as described herein or a vaccine as described herein.

[0024] In one aspect, the present invention provides a method for reducing the severity and / or duration of coronavirus (CoV) infection in a subject, the method comprising delivering to the subject a vaccine antigen as described herein or a vaccine as described herein.

[0025] In one aspect, the present invention provides a method for preventing or reducing viral shedding in human individuals infected with coronavirus (CoV), the method comprising delivering to the subject a vaccine antigen as described herein or a vaccine as described herein.

[0026] In one aspect, the present invention provides a vaccine antigen as described herein, or a vaccine as described herein, for use in one or more of the following: i) inducing an immune response against CoV in a subject; ii) enhancing an immune response against CoV in a subject; iii) preventing CoV infection or reducing the likelihood of CoV infection in a subject; iv) preventing CoV symptoms or reducing the likelihood or severity of CoV symptoms in a subject; v) reducing the severity and / or duration of CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating CoV infection in a subject.

[0027] In one aspect, the present invention provides kits, devices, surfaces, or test strips that contain coronavirus (CoV) vaccine antigens as described herein.

[0028] In one aspect, the present invention provides the use of coronavirus (CoV) vaccine antigens as described herein in the preparation of medicaments for one or more of: i) inducing an immune response against CoV in a subject; ii) enhancing an immune response against CoV in a subject; iii) preventing or reducing the likelihood of CoV infection in a subject; iv) preventing or reducing the likelihood or severity of CoV symptoms in a subject; v) reducing the severity and / or duration of CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and

[0029] vii) Treating CoV infection in subjects.

[0030] In one aspect, the present invention provides a method for increasing the yield of S protein trimer, comprising modifying CoV S protein trimer to include a stem region C-terminus truncated.

[0031] In one aspect, the present invention provides a method for stabilizing the CoV S protein trimer in a pre-fusion conformation, comprising modifying the CoV S protein trimer to include at least one non-endogenous protomer disulfide bond.

[0032] In one aspect, the present invention provides a method for increasing the melting temperature of the CoV S protein trimer, comprising modifying the CoV S protein trimer to include a stem region C-terminus truncation.

[0033] In one aspect, the present invention provides a method for increasing the melting temperature of a CoV S protein trimer that is stable in a pre-fusion conformation, comprising modifying the CoV S protein trimer to include a C-terminal truncation of the stem region.

[0034] In one aspect, the present invention provides a method for enhancing a neutralizing antibody response, comprising modifying a CoV S protein trimer to include at least one interprotomeric disulfide bond and / or modifying the CoV S protein trimer to include a C-terminal truncation of the stem region.

[0035] In one aspect, the present invention provides ribonucleic acid encoding a monomer of the S protein of a coronavirus (CoV) vaccine antigen, wherein the vaccine antigen comprises a CoV S protein trimer having at least one non-endogenous interprotomeric disulfide bond.

[0036] In one embodiment, the non-endogenous protopolymer disulfide bond is formed between cysteines selected from: i) cysteine ​​at positions corresponding to amino acid numbers 914 and 1123 of SEQ ID NO: 1 or SEQ ID NO: 2 (L23); ii) cysteine ​​at positions corresponding to amino acid numbers 571 and 967 of SEQ ID NO: 1 or SEQ ID NO: 2 (D17); and iii) cysteine ​​at positions corresponding to amino acid numbers 570 and 967 of SEQ ID NO: 1 or SEQ ID NO: 2 (I1).

[0037] In one aspect, the present invention provides ribonucleic acid encoding an S protein monomer of a coronavirus (CoV) vaccine antigen, wherein the vaccine antigen is a CoV S protein trimer, and wherein the S protein monomer of the CoV S protein trimer has a C-terminal truncation in the stem region.

[0038] In one aspect, the present invention provides a carrier comprising ribonucleic acid as described herein.

[0039] In one aspect, the present invention provides lipid nanoparticles comprising ribonucleic acid as described herein.

[0040] In one aspect, the present invention provides a host cell comprising ribonucleic acid as described herein or a vector as described herein.

[0041] In one aspect, the present invention provides a method for producing a coronavirus vaccine, comprising culturing host cells as described herein in a culture medium to produce ribonucleic acid as described herein.

[0042] In one aspect, the present invention provides an RNA vaccine comprising ribonucleic acid as described herein, or a vector as described herein, or lipid nanoparticles as described herein.

[0043] In one aspect, the present invention provides a method for inducing an immune response against CoV in a subject, the method comprising delivering to the subject a ribonucleic acid vaccine as described herein or an RNA vaccine as described herein.

[0044] In one aspect, the present invention provides a method for enhancing an immune response against CoV in a subject, the method comprising delivering to the subject a ribonucleic acid vaccine as described herein or an RNA vaccine as described herein.

[0045] In one aspect, the present invention provides a method for preventing or reducing the possibility of CoV infection in a subject, the method comprising delivering to the subject a ribonucleic acid vaccine as described herein or an RNA vaccine as described herein.

[0046] In one aspect, the present invention provides a method for preventing symptoms of CoV infection or reducing the likelihood or severity of symptoms of CoV infection in a subject, the method comprising delivering to the subject a ribonucleic acid vaccine as described herein or an RNA vaccine as described herein.

[0047] In one aspect, the present invention provides a method for reducing the severity and / or duration of CoV infection in a subject, the method comprising delivering to the subject an RNA vaccine ribonucleic acid as described herein or an RNA vaccine as described herein.

[0048] In one aspect, the present invention provides a method for preventing or reducing viral shedding in human individuals infected with CoV, the method comprising delivering to the subject an RNA vaccine ribonucleic acid as described herein or an RNA vaccine as described herein.

[0049] In one aspect, the present invention provides one or more of the following ribonucleic acid or RNA vaccines as described herein: i) inducing an immune response against CoV in a subject; ii) enhancing an immune response against CoV in a subject; iii) preventing or reducing the likelihood of CoV infection in a subject; iv) preventing or reducing the likelihood or severity of CoV symptoms in a subject; v) reducing the severity and / or duration of CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating CoV infection in a subject.

[0050] In one aspect, the present invention provides kits, devices, surfaces, or test strips that contain coronavirus (CoV) ribonucleic acid as described herein.

[0051] In one aspect, the present invention provides the use of CoV ribonucleic acid as described herein in the preparation of medicaments for one or more of: i) inducing an immune response against CoV in a subject; ii) enhancing an immune response against CoV in a subject; iii) preventing or reducing the likelihood of CoV infection in a subject; iv) preventing or reducing the likelihood or severity of CoV symptoms in a subject; v) reducing the severity and / or duration of CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating CoV infection in a subject.

[0052] Unless otherwise expressly stated, any embodiment described herein should be adapted to any other embodiment with the necessary modifications. For example, as those skilled in the art will understand, the examples of non-endogenous protomeric disulfide bonds outlined above for the vaccine antigens of the present invention are equally applicable to the ribonucleic acid of the S protein monomer encoding coronavirus (CoV) vaccine antigens.

[0053] The scope of this invention is not limited to the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this invention, as described herein.

[0054] Throughout this specification, unless otherwise expressly stated or required by the context, references to a single step, composition of matter, group of steps, or group of composition of matter shall be regarded as encompassing one or more (i.e., one or more) of such steps, compositions of matter, groups of steps, or groups of composition of matter.

[0055] The invention is described below with reference to the following non-limiting embodiments and the accompanying drawings. Attached Figure Description

[0056] Figure 1 The alignment of the S2P.BA45-1273 amino acid sequence (SEQ ID NO: 2) with the corresponding ancestral Hu-1 reference sequence (SEQ ID NO: 1) is shown. The amino acid numbering convention is the same as that used for Hu-1 and is followed throughout this document. The N-terminal domain (NTD) is highlighted in light gray. The receptor-binding domain (RBD) is highlighted in black. ACE2 receptor-binding residues within the RBD (i.e., the receptor-binding motif, RBM) are in bold text and highlighted in white. The stem region is highlighted in dark gray.

[0057] Figure 2A linear schematic diagram showing the spike protein, highlighting key structural and functional elements. A) Full-length SARS-CoV-2 spike protein. B) Extracellular domains of the spike protein comprising a head (amino acids 16 to 1139) and a stem region (amino acids 1140 to 1207). L: Leader peptide; NTD: N-terminal domain; RBD: Receptor-binding domain; RBM: ACE2 receptor-binding motif; H681RRAR: furin cleavage site; FP: Fusion peptide; HR1: Heptapeptide repeat 1; CH: Central helix; MSS: Transmembrane sequence; tPAL: tissue-plasminogen activator leader; P681GSAS: furin ablation mutation; 2P: K986P / V987P mutation; VI: A1016V / A1020I mutation.

[0058] Figure 3 The expected amino acid sequence of the mature S2P.BA45.VI-1208 glycoprotein (amino acids 16 to 1208) is shown. The non-natural N-terminal tPAL and linking amino acid (AlaSer), as well as the C-terminal Gly-Ser-Gly-Ser-His8 sequence added to spike amino acid 1208, are not included.

[0059] Figure 4 The DNA sequence corresponding to the expected mature S2P.BA45.VI-1208 glycoprotein is shown. The DNA sequence encoding the non-natural N-terminal tPAL and linking amino acid (AlaSer) and the C-terminal Gly-Ser-Gly-Ser-His8 sequence is not included.

[0060] Figure 5 The 3D structure of the SARS-CoV-2 S trimer in the “3RBD down” conformation is shown (plotted using coordinates in PDB ID 6XR8). The trimer head domain, linked to a transmembrane sequence via a stem, is shown. An external trimerizing clamp derived from a foreign protein (e.g., T4 foldon) is typically added to Q1208 (thus replacing the native membrane anchor) to stabilize the trimer spike extracellular domain in a soluble form. In the cryo-EM (cryo-EM) structure of the spike trimer, two lengths of the coiled helix at the top of the stem have been resolved. The last residues resolved were S1147 (e.g., PDB ID 6VSB) or P1162 (e.g., PDB ID 6XR8). The remainder of the stem is flexible and therefore not resolved at high resolution.

[0061] Figure 6The sequence of the stem region of coronaviruses is shown. A) Amino acid sequence of the SARS-CoV-2 spike glycoprotein stem, amino acids 1140 to 1207 (SEQ ID NO: 137). Hydrophobic repeating residues are shaded in gray, and potential N-linked glycosylation sites are in black. Stem truncations described in this document are indicated by vertical lines, and C-terminal amino acids are underlined and numbered. B) Alignment of the stem sequence of β-coronavirus (SEQ ID NO: 137 to 142). Identical amino acids are shaded in dark gray, and similar amino acids are shaded in light gray. Potential N-linked glycosylation sites are in black. SARS-CoV-2 stem truncations analyzed in this study are indicated by vertical lines, and C-terminal amino acids are numbered.

[0062] Figure 7 The amino acid sequence of the expected mature S2P.BA45.VI glycoprotein stem-truncation mutant is shown. The Gly-Ser-His6 sequence, which is added to the C-terminal amino acid of the spike-truncation mutant, is not included.

[0063] Figure 8 The DNA sequence corresponding to the expected mature S2P.BA45.VI glycoprotein stem-truncation mutant is shown. DNA sequences encoding the non-natural N-terminal tPAL and linking amino acid (AlaSer) and the C-terminal Gly-Ser-His6 sequence are not included.

[0064] Figure 9 Biochemical characterization of the pre-fusion stable omicron BA.4 / BA.5 trimer (S2P.BA45.VI) containing the VI mutation, terminating at S1147, K1157, D1165, N1192, D1199, L1200, Q1201, G1204, and Q1208, is shown. A) Superose 6 size-exclusion chromatography (SEC) of total secretory spike protein obtained from 50 mL Expi293F culture by affinity purification on TALON resin. Molecular weight markers are thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), and ovalbumin (43 kDa). B) SEC of purified trimer spikes. Fractions within the vertical dashed line in A) were combined, concentrated, and subsequently re-analyzed after freeze (-80°C)-thaw cycles. The vertical dashed line represents the elution volume. C) The melting temperature of the purified spike trimer, determined by differential scanning fluorimetry (DSF). D) The yield, elution volume, and Tm of the purified trimer.

[0065] Figure 10The diagram shows the results in the absence of (left) and presence of (right) a reducing agent (β-mercaptoethanol). Figure 9 The purified spike trimer is shown in the SDS-PAGE image. The number of C-terminal amino acids for each spike construct is shown above each lane. S, spike protein.

[0066] Figure 11 The graph shows the trimer yield and Tm as a function of C-terminal length. Data from... Figure 9 .

[0067] Figure 12 The antigenic characterization of purified spike trimers, determined by biolayer interferometry using neutralizing ligands targeting: NTD: C1520, RBM: ACE2-Fc, Omi-18, and Omi-42; RBM-free RBD: S2H97, SP1-77; FP: COV44-79; and stem residues 1149 to 1167: CV3-25. The neutralizing ligands were immobilized on an anti-human IgG Fc capture biosensor, and the spike trimer is shown in the analyte phase. The first 300 seconds represent association, and the next 300 seconds represent dissociation. The C-terminal amino acids of the spike construct are shown on the left side of each figure. Top line: 30 nM spike. Middle line: 10 nM spike. Bottom line: 3 nM spike.

[0068] Figure 13 The relative binding of the spike ligand to the S2P.BA45.VI stem truncated mutant is shown. The response (nm) (Req) at binding equilibrium is plotted as a function of C-terminal length. The C-terminal amino acids of the spike construct are shown at the bottom of each figure. Data from Figure 12 .

[0069] Figure 14 The locations of paired Cys substitutions in the omicron BA.4 S6P trimer are shown. The model was plotted using PYMOL with coordinates PDB ID7XNQ. The amino acid pairs substituted by Cys are shown in CPK and identified by the codes used in Table 2.

[0070] Figure 15 The amino acid sequence of the expected mature S2P.BA45.VI-1147 Cys substitution mutant is shown. (Based on Table 2 and...) Figure 14 Encoding mutant. The SARS CoV-2 omicron Ba.4 / omicron BA.5 sequence is shown. The non-natural N-terminal tPAL and linking amino acid (AlaSer) and the C-terminal Gly-Ser-His6 sequence are not included.

[0071] Figure 16The DNA sequence corresponding to the expected mature S2P.BA45.VI-1147 Cys substitution mutant is shown. (Based on Table 2 and...) Figure 13 Encoding mutants. Only the SARS CoV-2 omicron BA.4 / omicron BA.5 sequence is shown. DNA sequences encoding the non-natural N-terminal tPAL and linking amino acid (AlaSer) and the C-terminal Gly-Ser-His6 sequence are not included.

[0072] Figure 17 Biochemical characterization of secretory Cys-substituted S2P.BA45.VI-1147 protein is shown. A) Superose 6 size exclusion chromatography (SEC) of total secretory S2P.BA45.VI-1147 protein after affinity purification using TALON resin. B) SEC of purified trimer spikes. Fractions within the vertical dashed line in A) were combined, concentrated, and subsequently re-analyzed after freeze-thaw cycles (-80°C). Molecular weight markers are thyroglobulin (669 kDa) and ferritin (440 kDa). C) Demothermation temperature of purified spike trimers determined in DSF. D) Trimer yield and Tm.

[0073] Figure 18 The SDS-PAGE of purified S2P.BA45.VI-1147, I1, D17, and L23 spike trimers are shown in the absence of a reducing agent (β-mercaptoethanol) (left panel) and in the presence of a reducing agent (right panel). S2P3: 3 spike monomers linked by disulfide bonds; S2P2: 2 spike monomers linked by disulfide bonds; S2P1: spike monomer.

[0074] Figure 19 The amino acid sequences of the expected mature S2P.BA45.VI-1192 and S2P.BA45.VI-1204 Cys substitution mutants are shown. (Based on Table 2 and...) Figure 14 Encoding mutant. Non-natural N-terminal tPAL and linking amino acid (AlaSer) and C-terminal Gly-Ser-His6 sequence are not included.

[0075] Figure 20 DNA sequences corresponding to the expected mature S2P.BA45.VI-1192 and S2P.BA45.VI-1204 Cys substitution mutants are shown. (Based on Table 2 and...) Figure 13 Encoding mutants. DNA sequences encoding non-natural N-terminal tPAL and linking amino acids (AlaSer) and the C-terminal Gly-Ser-His6 sequence are not included.

[0076] Figure 21Biochemical characterization of secretory S2P.BA45.VI-1192 and S2P.BA45.VI-1204 proteins carrying D17, I1, and L23 mutations is shown. A) Superose 6 SEC of total secretory S2P.BA45.VI-1192 spike mutant after affinity purification using TALON resin. Molecular weight markers are thyroglobulin (669 kDa), ferritin (440 kDa), and aldolase (158 kDa). B) Fractions within the vertical dashed line in A) were combined, concentrated, and subsequently subjected to DSF to obtain Tm. Inset: Trimer yield, elution volume, and Tm are shown from 50 mL culture. C) SDS-PAGE under non-reducing and reducing conditions. dsl - spike, disulfide-linked spike; mon - spike; spike monomer. D) Superose 6 SEC of total secretory S2P.BA45.VI-1204 spike mutant after affinity purification using TALON resin. E) DSF plot of S2P.BA45.VI-1204 spike trimer obtained from D) : Trimer yield / 50 mL culture, elution volume, and Tm are listed. F) Non-reducing and reducing SDS-PAGE of purified S2P.BA45.VI-1204 spike mutant trimer. dsl-spike: disulfide-linked spike. mon-spike: spike monomer.

[0077] Figure 22 The antigenic characterization of purified S2P.BA45.VI-1192 spike trimer with D17 and I1 mutations, as determined by biolayer interferometry using neutralizing ligands targeting: NTD: C1520, RBM: ACE2-Fc, Omi-18, and Omi-42; RBM excluding RBM: S2H97, SP1-77; FP: COV44-79; and stem residues 1149 to 1167: CV3-25. The neutralizing ligands were immobilized on an anti-human IgG Fc capture biosensor, and the spike trimer was visualized in the analyte phase. The first 300 seconds represent association, and the next 300 seconds represent dissociation. The top line represents a 30 nm spike, the middle line represents a 10 nm spike, and the bottom line represents a 3 nm spike. The orientation of the ACE2 extracellular domain (ECD) and the Fab region of the IgG molecule relative to the RBM (shown as CPK) and the flanking region of the RBD is shown on the left adjacent to the corresponding ligand name.

[0078] Figure 23The amino acid sequence of the expected mature S2P.BA45.AA-1192 glycoprotein, carrying the D17, I1, and L23 mutations, is shown, with the A1016V / A1020I(VI) mutation restored to Ala1016 / Ala1020(AA). The non-natural N-terminal tPAL and linking amino acid (AlaSer), as well as the C-terminal Gly-Ser-His6 sequence, are not included.

[0079] Figure 24 The DNA sequence corresponding to the expected mature S2P.BA45.AA-1192 glycoprotein, which carries the D17, I1, and L23 mutations, with the A1016V / A1020I(VI) mutation restored to Ala1016 / Ala1020(AA). The DNA sequence encoding the C-terminal Gly-Ser-His6 sequence is not included.

[0080] Figure 25 Biochemical characterization of the secretory S2P.BA45.AA-1192 glycoprotein carrying D17, I1, and L23 mutations, with the A1016V / A1020I(VI) mutation restored to Ala1016 / Ala1020, is shown. A) Superose 6 SEC of total secretory S2P.BA45.AA-1192 spike mutants after affinity purification using TALON resin. B) SEC of purified trimer spikes. Fractions within the vertical dashed line in A were combined, concentrated, and subsequently re-analyzed after freeze-thaw cycles (-80°C). C) Demotherm temperature of purified spike trimers determined by DSF. Inset: Trimer yield and Tm from 50 mL culture are listed. D) SDS-PAGE under non-reducing and reducing conditions. m, label; dsl - spike, disulfide-linked spike; mon - spike; spike monomer.

[0081] Figure 26 The effect of β-mercaptoethanol, as determined by DSF, on Tm of D17 and I1-1192 constructs with and without the VI mutation is shown.

[0082] Figure 27 The reactivity of purified S2P.BA45.VI-1192 and S2P.BA45.AA-1192 spike trimers with D17 and I1 in BLI with neutralizing ligands is shown. The neutralizing ligands were immobilized on an anti-human IgG Fc capture biosensor, and the spike trimer (30 nM) was in the analyte phase. Association occurred for the first 300 seconds, followed by dissociation for the next 300 seconds.

[0083] Figure 28The expected amino acid sequence of the mature S2P.BA45-1192 spike glycoprotein is shown, where the D17 and I1 mutations are combined with L23 to yield DL and IL, respectively. The non-natural N-terminal tPAL and linking amino acid (AlaSer) and the C-terminal Gly-Ser-His6 sequence are not included.

[0084] Figure 29 The DNA sequence corresponding to the expected mature S2P.BA45-1192 spike glycoprotein is shown, with the D17 and I1 mutations combined with L23 (DL and IL, respectively). The DNA sequences encoding the non-natural N-terminal tPAL and linking amino acid (AlaSer) and the C-terminal Gly-Ser-His6 sequence are not included.

[0085] Figure 30 Biochemical characterization of secretory S2P.BA45-1192 glycoprotein is shown, with D17 and I1 mutations combined with L23 in the presence and absence of the VI mutation. A) Superose 6 SEC of total secretory S2P.BA45.VI-1192 spike mutant after affinity purification using TALON resin. B) SEC of purified trimer spikes. Fractions within the vertical dashed line in A) were combined, concentrated, and subsequently re-chromatographically analyzed after freeze-thaw cycles (-80°C). nd: undetermined. C) Demotherm temperature of purified spike trimers determined by DSF. nd: undetermined. Inset: Trimer yield and Tm from 50 mL culture are listed. D) SDS-PAGE under non-reducing and reducing conditions. m, label; dsl - spike, disulfide-linked spike; mon - spike; spike monomer.

[0086] Figure 31 The amino acid sequence of the expected mature spike glycoprotein, in which one of the 2P mutations carrying D17 and I1 mutations has been restored to the native amino acid Lys986Val987 (SnoP), is shown. The non-native N-terminal tPAL and linking amino acid (AlaSer), as well as the C-terminal Gly-Ser-His6 sequence, are not included.

[0087] Figure 32 The DNA sequence corresponding to the expected mature spike glycoprotein carrying the D17 and I1 mutations, one of which has been restored to the native amino acid Lys986Val987 (SnoP), is shown. DNA sequences encoding the non-native N-terminal tPAL and linking amino acid (AlaSer), as well as the C-terminal Gly-Ser-His6 sequence, are not included.

[0088] Figure 33Biochemical signatures of secretory omicron BA45.VI-1192 and BA45.AA-1192 glycoproteins, in which the 2P mutation has been restored to the native amino acid Val986Lys987 (referred to as SnopP), are shown. A) Superose 6 SEC of total secretory omicron BA4 / 5 S2P and SnopP glycoproteins after affinity purification using TALON resin. B) SEC of purified trimer spikes. Fractions within the vertical dashed line in A) were combined, concentrated, and subsequently re-analyzed after freeze-thaw cycles (-80°C). C) Melting temperature of purified spike trimers determined by DSF. D) Inset: Trimer yield and Tm from 50 mL culture are listed.

[0089] Figure 34 The SDS-PAGE of omicronBA45.VI-1192 and BA45.AA-1192 glycoproteins, in which the 2P mutation has been restored to the native amino acid Val986Lys987 (SnoP), is shown under non-reducing and reducing conditions. m, label; dsl - spike, disulfide-linked spike; mon - spike; spike monomer.

[0090] Figure 35 The amino acid sequence of the full-length spike glycoprotein (amino acids 1 to 1273) carrying D17, I1, L23, DL, and IL is shown, in addition to mutations at the 2P and furin protease sites. “VI” indicates the presence of the VI mutation.

[0091] Figure 36 The DNA sequence corresponding to the full-length spike glycoprotein (amino acids 1 to 1273) carrying D17, I1, L23, DL, and IL in addition to mutations at the 2P and furin sites is shown. “VI” indicates the presence of the VI mutation.

[0092] Figure 37 SDS-PAGE / Western blot analysis of the S2P.BA45.VI-1273 glycoprotein containing D17, I1, L23, DL, and IL Cys mutations from lysates of transfected 293T cells is shown under non-reducing (left) and reducing (right) conditions. VI: Constructs containing Val and Ile at amino acid positions 1016 and 1020, respectively. If VI is not indicated, the protein has Ala at positions 1016 and 1020. Samples were electrophoresed on 3% to 8% SDS-PAGE gels, transferred to nitrocellulose, and blotted with rabbit anti-S1 and anti-rabbit IRDye 800. m, label; dsl - spike, disulfide-linked spike; mon - spike; spike monomer.

[0093] Figure 38The binding of ACE2-Fc and human monoclonal NAbs, as determined by FACS, to the S2P.BA45.VI-1273, D17.VI-1273, and I1.VI-1273 glycoproteins expressed on the surface of transfected 293T cells is shown. A) Transfected cells were gently separated from the culture plate, and intact cells were stained with ACE2-Fc and various human monoclonal NAbs, as well as AlexaFluor-conjugated anti-human immunoglobulins. Cells were counterstained with a live / dead staining agent to exclude dead cells from the analysis. Fluorescence intensity was analyzed for EGFP / S2P.BA45.VI-1273 variant glycoprotein double-positive cells outside the negative cell population. HCV1 is an HCV-specific NAb and was used as an isotype (IgG1) control. B) Shown Figure 38 The geometric mean of fluorescence intensity shown in the histogram in Figure A.

[0094] Figure 39 The amino acid sequence of the expected mature S6P.BA45.AA-1192 glycoprotein carrying the D17 and I1 mutations is shown. “VI” indicates the presence of the VI mutation. The non-natural N-terminal tPAL and linking amino acid (AlaSer) and the C-terminal Gly-Ser-His6 sequence are not included.

[0095] Figure 40 The amino acid sequence of the expected mature S6P.BA45.AA-1192 glycoprotein carrying the D17 and I1 mutations is shown. “VI” indicates the presence of the VI mutation. The non-natural N-terminal tPAL and linking amino acid (AlaSer) and the C-terminal Gly-Ser-His6 sequence are not included.

[0096] Figure 41 Biochemical characterization of secretory S2P.BA45-1192 and S6P.BA45-1192 glycoproteins carrying D17 and I1 mutations is shown. A) Superose 6 SEC of total secretory S2P.BA45-1192 and S6P.BA45-1192 spike mutants after affinity purification using TALON resin. B) SEC of purified trimer spikes. Fractions within the vertical dashed line in A were combined, concentrated, and subsequently re-analyzed after freeze-thaw cycles (-80°C). C) Melting temperatures of purified S2P.BA45-1192 (dashed line) and S6P.BA45-1192 (solid line) trimers determined by DSF. Melting temperatures are shown at the top of the figure. D) SDS-PAGE under non-reducing (top) and reducing (bottom) conditions. m, mark; dsl - spike, disulfide-linked spike; mon - spike; spike monomer.

[0097] Figure 42The antigenic characteristics of purified S6P.BA45-1192 spike trimer, as determined by biolayer interferometry using neutralizing ligands targeting: NTD: C1520, RBM: ACE2-Fc, Omi-18, Omi-42, SA55; RBM excluding RBM: S2H97, SP1-77, S309; ​​and stem: CV3-25, CC99-103, CC95-108. The neutralizing ligands were immobilized on an anti-human IgG Fc capture biosensor, and the spike trimer was observed in the analytical phase. The first 300 seconds represent association, and the next 300 seconds represent dissociation. The top black line represents a 30 nM spike. The middle line represents a 10 nM spike. The bottom gray line represents a 3 nM spike.

[0098] Figure 43 Projection of NAb epitopes for ACE2 and RBM onto open and closed spike trimers. The amino acids contributing to the epitopes are shown in CPK mode. The coordinates used to plot the open and closed structures are PDB ID 6VSB and 6XR8, respectively. The 3D models were drawn using PyMOL.

[0099] Figure 44 The K18-hACE2 mouse immunization-viral challenge protocol is shown.

[0100] Figure 45 It shows that in the use of 10 4 Omicron BA.5 virus replication in mice immunized three times with an experimental vaccine following infection with SARS-CoV-2 BA.5 virus of TCID50. Viral titers in the nasal turbinate (NT) and lungs of eight mice in each group, obtained on day 4 post-infection, are expressed as log [log value missing]. 10 TCID 50 (50% tissue culture infection dose) / mL (NT) and log 10 TCID 50 / Organ (lung). Horizontal bars represent the geometric mean titer, and symbols indicate the titer from a single mouse. The detection limit for NT is 100.5 TCID. 50 / mL and the detection limit for lung cancer is 100.8 TCID. 50 / organ. Relative to the carrier group; Kruskal-Wallis test and Dunn post-hoc test.

[0101] Figure 46The antibody response in mice following two (week 6) and three (week 8) immunizations with the experimental vaccine shown in the figure is illustrated. A) Neutralization assays were performed in high-throughput using real SARS-CoV-2 omicron BA.5 virus. Geometric mean neutralization ID of the control group receiving three doses of 50% Addavax-PBS. 50 <1 / 20 (dashed line). B) ELISA binding titers against BA.5 RBD and C) against the S6P.BA45.AA-1192 trimer. The endpoint was defined as 50-fold background luciferase activity obtained in the absence of primary antibody. The horizontal dashed line represents the geometric mean binding titer of the control group receiving three doses of 50% Addavax-PBS. The ID observed in serum at weeks 6 and 8 was determined using the Wilcoxon rank test. 50 Is the difference in binding titer significant? (ns), not significant; .

[0102] Figure 47 The amino acid changes present in the omicron BA.5, XBB.1.5, BA.2.86, and JN.1 spike glycoproteins relative to the ancestral Hu-1 spike glycoprotein are listed. Del: deletion; ins: insertion. Amino acid numbering is based on the Hu-1 reference sequence.

[0103] Figure 48 The neutralizing activity of serum obtained after three immunizations with the experimental vaccine is shown. The real SARS-CoV-2 variant used in the neutralization assay is shown in the figure above. The drug-loaded control and vaccine groups are shown below the x-axis. The neutralization assay was performed in high-throughput mode. The horizontal bars are the geometric mean ID for each immunogen group. 50 For all viral variants, the geometric mean neutralization ID in the control (loador) group receiving three doses of 50% Addavax-PBS was... 50 <1 / 20 (dashed line). The Kruskal-Wallis test was used to determine the ID observed between animal groups. 50 Is the difference significant? (ns), not significant; .

[0104] Figure 49The ELISA binding titers of serum obtained after three immunizations with the experimental vaccine against RBDs derived from ancestral, Omicron BA.5, XBB, and JN.1 variants are shown. Variant RBDs are shown at the top of the graph, and the drug-loaded control and vaccine groups are shown below the x-axis. The endpoint was defined as 50-fold background luciferase activity obtained in the absence of primary antibody. Horizontal bars are geometric means. The Kruskal-Wallis test was used to determine whether differences in binding titers were significant. (ns, not significant;) .

[0105] Figure 50 The ELISA binding titers of serum obtained after three immunizations with the experimental vaccine against NTDs derived from ancestor, Omicron BA.5, and JN.1 variants are shown. Variant NTDs are shown at the top of the graph, and the carrier control and vaccine group are shown below the x-axis. The endpoint was defined as 50-fold background luciferase activity obtained in the absence of primary antibody. The horizontal bars are geometric means. The Kruskal-Wallis test was used to determine whether differences in binding titers were significant. (ns, not significant;) .

[0106] Figure 51 The ELISA binding titers of serum against spike protein fragments obtained after three immunizations with the experimental vaccine are shown. A) Stem synthetic peptide (S amino acids 1138 to 1165); B) Maltose-binding protein-(S amino acids 1138 to 1208) chimeric protein; C) Synthetic fusion peptide (S amino acids 808 to 832). The carrier control and vaccine groups are shown below the x-axis. The endpoint was defined as 50-fold background luciferase activity obtained in the absence of primary antibody. The horizontal bars are geometric means. The Kruskal-Wallis test was used to determine whether differences in binding titers were significant. ns, not significant; .

[0107] Figure 52 Summary Figures 45 to 51 (excluding) Figure 46 The data presented in the figure. The lines in each figure connect the geometric mean TCID. 50 (Viral load in the organization), ID 50 (Virus neutralization titer) or binding titer (right-hand side plot). A single symbol represents the titer of a single animal. The Kruskal-Wallis test and Dunn post-hoc test were used to determine whether the difference in binding titers between the glycoprotein vaccine-immunized animal groups and the load-controlled group was significant. ns, not significant; .

[0108] Figure 53 The amino acid sequence of the expected mature omicron BA.2.86 spike glycoprotein, carrying D17 and I1 mutations in addition to the 6P mutation, is shown. The non-natural N-terminal tPAL and linking amino acid (AlaSer), as well as the C-terminal Gly-Ser-His6 sequence, are not included.

[0109] Figure 54 The DNA sequence of the expected mature omicron BA.2.86 spike glycoprotein, carrying D17 and I1 mutations in addition to the 6P mutation, is shown. The non-natural N-terminal tPAL and linking amino acid (AlaSer), as well as the C-terminal Gly-Ser-His6 sequence, are not included.

[0110] Figure 55 Biochemical characterization of secretory S6P.BA286-1192 glycoprotein carrying D17 and I1 mutations is shown. A) Superose 6SEC of total secretory S6P.BA286-1192 spike mutants after affinity purification using TALON resin. B) SEC of purified trimer spikes after freeze-thaw cycles (-80°C). C) Melting temperature of purified S6P.BA286-1192 trimers determined by DSF. Melting temperatures are shown above the figure. D) SDS-PAGE under non-reducing and reducing conditions. m, label; dslS, disulfide-linked spike; monS, spike monomer.

[0111] Figure 56 The antigenic characterization of purified S6P.BA286-1192 spike trimer with D17 and I1 mutations, as determined in BLI using neutralizing ligands, is shown. Ligands target: NTD: C1520, RBM: ACE2-Fc, Omi-18, and Omi-42; RBM-free RBM: S2H97, SP1-77; and stem residues 1149 to 1167: CV3-25, CC95-108, and CC99-103. Neutralizing ligands were immobilized on an anti-human IgG Fc capture biosensor, and the spike trimer (30 nM) was in the analytical phase. The first 300 seconds were for association, and the next 300 seconds for dissociation. Gray dashed line: S6P.BA286.AA-1192; Black solid line: S6P.BA286.D17.AA-1192; Gray solid line: S6P.BA286.I1.VI-1192.

[0112] Figure 57A maximum likelihood phylogenetic tree of the sarbecovirus spike amino acid sequence is shown. The spike sequence is segregated into four clades: 1a, which includes SARS CoV; 1b, which includes SARS CoV-2, 2, and 3. The amino acid identity of representative isolates from each clade (enclosed in gray boxes) relative to omicron BA.5 is shown. Clades 1a, 1b, and 3 utilize ACE2 as an entry receptor; however, clade 2 virus does not utilize ACE2 due to a deletion in the RBD. This tree was constructed using MEGA X.

[0113] Figure 58 The locations of equivalent D17 and I1 mutation targets in the 3D structure of the spike protein are shown. A) D17 and I1 mutation targets in the SARS CoV-2 spike protein (PDB ID 6XR8). B), C), and D) Close-up views of D17 and I1 mutation targets in PRD-0038 (PDB ID 8U29), WIV1 (PDB ID 8TC0), and BANAL-20-236 (PDB ID 8I3W), respectively.

[0114] Figure 59 The amino acid sequence of the expected mature omicron PRD-0038 spike glycoprotein, carrying D17 and I1 mutations in addition to the 6P mutation, is shown. The non-natural N-terminal tPAL and linking amino acid (AlaSer) and the C-terminal Gly-Ser-His6 sequence are not included.

[0115] Figure 60 The DNA sequence of the expected mature PRD-0038 spike glycoprotein, carrying D17 and I1 mutations in addition to the 6P mutation, is shown. The non-natural N-terminal tPAL and linking amino acid (AlaSer) and the C-terminal Gly-Ser-His6 sequence are not included.

[0116] Figure 61 Biochemical characteristics of secretory S6P.PRD-1192 glycoprotein carrying D17 and I1 mutations are shown. A) Superose 6SEC of total secretory S6P.BA286-1192 spike mutants after affinity purification using TALON resin. B) Melting temperature of purified S6P.BA286-1192 trimer determined by DSF. Melting temperatures are shown above the figure. C) SDS-PAGE under non-reducing and reducing conditions. m, label; dslS, disulfide-linked spike; monS, spike monomer.

[0117] Figure 62The antigenic characterization of purified S6P.PRD-1192 spike trimer with D17 and I1 mutations, as determined in BLI using neutralizing ligands, is shown. Ligand targets: RBD: CR3022; and stems: CV3-25, S2P6, CC40.8, CC95-108, CC99-103. Neutralizing ligands were immobilized on an anti-human IgG Fc capture biosensor, and the spike trimer was in the analyte phase. The first 300 seconds represent association, and the next 300 seconds represent dissociation. Thick black lines: 30 nm analyte, thick gray lines: 10 nm analyte, thin gray lines: 3 nm analyte. KD values ​​are shown in the upper right of each sensor plot.

[0118] Figure 63 Biochemical characteristics of secretory S6P.BA286-1192 glycoprotein expressed by mRNA carrying D17 and I1 mutations are shown. A) Superose 6 SEC of total secretory S6P.BA286-1192 spike mutants after affinity purification using TALON resin. B) Melting temperature of purified S6P.BA286-1192 trimers determined by DSF. Melting temperatures are shown above the figure. C) SDS-PAGE under non-reducing and reducing conditions. m, label; dslS, disulfide-linked spike; monS, spike monomer.

[0119] Figure 64 The antigenic characterization of purified S6P.BA286-1192 spike trimer with D17 and I1 mutations, expressed by mRNA and identified in BLI using neutralizing ligands, is shown. Ligands target: NTD: C1520, RBM: ACE2-Fc, Omi-18, and Omi-42; RBM-free RBM: S2H97, SP1-77; and stem residues 1149 to 1167: CV3-25. Neutralizing ligands were immobilized on an anti-human IgG Fc capture biosensor, and the spike trimer is shown in the analyte phase. The first 300 seconds represent association, and the next 300 seconds represent dissociation. Dashed gray line: S6P.BA286.AA-1192; solid black line: S6P.BA286.D17.AA-1192; solid gray line: S6P.BA286.I1.VI-1192.

[0120] Figure 65 The amino acid sequence of the full-length spike glycoprotein (amino acids 1 to 1273) carrying mutations in addition to those at the 6P and furin protease sites is shown. “VI” indicates the presence of the VI mutation.

[0121] Figure 66The DNA sequence of the full-length spike glycoprotein (amino acids 1 to 1273) carrying mutations in addition to those at the 6P and furin protease sites is shown. “VI” indicates the presence of the VI mutation.

[0122] Figure 67 Expression of the full-length membrane-anchored S6P-1273 spike protein from mRNA and DNA, as shown by Western blotting. mRNA encoding the S6P.BA286-1273 glycoprotein carrying D17 and I1 mutations and DNA encoding the S6P.BA45-1273 glycoprotein carrying D17 and I1 mutations were transfected into 293T cells. Forty-eight hours post-infection, cells were lysed and subjected to SDS-PAGE under non-reducing or reducing conditions, followed by Western blotting with serum from guinea pigs immunized with spike trimers. The blots were visualized using peroxidase-conjugated anti-guinea pig immunoglobulin and chemiluminescence. m, label; dslS, disulfide-linked spike; monS, spike monomer.

[0123] Figure 68 The binding of ACE2-Fc and human monoclonal NAbs to S6P.BA45.AA-1273, S6P.BA45.D17.AA-1273, and S6P.BA45.I1.VI-1273 glycoproteins expressed on the surface of transfected 293T cells is shown, as determined by flow cytometry. A) Transfected cells were gently separated from the culture plate, and intact cells were stained with ACE2-Fc and various human monoclonal NAbs, as well as AlexaFluor-conjugated anti-human immunoglobulins. Cells were counterstained with a live / dead staining agent to exclude dead cells from the analysis. Fluorescence intensity was analyzed for EGFP / S6P.BA45-1273 variant glycoprotein double-positive cells outside the negative cell population. HC33.1 is an HCV-specific NAb and was used as an isotype (IgG1) control. B) Shown Figure 60 The geometric mean of fluorescence intensity in the histogram shown in Figure A.

[0124] Figure 69The binding of ACE2-Fc and human monoclonal NAbs, as determined by flow cytometry, to the S6P.BA45.AA-1273, S6P.BA45.D17.AA-1273, and S6P.BA45.I1.VI-1273 glycoproteins expressed by mRNA on the surface of transfected 293T cells is shown. A) Cells transfected with mRNA were gently separated from the culture plate, and intact cells were stained with ACE2-Fc and various human monoclonal NAbs, as well as AlexaFluor-conjugated anti-human immunoglobulins. Cells were counterstained with a live / dead staining agent to exclude dead cells from the analysis. Fluorescence intensity was analyzed for EGFP / S6P.BA45-1273 variant glycoprotein double-positive cells outside the negative cell population. HC33.1 is an HCV-specific NAb and was used as an isotype (IgG1) control. B) Shown Figure 61 The geometric mean of fluorescence intensity in the histogram shown in Figure A.

[0125] Figure 70 This study demonstrates how D17 and I1 mutations affect the ability of S6P.BA45-1273 to mediate HIV pseudotyped entry into 293-ACE2 cells via luciferase reporter HIV. Luciferase reporter HIV particles were pseudotyped using either wild-type or S6P variants after transfection of 293T cells. Forty-eight hours post-transfection, virus-containing supernatant was used to infect 293-ACE2 cells. 293-ACE2 cells were lysed after 72 hours, and luciferase activity was determined. Data are presented as mean ± standard error of mean from three independent transfections. ns, not significant; S6P.BA45.AA-1273 (S6P) relative to S6P.BA45.D17.AA-1273 (S6P.D17.AA), S6P.BA45.D17.VI-127 3 (S6P.D17.VI), S6P.BA45.I1.AA-1273 (S6P.I1.AA), S6P.BA45.I1.VI-1273 (S6P.I1.VI).

[0126] Sequence List Description

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] Detailed Implementation

[0139] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Any materials and methods similar to or equivalent to those described herein may be used to practice or test the contents of this disclosure. For definitions and terms in the art, as well as other methods known to those skilled in the art, practitioners shall refer in particular to Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons, New York, 1999; Colowick and Kaplan, eds., Methods In Enzymology, Academic Press, Inc.; Weir and Blackwell et al., Handbook of Experimental Immunology, Volumes I-IV, Blackwell Scientific Publications, 1986; Kontermann and Dubel (eds.), Antibody Engineering, Volumes 1-2, eds., Springer Press, 2010.

[0140] Any reference to prior art in this specification is not, and should not be construed as, an acknowledgment or any form of implication that such prior art is part of the common general knowledge of any country.

[0141] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y” and should be regarded as providing explicit support for both or either meaning. Unless otherwise indicated, the term “about” as used herein means + / - 10% of a specified value, more preferably + / - 5%, and even more preferably + / - 1%.

[0142] Throughout this specification, the term "including / comprises" or variations thereof will be understood to mean including the stated element, whole or step, or group of elements, integers or steps, but does not exclude any other element, integer or step, or group of elements, integers or steps.

[0143] Unless the context otherwise indicates, nouns without quantifiers as used herein include both singular and plural referents. Unless otherwise expressly stated, each embodiment in this specification, with necessary modifications, will be applicable to all other embodiments.

[0144] Nucleotide and amino acid sequences are identified by sequence identifier numbers (SEQ ID NO: ). SEQ ID NO: numerically corresponds to the sequence identifier. <400> 1 (SEQ ID NO: 1) <400> 2 (SEQ ID NO: 2), etc. A sequence list is provided after the claims.

[0145] The sequence of the coronavirus spike (S) protein from the ancestral Hu-1 strain is described in Wu et al., 2020, and in NCBI reference sequence: YP_009724390.1. This strain may also be referred to herein as the “wild-type,” “ancestral,” and “parental” strain.

[0146] The term "antigen" as used in this article refers to a substance that can stimulate an immune response.

[0147] As used herein, "protopolymer" refers to the basic structural unit of an oligomeric protein (e.g., the S protein monomer of an S protein trimer). In some embodiments, protopolymers may be chemically linked (e.g., via disulfide bonds) to form or stabilize a portion of an oligomeric structure (e.g., three S protein monomers may be linked to form an S protein trimer oligomeric structure). Those skilled in the art will appreciate that an S protein trimer comprises three protopolymers.

[0148] As used in this article, "endogenous" refers to something that develops or originates within a virus.

[0149] As used in this article, “non-endogenous” refers to something that has not yet developed or originated within a virus (e.g., something that has been modified to include, such as something artificial).

[0150] As used herein, "unfolding temperature" refers to the temperature at which 50% of a protein unfolds. Temperature can disrupt the chemical interactions that maintain the structural shape of a protein. Therefore, unfolding temperature can be used to reflect or indicate protein stability. In one embodiment, the unfolding temperature can be determined using differential scanning fluorometry (DSF).

[0151] As used herein, “signal sequence” refers to residues 1 to 15 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.

[0152] As used in this article, “N-terminal domain” or “NTD” refers to residues 16 to 305 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.

[0153] As used herein, “receptor-binding domain” or “RBD” refers to residues 335 to 415, 418 to 433, and 507 to 520 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.

[0154] As used in this article, “receptor-binding motif” or “RBM:” refers to residues 417, 446, 449, 453, 455, 456, 475, 486, 487, 489, 493, 496, 498, 500, 501, 502, and 505 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.

[0155] As used in this article, “flank of RBD” refers to a portion of the RBD that supports the RBM at its apex.

[0156] As used herein, “stem region” refers to residues 1140 to 1207 corresponding to SEQ ID: NO: 1. In one embodiment, the stem region contains residues 1140 to 1204 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.

[0157] As used herein, the term "trimerizing sequence" refers to a sequence present in the C-terminal region of the S protein monomer that promotes the trimerization of the S protein. In some embodiments, the trimerizing domain is a heterologous sequence within a coronavirus. In one embodiment, the trimerizing sequence is a heterologous sequence not present in SARS-CoV-2. In some embodiments, the trimerizing sequence is the trimerizing foldon domain of phage T4 fibrin or a modified form thereof. In some embodiments, the trimerizing sequence is a coiled helix, an artificially coiled helix, or a modified coiled helix. In some embodiments, the trimerizing sequence is de novo designed. In some embodiments, the trimerizing sequence is the trimerizing foldon domain of phage T4 fibrin or a modified form thereof. In some embodiments, the trimerizing sequence is a native CoV trimerizing sequence (in some embodiments, it is a native transmembrane domain). In some embodiments, the trimerizing sequence comprises residues 1209 to 1256 of the S protein monomer or is composed of residues 1209 to 1256 of the S protein monomer. In some embodiments, the trimer sequence comprises residues 1217 to 1237 of the S protein monomer or consists of residues 1217 to 1237 of the S protein monomer.

[0158] As used herein, "truncation" refers to shortening a molecule by removing a portion of it. In one embodiment, the molecule is a protein and is truncated at the C-terminus of the protein sequence. In some embodiments, truncation removes at least a portion of the trimerizing sequence.

[0159] The term “increase / enhancement” and its variations as used in this article refer to a given parameter that is at a higher or greater level than the baseline level or the control level.

[0160] As used herein, "control" refers to a comparative standard used to examine the results of a survey or experiment. In one embodiment, a control is a vaccine antigen lacking one or more of the following: i) a non-endogenous promoter disulfide bond, ii) a C-terminal truncation in the stem region, and iii) a structural modification that reduces the size of the alanine cavity in the coiled-coil region of the S protein trimer. In one embodiment, a control is a vaccine antigen lacking a non-endogenous promoter disulfide bond. In one embodiment, a control is a vaccine antigen lacking a C-terminal truncation in the stem region. In one embodiment, a control is a vaccine antigen lacking a structural modification that reduces the size of the alanine cavity in the coiled-coil region of the S protein trimer.

[0161] The term “reduction / decrease” and its variations as used in this article refer to a given parameter that is at a lower or lesser level compared to the baseline level or compared to a control.

[0162] As used in this article, the term "epitope" refers to a specific antigenic peptide sequence on a molecule that triggers a specific immune response. An epitope is a region of an antigen that elicits a B cell and / or T cell response. Antibodies can bind to specific antigenic epitopes that can be formed from either continuous or non-continuous amino acids.

[0163] As used in this article, “trimer yield” or “trimer production” refers to the number of micrograms of pure trimeric spike protein obtained from 50 mL of cell culture.

[0164] coronavirus

[0165] "Coronavirus" or "CoV" is an enveloped, positive-sense, single-stranded RNA virus. The Coronaviridae family has two subfamilies: Letovirinae and Orthocoronavirinae. In one embodiment, CoV is selected from the genera α-coronavirus (αCoV), β-coronavirus (βCoV), γ-coronavirus (γCoV), and δ-coronavirus (δCoV). In one embodiment, αCoV is selected from coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), feline infectious peritonitis virus (FIPV), and canine coronavirus (CCoV). In one embodiment, CoV is a β-coronavirus. In one embodiment, βCoV is selected from human coronavirus HKU1 (HCoV-HKU1), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome-associated coronavirus (SARS-CoV), severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2), Middle East respiratory syndrome-associated coronavirus (MERS-CoV), murine hepatitis virus (MHV), and / or bovine coronavirus (BCoV). In one embodiment, the CoV is capable of infecting humans. In one embodiment, the CoV capable of infecting humans is selected from SARS-CoV-2, HCoV-OC43, HCoV-HKU1, HCoV-229E, HCoV-NL63, SARS-CoV, and MERS-CoV, or their subtypes or variants.

[0166] In one implementation, CoV is SARS-CoV-2 or its subtypes or variants. In one implementation, SARS-CoV-2 is SARS-CoV-2 hCoV-19 / Australia / VIC01 / 2020. In one implementation, SARS-CoV-2 comprises a sequence as described in NCBI Reference Sequence: NC_045512.2. In one implementation, SARS-CoV-2 comprises a sequence as described in GenBank: MN908947.3 or a variant thereof. For example, some examples of SARS-CoV-2 variants are described in: Shen et al., 2020, Tang et al., 2020, Phan et al., 2020, Khan et al., 2020, Foster et al., 2020, Vasireddy et al., 2021, Winger et al., 2021, Sanyaolu et al., 2021, Ou et al., 2022, and Fernandes et al., 2022.

[0167] In one embodiment, SARS-CoV-2 is an omicron variant or its subtype or variant. In one embodiment, SARS-CoV-2 is a non-omicron variant. In one embodiment, SARS-CoV-2 is a delta variant or its subtype or variant.

[0168] In one embodiment, the CoV variant shares at least 90% identity with the parent strain. In one embodiment, the variant shares at least 92% identity with the parent strain. In one embodiment, the variant shares at least 93% identity with the parent strain. In one embodiment, the variant shares at least 94% identity with the parent strain. In one embodiment, the variant shares at least 95% identity with the parent strain. In one embodiment, the variant shares at least 96% identity with the parent strain. In one embodiment, the variant shares at least 97% identity with the parent strain. In one embodiment, the variant shares at least 98% identity with the parent strain. In one embodiment, the variant shares at least 99% identity with the parent strain. In one embodiment, the parent strain (also referred to as the ancestral strain) is the Hu-1 strain, described in Wu et al., 2020. In some embodiments, the parent strain is SARS-CoV-2 hCoV-19 / Australia / VIC01 / 2020. In some embodiments, the parent strain is βCoV / ancestor / WIV04 / 2019.

[0169] In one implementation, CoV is a “target variant,” also known as a “VOI.” As used herein, a VOI is an CoV variant associated with genetic changes predicted or known to affect viral characteristics such as transmissibility, disease severity, immune evasion, diagnostic or treatment evasion; and identified as causing significant community transmission or multiple disease clusters (in the case of SARS-CoV-2, COVID-19 clusters) in multiple regions and with a relative prevalence increasing over time with increasing case numbers; or other epidemiological effects that clearly indicate a new risk to global public health.

[0170] In one implementation, CoV is a “variant of concern,” also known as a “VOC.” As used herein, VOC refers to a variant of CoV that is associated with one or more of the following changes that have a degree of global public health significance: increased transmissibility or harmful changes in epidemiology (in the case of SARS-CoV-2, harmful changes are epidemiological aspects of COVID-19); increased virulence or changes in clinical disease presentation; or a decline in the effectiveness of public health and social measures or available diagnostics, vaccines, or therapeutics.

[0171] In one implementation, CoV is a VOC or VOI as described in Vasileddy et al., 2021, Winger et al., 2021, or Sanyaolu et al., 2021, Chavda et al., 2022. In one implementation, a health regulatory agency such as the World Health Organization (WHO), the US Centers for Disease Control and Prevention (CDC), the European Centre for Disease Prevention and Control (ECDC), or the corresponding local government health regulatory agency in a specific jurisdiction classifies CoV as a VOC, VOI, or VHC. In one implementation, CoV is classified as a VOC or VOI by the WHO. In one implementation, CoV is classified as a VOC, VOI, or VHC by the CDC. In one implementation, CoV is classified as a VOC or VOI by the ECDC.

[0172] In one implementation, CoV is a “Variant of High Consequence,” also known as a “VHC.” In one implementation, a VHC is defined as a clear indication that the effectiveness of preventative measures or medical countermeasures is significantly reduced relative to previously circulating variants. In addition to the characteristics of VOCs, VHCs can have one or more of the following effects on medical countermeasures: proven failure of diagnostic testing targets; evidence of significantly reduced vaccine effectiveness, a disproportionately high number of vaccine breakthrough cases, or very low vaccine-induced protection against severe disease; significantly reduced sensitivity to multiple emergency use authorized or approved therapeutics; and an increase in more severe clinical illness and hospitalizations.

[0173] In one implementation, when the CoV is a SARS-CoV-2 VOC, the VOC contains one or more of the following mutations: H69del, V70del, G142del / D, Y144del, V213G, S371F / L, D405N, R408S, E484K / Q / A, S494P, N501Y, A570D, D614G, P681H / R, T716I, S982A, D1118H, V1176F, K1191N, D80A, D215G, 241del, 242del, 243del, K417N, N501Y, D614G, A701V, T19R, V70F, T95I, E156-, F157-, R158G, , L452R, T478K, D614G, D950N, L18F, T20N, P26S, D138Y, R190S, K417T, N501Y, D614G, H655Y, T1027I, H655Y A67V, del69-70, T95I, del142-144, Y145D, N211del, L212I, ins214EPE, G339D, S373P, S375F, K417N, N440K, G446S, S477N, T478K, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, N764K, D796Y, N856K, Q954H, N969K, and L981F.

[0174] In one implementation, when the CoV is a SARS-CoV-2 VOC, the VOC contains one or more of the following RBD mutations: G339D / H, R346T, L368I, S371F / L, S373P, S375F, T376A, D405N, R408S, K417N / T, N440K, K444T, V445P, G446S, L452R, F456L, N460K, S477N, T478K / R, E484K / Q / A, F486V / S / P, F490S, Q493R, G496S, Q498R, S494P, N501Y, Y505H. In one implementation, the VOC contains one or more of the following NTD mutations: L18F, T19R / I, T20N, del24-26, P26S, A27S, Q52H, A67V, del69-70, H69del, V70del, V70F, D80A, V83A, T95I, D138Y, del142-144, G142del, G142D, Y144del, Y145D, K147Q, delE156, delF157, del157-158, R158G, E180V, Q183E, R190S, N211del, L212I, V213G / E, ins214EPE, D215G, 241del, 242del, 243del, G252V. .

[0175] In one embodiment, the VOC is B.1.1.7 or a variant thereof. In one embodiment, the VOC is B.1.351 or a variant thereof. In one embodiment, the VOC is B.1.351.2 or a variant thereof. In one embodiment, the VOC is B.1.351.2 or a variant thereof. In one embodiment, the VOC is B.1.351.3 or a variant thereof. In one embodiment, the VOC is P1 or a variant thereof. In one embodiment, the VOC is P1.1 or a variant thereof. In one embodiment, the VOC is P1.2 or a variant thereof. In one embodiment, the VOC is B.1.617.2 or a variant thereof. In one embodiment, the VOC is AY.1 or a variant thereof. In one embodiment, the VOC is AY.2 or a variant thereof. In one embodiment, the VOC is AY.3 or a variant thereof. In one embodiment, the VOC is B.1.1.529 or a variant thereof. In one embodiment, the VOC is BA.1 or a variant thereof. In one embodiment, the VOC is BA.1.1 or a variant thereof. In one embodiment, the VOC is BA.2 or a variant thereof. In one embodiment, the VOC is BA.2.74 or a variant thereof. In one embodiment, the VOC is BA.3 or a variant thereof. In one embodiment, the VOC is BA.4 or a variant thereof. In one embodiment, the VOC is BA.5 or a variant thereof. In one embodiment, the VOC is BA.4-5 (also known as BA.4 / 5) or a variant thereof. In one embodiment, the VOC is XBB 1.5 or a variant thereof. In one embodiment, the VOC is XBB 1.16 or a variant thereof. In one embodiment, the VOC is XBB 2.3 or a variant thereof. In one embodiment, the VOC is XBB 1.9.2 or a variant thereof. In one embodiment, the VOC is XBB 1.9.1 or a variant thereof. In one embodiment, the VOC is BA.2.86 or a variant thereof. In one embodiment, the VOC is JN.1 or a variant thereof. In one embodiment, the VOC is CH.1.1 or a variant thereof.

[0176] In one implementation, when the CoV is a SARS-CoV-2 VOI, the VOI contains one or more of the following mutations: L452R, D614G, S13I, W152C, A67V, 69del, 70del, 144del, E484K, Q677H, F888L, L5F, D80G, T95I, Y144, F157S, D253G, L452R, S477N, E484K, A701V, T859N, D950H, Q957R, N501Y, P681R, P681H, E484Q, P681R, S477N, L452Q, and F490S. In one implementation, the VOI is B.1.525 or a variant thereof. In one implementation, the VOI is B.1.526 or a variant thereof. In one embodiment, VOI is B.1.617.1 or a variant thereof. In one embodiment, VOI is C37 or a variant thereof. In one embodiment, VOI is B.1.427 or a variant thereof. In one embodiment, VOI is B.1.429 or a variant thereof. In one embodiment, VOI is P2 or a variant thereof. In one embodiment, VOI is B.1.525 or a variant thereof. In one embodiment, VOI is P3 or a variant thereof. In one embodiment, VOI is B.1.620 or a variant thereof. In one embodiment, VOI is B.1.621 or a variant thereof. In one embodiment, VOI is C.37 or a variant thereof. In one embodiment, VOI is BA2.75 or a variant thereof. In one embodiment, VOI is BQ.1 or a variant thereof.

[0177] CoV infection can cause respiratory, intestinal, hepatic, and neurological diseases in various animal species, including camels, cattle, cats, and bats. CoV can be transmitted from one individual to another through contact with mucous membranes via viral droplets. Typically, viral droplets are airborne and inhaled through the respiratory tract, including the nasal airway. Typically, the individual is a human. In some embodiments, the individual is a live animal or domesticated animal. Typically, CoV can be found in the upper respiratory tract (e.g., the nasal passages) during infection. In some instances, CoV can be found in the lower respiratory tract (e.g., the bronchi and / or alveoli).

[0178] In one embodiment, CoV infection causes one or more symptoms selected from the following: fever, cough, sore throat, shortness of breath, viral shedding, respiratory insufficiency, runny nose, nasal congestion, malaise, bronchitis, headache, muscle pain, dyspnea, moderate pneumonia, severe pneumonia, acute respiratory distress syndrome (ARDS). In one embodiment, ARDS is selected from mild ARDS (defined as 200 mmHg < PaO2 / FiO2 ≤ 300 mmHg), moderate ARDS (defined as 100 mmHg < PaO2 / FiO2 ≤ 200 mmHg), and severe ARDS (defined as PaO2 / FiO2 ≤ 100 mmHg). In one embodiment, SARS-CoV-2 infection can cause one or more symptoms selected from the following: fever, cough, sore throat, shortness of breath, viral shedding, respiratory insufficiency, runny nose, nasal congestion, malaise, bronchitis, headache, muscle pain, dyspnea, moderate pneumonia, severe pneumonia, acute respiratory distress syndrome (ARDS). In one embodiment, CoV infection is asymptomatic.

[0179] SARS-CoV-2

[0180] SARS-CoV-2 has four main structural proteins: spike (S), membrane (M), and envelope (E) proteins, and nucleocapsid (N) protein. S, M, and E are embedded in the viral surface envelope and N is located in the ribonucleoprotein. The S protein recognizes host cell receptors to initiate virus entry.

[0181] The viral S glycoprotein mediates receptor attachment and virus-cell membrane fusion and is a target of NAbs (Duan et al., 2020; Finkelstein et al., 2021; Walls et al., 2020; Hoffmann et al., 2020). The mature spike contains two functional subunits, S1 and S2, derived from the multiprotein precursor S, which cleaves the oligobasic motif with furin as it passes through the Golgi apparatus. ACE2 receptor attachment is mediated by the RBD within the large subunit S1, while membrane fusion is mediated by the small subunit S2, which contains the fusion peptide. S1 and S2 form a heterodimer via non-covalent interactions; the coiled helix of S2 forms an α-helix (amino acids 986 to 1033; referred to as CH) forming the core of the trimer (Wrapp et al., 2020). The transmembrane sequence at the C-terminus of S2 stabilizes the trimer and anchors it to the viral or cell membrane (Fu et al., 2021). The ACE2 RBD is located at the top of the S1 glycoprotein trimer and exists with an "up" ACE2-binding ready and "down" inert orientation (Ke et al., 2020). After receptor attachment, S2 is cleaved on the cell surface by the TMPRSS2 protease to release the fusion peptide and complete fusion activation. The S glycoprotein mediates membrane fusion through a class I mechanism, where activation triggers (via ACE2 binding of S1 and TMPRSS2 cleavage of S2) that causes the S2 subunit of the metastable pre-fusion trimer to refold into a stable hairpin trimer, bringing together the N-terminal fusion peptide and the C-terminal transmembrane sequence, enabling their associated membrane fusion (Cai et al., 2020).

[0182] Before encountering cellular ACE2 and TMPRSS2, the pre-fusion spike conformation, the major spike conformation present on the virion surface (Ke et al., 2020, Turunova et al., 2020), comprises a trimer of the outer S1 subunit, which associates with a trimer of the inner S2 fusion / transmembrane subunit to form the core of the S1-S2 complex. In this conformation, three copies of the RBD are located at the top of the spike and surrounded by three copies of the NTD, while the N-terminal fusion peptide of S2 is largely isolated within the trimer. In the closed conformation, the three RBDs lie flat, occluding the RBM, while in the open conformation, one or more RBDs are raised to expose the RBM, enabling ACE2 binding.

[0183] Type I viral fusion glycoproteins (such as S of β-coronaviruses, Env of retroviruses, and HA of orthomyxoviruses) contain a central coiled helix that acts as a scaffold for the conformational changes required for membrane fusion (Bullough et al., 1994; Cai et al., 2020; Chan et al., 1997; Julien et al., 2013; Walls et al., 2017; Walls et al., 2020; Weissenhorn et al., 1997; Wilson et al., 1981; Wrapp et al., 2020). In the case of SARS-CoV-2, prior to fusion, the coiled helix contains amino acids 988 to 1031 listed in SEQ ID NO: 1 (NCBI reference sequence YP_009724390.1). Upon fusion, the coiled-coil sequence is extended to contain amino acids 913 to 1031 listed in SEQ ID NO: 1 (NCBI reference sequence YP_009724390.1). In some embodiments, residues 986 and 987 are modified with proline (K986P; V987P).

[0184] The in-plane positions of the coiled helix are typically occupied by 3 to 4 repeating hydrophobic residues. In the case of SARS-CoV and SARS-CoV-2 S, these positions are predominantly occupied by polar residues, which mediate the few interhelical contacts in the pre-fusion trimer (see PCT / AU2022 / 050429 and PCT / AU2022 / 050880). Figure 1 B. In the fused trimer, the N-terminal two-thirds of the coiled helix are joined together by the stacking of HR1 helices, extending the coiled helix 110 Å along the N-terminal direction. In this conformation, the in-plane residues are close enough to form hydrogen bonds (see [link to original text]). Figure 1 C PCT / AU2022 / 050429 and PCT / AU2022 / 050880. Ile1013 forms small hydrophobic cores through interhelical contact with I1013 and L1012. These interactions form a hydrophobic ceiling above the cavity formed by A1016 and A1020 occupying the central position of the coiled helix (PCT / AU2022 / 050429 and PCT / AU2022 / 050880). Figure 1 (A to D).

[0185] vaccine antigen

[0186] S protein monomer of coronavirus vaccine antigen

[0187] In one embodiment, the S protein monomer in the S protein trimer may be an ancestral SARS-CoV-2 sequence as described herein (e.g., NCBI reference sequence: YP_009724390.1) or a more recent variant, such as VOC, VOI, or VHC as described herein (e.g., delta (δ), beta (β), omicron (ο), alpha (α), gamma (γ), epsilon (ε), eta (η), iota (ι), kappa (κ), zeta (ζ), mu (μ)). In one embodiment, the S protein monomer is SEQ ID NO: 2 or a variant or modified form thereof. In one embodiment, the S protein monomer is modified to contain one or more mutations present in the ancestral SARS-CoV-2 sequence as described herein in VOC, VO1, or VHC. In one embodiment, the modification is selected from one or more of the following: S13I, L18F, T19R, T20N, P26S, A67V, delH69-V70, D80A, T95I, D138Y, G142D, delY144, W152C, E154K, E156del, F157del, R158G, R190S, D215G, del242-245, D253G, R 246I, K417N / T, N439K, L452R / Q, Y453F, S477N, T478K, E484K / Q, N501Y, F565L, A570D, D614G, H655Y, Q677H, P681H / R, I692V, A701V, T716I, F888L, D950N, S982A, T1027I, Q1071H and D1118H;

[0188] In one embodiment, the S protein monomer belongs to the Omecron lineage. In one embodiment, the S protein monomer belongs to the Alpha lineage. In one embodiment, the S protein monomer belongs to the Gamma lineage. In one embodiment, the S protein monomer belongs to the Epsilon lineage. In one embodiment, the S protein monomer belongs to the Ita lineage. In one embodiment, the S protein monomer belongs to the Yota lineage. In one embodiment, the S protein monomer belongs to the Kappa lineage. In one embodiment, the S protein monomer belongs to the Zeta lineage. In one embodiment, the S protein monomer belongs to the Mü lineage. In one embodiment, the S protein monomer belongs to the Pangolin lineage.

[0189] In one embodiment, the S protein monomer comprises residues corresponding to positions 16 to 1207 of the amino acid sequence SEQ ID NO: 1, or a sequence having at least 90% identity with it. In one embodiment, the S protein monomer comprises residues corresponding to positions 16 to 1207 of the amino acid sequence SEQ ID NO: 2, or a sequence having at least 90% identity with it. In one embodiment, the S protein monomer comprises residues corresponding to positions 16 to 1207 of the amino acid sequence SEQ ID NO: 3, or a sequence having at least 90% identity with it.

[0190] In one embodiment, the S protein monomer comprises residues corresponding to positions 16 to 1237 of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 90% identity with it. In one embodiment, the S protein monomer comprises residues corresponding to positions 16 to 1237 of the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 90% identity with it. In one embodiment, the S protein monomer comprises residues corresponding to positions 16 to 1237 of the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 90% identity with it.

[0191] In one embodiment, the S protein monomer comprises residues from positions 16 to 1256 of the amino acid sequence corresponding to SEQ ID NO: 1, or a sequence having at least 90% identity with it. In one embodiment, the S protein monomer comprises residues from positions 16 to 1256 of the amino acid sequence corresponding to SEQ ID NO: 2, or a sequence having at least 90% identity with it. In one embodiment, the S protein monomer comprises residues from positions 16 to 1256 of the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 90% identity with it.

[0192] In one embodiment, the S protein monomer contains an endogenous or exogenous signal sequence. In one embodiment, the S protein monomer lacks an endogenous or exogenous signal sequence. In one embodiment, the endogenous signal sequence contains residues 1 to 15 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0193] In one embodiment, the S protein monomer comprises a sequence encoding a transmembrane domain of a coronavirus. In one embodiment, the S protein monomer comprises a sequence encoding a transmembrane domain of SARS-CoV-2. In one embodiment, the transmembrane domain comprises residues corresponding to positions 1217 to 1237 of the amino acid sequence SEQ ID NO: 1, or a sequence having at least 90% identity with it. In one embodiment, the transmembrane domain comprises residues corresponding to positions 1217 to 1237 of the amino acid sequence SEQ ID NO: 2, or a sequence having at least 90% identity with it. In one embodiment, the transmembrane domain comprises residues corresponding to positions 1217 to 1237 of the amino acid sequence SEQ ID NO: 3, or a sequence having at least 90% identity with it. In one embodiment, the transmembrane domain comprises residues corresponding to positions 1209 to 1256 of the amino acid sequence SEQ ID NO: 1, or a sequence having at least 90% identity with it. In one embodiment, the transmembrane domain comprises residues corresponding to positions 1209 to 1256 of the amino acid sequence SEQ ID NO: 2, or a sequence having at least 90% identity with it. In one embodiment, the transmembrane domain comprises residues corresponding to positions 1209 to 1256 of the amino acid sequence SEQ ID NO: 3, or a sequence having at least 90% identity with it.

[0194] In one embodiment, the S protein monomer lacks an endogenous or exogenous transmembrane domain. In one embodiment, the S protein monomer does not contain a sequence encoding a transmembrane domain of a coronavirus.

[0195] In one embodiment, the S protein monomer contains the 2P mutation as described herein. In another embodiment, the S protein monomer does not contain the 2P mutation as described herein.

[0196] In one embodiment, the S protein monomer contains the 6P mutation (F817P, A892P, A899P, A942P, K986P, V987P) as described by Hsieh et al., 2020. In another embodiment, the S protein monomer does not contain the 6P mutation.

[0197] In one embodiment, the S protein monomer comprises one or more VOC and / or VOI mutant amino acid sequences as described herein.

[0198] In one embodiment, the S protein monomer does not contain a trimerizing sequence. In one embodiment, the S protein monomer does not contain a transmembrane domain sequence. In one embodiment, the S protein monomer does not contain a folded sequence. In one embodiment, the S protein monomer does not contain a ferritin folded sequence. In one embodiment, the S protein monomer does not contain FHA.

[0199] Coronavirus vaccine antigen

[0200] In one aspect, the coronavirus (CoV) vaccine antigen of the present invention comprises a CoV S protein trimer having at least one non-endogenous protomer disulfide bond.

[0201] In one aspect, the coronavirus (CoV) vaccine antigen of the present invention comprises a CoV S protein trimer having a C-terminus truncated in the stem region.

[0202] In one aspect, the coronavirus (CoV) vaccine antigen of the present invention comprises a CoV S protein trimer having at least one non-endogenous protomeric disulfide bond and a C-terminus truncated in the stem region.

[0203] In one aspect, the coronavirus (CoV) vaccine antigen of the present invention comprises a CoV S protein trimer having at least one non-endogenous protomer disulfide bond and a structural modification that reduces the size of the alanine cavity.

[0204] In one aspect, the coronavirus (CoV) vaccine antigen of the present invention comprises a CoV S protein trimer having a C-terminal truncation and a reduced alanine cavity size in the stem region.

[0205] In one aspect, the coronavirus (CoV) vaccine antigen of the present invention comprises a CoV S protein trimer having at least one non-endogenous protomer disulfide bond, a C-terminal truncation in the stem region, and structural modifications that reduce the size of the alanine cavity.

[0206] In one implementation, the S protein monomer of the S protein trimer lacks a signal sequence.

[0207] In one embodiment, the S protein monomer of the S protein trimer lacks at least a portion of the trimerization sequence.

[0208] In one implementation, the S protein monomer of the S protein trimer does not contain a sequence encoding a functional endogenous trimer sequence.

[0209] In one implementation, the S protein monomer of the S protein trimer does not contain a sequence encoding a functional non-endogenous trimerization sequence.

[0210] In one implementation, the S protein trimer is soluble.

[0211] In one implementation, the CoV vaccine antigen additionally includes a 2P modification.

[0212] In one implementation, the CoV vaccine antigen additionally includes a 6P modification.

[0213] In one embodiment, the CoV vaccine antigen is a pan-coronavirus vaccine antigen. In one embodiment, the CoV vaccine antigen is a SARS-CoV-2 vaccine antigen. In one embodiment, the CoV vaccine antigen is a SARS-CoV-2 Omeprón vaccine antigen. In one embodiment, the CoV vaccine antigen is a SARS-CoV-2 non-Omeprón vaccine antigen.

[0214] In one implementation, the S protein trimer is stabilized in its pre-fusion conformation.

[0215] In one implementation, the S protein trimer has modified antigenicity compared to the S protein trimer which lacks interprotomer disulfide bonds.

[0216] In one implementation, the S protein trimer exhibits modified immunogenicity compared to the S protein trimer, which lacks interprotomer disulfide bonds.

[0217] In one implementation, when applied to a subject, the S protein trimer elicits a neutralizing antibody response as described herein.

[0218] In one embodiment, the S protein trimer further comprises a structural modification that reduces the size of the alanine cavity in the coiled-coil region of the S protein trimer, and wherein the S protein trimer is...

[0219] Ribonucleic acid encoding the S protein monomer of coronavirus (CoV) vaccine antigen

[0220] In one aspect, the present invention provides ribonucleic acid encoding coronavirus vaccine antigens as described herein.

[0221] In one aspect, the present invention provides ribonucleic acid encoding a monomer of the S protein of a coronavirus (CoV) vaccine antigen, wherein the vaccine antigen comprises a CoV S protein trimer having at least one non-endogenous interprotomeric disulfide bond.

[0222] In one aspect, the present invention provides ribonucleic acid encoding an S protein monomer of a coronavirus (CoV) vaccine antigen, wherein the vaccine antigen is a CoV S protein trimer, and wherein the S protein monomer of the CoV S protein trimer has a C-terminal truncation in the stem region.

[0223] In one aspect, the present invention provides ribonucleic acid encoding a monomer of the S protein of a coronavirus (CoV) vaccine antigen, wherein the vaccine antigen comprises a CoV S protein trimer having at least one non-endogenous interprotomeric disulfide bond and a C-terminus truncated in the stem region.

[0224] In one aspect, the present invention provides ribonucleic acid encoding an S protein monomer of a coronavirus (CoV) vaccine antigen, wherein the vaccine antigen comprises a CoV S protein trimer having at least one non-endogenous interprotomeric disulfide bond and a structural modification that reduces the size of the alanine cavity.

[0225] In one aspect, the present invention provides ribonucleic acid encoding an S protein monomer of a coronavirus (CoV) vaccine antigen, wherein the vaccine antigen is a CoV S protein trimer, and wherein the S protein monomer of the CoV S protein trimer has structural modifications including C-terminal truncation and reduced alanine cavity size in the stem region.

[0226] In one aspect, the present invention provides ribonucleic acid encoding a monomer of the S protein of a coronavirus (CoV) vaccine antigen, wherein the vaccine antigen comprises a CoV S protein trimer having at least one non-endogenous protomer disulfide bond and a C-terminal truncation in the stem region, and structural modifications that reduce the size of the alanine lumen.

[0227] In one embodiment, the ribonucleic acid comprises an RNA sequence corresponding to a sequence selected from the following: SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61. SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90. SEQ ID NO: 91, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 125. SEQ ID NO: 126. SEQ ID NO: 127. SEQ ID NO: 135. SEQ ID NO: 136, or a sequence that has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identity with it.

[0228] In one embodiment, the ribonucleic acid (RNA) comprises a sequence corresponding to SEQ ID NO: 5 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 14 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 15 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 16 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 17 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 18 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 19 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 20 or a sequence having at least 70% identity with it. In one embodiment, the ribonucleic acid (RNA) comprises a sequence corresponding to SEQ ID NO: 21 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 33 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 34 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 35 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 36 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 37 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 38 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 39 or a sequence having at least 70% identity with it. In one embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 40 or a sequence having at least 70% identity with it. In another embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 41 or a sequence having at least 70% identity with it.In one embodiment, the ribonucleic acid (RNA) comprises a sequence corresponding to SEQ ID NO: 42 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 43 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 50 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 51 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 52 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 53 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 54 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 55 or a sequence having at least 70% identity with it. In one embodiment, the ribonucleic acid (RNA) comprises a sequence corresponding to SEQ ID NO: 56 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 57 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 58 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 59 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 60 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 61 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 62 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 63 or a sequence having at least 70% identity with it. In one embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 64 or a sequence having at least 70% identity with it. In one embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 65 or a sequence having at least 70% identity with it.In one embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 66 or a sequence having at least 70% identity with it. In another embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 67 or a sequence having at least 70% identity with it.

[0229] In one embodiment, the ribonucleic acid (RNA) comprises a sequence corresponding to SEQ ID NO: 68 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 69 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 74 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 75 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 76 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 77 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 86 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 87 or a sequence having at least 70% identity with it. In one embodiment, the ribonucleic acid (RNA) comprises a sequence corresponding to SEQ ID NO: 88 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 90 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 91 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 103 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 105 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 111 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 112 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 113 or a sequence having at least 70% identity with it. In one embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 114 or a sequence having at least 70% identity with it. In another embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 115 or a sequence having at least 70% identity with it.In one embodiment, the ribonucleic acid (RNA) comprises a sequence corresponding to SEQ ID NO: 119 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 120 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 121 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 125 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 126 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 127 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 135 or a sequence having at least 70% identity with it. In one embodiment, the RNA comprises a sequence corresponding to SEQ ID NO: 136 or a sequence having at least 70% identity with it.

[0230] In one embodiment, the ribonucleic acid encodes an amino acid sequence selected from the following: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 132, and SEQ ID NO: 133.

[0231] In one embodiment, the ribonucleic acid encodes an amino acid sequence selected from the following sequences: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

[0232] In one embodiment, the ribonucleic acid encodes an amino acid sequence selected from the following sequences: SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49.

[0233] In one embodiment, the ribonucleic acid encodes an amino acid sequence selected from the following: SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 102, and SEQ ID NO: 104.

[0234] In one embodiment, the ribonucleic acid encodes an amino acid sequence selected from the following sequences: SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO: 124.

[0235] Cysteine ​​modification

[0236] As used herein, a "disulfide bond" refers to a covalent bond formed between the thiol or mercaptan (R-SH) side chains of two cysteine ​​residues in one or more proteins. In some embodiments, the disulfide bond is formed between two cysteine ​​residues within the protein.

[0237] As used herein, "interpromerinogenic disulfide bond" refers to a disulfide bond between two protomers of an oligomeric structure. In some embodiments, the interpromerinogenic disulfide bond is formed between two protomers (S protein monomers) of the S protein trimer (oligomeric structure). In one embodiment, the non-endogenous interpromerinogenic disulfide bond is formed between cysteine ​​residues selected from: i) cysteine ​​at positions corresponding to amino acid numbers 914 and 1123 of SEQ ID NO: 1 or SEQ ID NO: 2 (L23), ii) cysteine ​​at positions corresponding to amino acid numbers 571 and 967 of SEQ ID NO: 1 or SEQ ID NO: 2 (D17), and iii) cysteine ​​at positions corresponding to amino acid numbers 570 and 967 of SEQ ID NO: 1 or SEQ ID NO: 2 (I1).

[0238] In some embodiments, non-endogenous protopolymer disulfide bonds are formed between cysteine ​​residues at positions corresponding to amino acid numbers 914 and 1123 of SEQ ID NO: 1 (L23). In some embodiments, non-endogenous protopolymer disulfide bonds are formed between cysteine ​​residues at positions corresponding to amino acid numbers 914 and 1123 of SEQ ID NO: 2 (L23). In some embodiments, non-endogenous protopolymer disulfide bonds are formed between cysteine ​​residues at positions corresponding to amino acid numbers 571 and 967 of SEQ ID NO: 1 (D17). In some embodiments, non-endogenous protopolymer disulfide bonds are formed between cysteine ​​residues at positions corresponding to amino acid numbers 571 and 967 of SEQ ID NO: 2 (D17). In some embodiments, non-endogenous protopolymer disulfide bonds are formed between cysteine ​​residues at positions corresponding to amino acid numbers 570 and 967 of SEQ ID NO: 1 (I1). In some embodiments, non-endogenous protomer disulfide bonds are formed between cysteine ​​residues at positions corresponding to amino acid numbers 570 and 967 of SEQ ID NO: 2 (II).

[0239] In some embodiments, the interprotopolymer disulfide bond is formed by the substitution of residues 914 and 1123 corresponding to SEQ ID NO: 1 with cysteine ​​(L23).

[0240] In some embodiments, the interprotopolymer disulfide bonds are formed by the substitution of residues corresponding to SEQ ID NO: 2 (914 and 1123) with cysteine ​​residues (L23). In some embodiments, the interprotopolymer disulfide bonds are formed by the substitution of residues corresponding to SEQ ID NO: 1 (571 and 967) with cysteine ​​residues (D17). In some embodiments, the interprotopolymer disulfide bonds are formed by the substitution of residues corresponding to SEQ ID NO: 2 (571 and 967) with cysteine ​​residues (D17). In some embodiments, the interprotopolymer disulfide bonds are formed by the substitution of residues corresponding to SEQ ID NO: 1 (570 and 967) with cysteine ​​residues (I1). In some embodiments, the interprotopolymer disulfide bonds are formed by the substitution of residues corresponding to SEQ ID NO: 2 (570 and 967) with cysteine ​​residues (I1).

[0241] In some embodiments, each protopolymer of the CoV S protein trimer may be modified with one or more of the aforementioned interproton disulfide bonds. The interproton disulfide bond modification introduces one or more of the following into the molecule's structure, stability, or function. In some embodiments, the stability modification increases the melting temperature of the S protein trimer compared to the same S protein trimer lacking interproton disulfide bonds. In some embodiments, the functional modification increases the immunogenicity of the S protein trimer compared to the same S protein trimer lacking interproton disulfide bonds. In one embodiment, the S protein trimer is modified to include one interproton disulfide bond. In one embodiment, the S protein trimer, as described herein, is modified to include one or more interproton disulfide bonds. In one embodiment, the S protein trimer is modified to include two interproton disulfide bonds. In one embodiment, the S protein trimer is modified to include three interproton disulfide bonds. In one embodiment, the S protein trimer is modified to include four interproton disulfide bonds.

[0242] In some embodiments, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by at least about 1.5°C compared to the same trimer lacking inter-protopolymer disulfide bonds. In some embodiments, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by at least about 2°C compared to the same trimer lacking inter-protopolymer disulfide bonds. In some embodiments, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by at least about 3°C ​​compared to the same trimer lacking inter-protopolymer disulfide bonds. In some embodiments, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by at least about 4°C compared to the same trimer lacking inter-protopolymer disulfide bonds. In some embodiments, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by at least about 5°C compared to the same trimer lacking inter-protopolymer disulfide bonds. In some embodiments, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by at least about 6°C compared to the same trimer lacking inter-protopolymer disulfide bonds. In some embodiments, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by at least about 1°C compared to the same trimer lacking inter-protopolymer disulfide bonds. In some embodiments, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by at least about 8°C compared to the same trimer lacking inter-protopolymer disulfide bonds. In some embodiments, the presence of at least one interprotomeric disulfide bond in the CoV S protein trimer increases the trimer melting temperature by at least about 10°C compared to the same trimer lacking interprotomeric disulfide bonds. In some embodiments, the presence of at least one interprotomeric disulfide bond in the CoV S protein trimer increases the trimer melting temperature by at least about 12°C compared to the same trimer lacking interprotomeric disulfide bonds. In some embodiments, the presence of at least one interprotomeric disulfide bond in the CoV S protein trimer increases the trimer melting temperature by at least about 15°C compared to the same trimer lacking interprotomeric disulfide bonds.

[0243] In some embodiments, the melting temperature of the CoV vaccine antigen is about 38°C to about 71°C, or about 40°C to about 71°C, or about 42°C to about 71°C, or about 45°C to about 71°C, or about 50°C to about 71°C, or about 55°C to about 71°C, or about 60°C to about 71°C, or about 51°C to about 58°C, or about 51°C to about 57°C, or about 51°C to about 55°C, or about 52°C to about 58°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 38°C to about 71°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 40°C to about 71°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 42°C to about 71°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 45°C to about 71°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 50°C to about 71°C. In some embodiments, the melting temperature of the CoV vaccine antigen is from about 51°C to about 58°C. In some embodiments, the melting temperature of the CoV vaccine antigen is from about 51°C to about 57°C. In some embodiments, the melting temperature of the CoV vaccine antigen is from about 51°C to about 55°C. In some embodiments, the melting temperature of the CoV vaccine antigen is from about 52°C to about 58°C. In some embodiments, the melting temperature of the CoV vaccine antigen is from about 55°C to about 71°C. In some embodiments, the melting temperature of the CoV vaccine antigen is from about 60°C to about 71°C.

[0244] In some embodiments, the melting temperature of the CoV vaccine antigen is about 51°C, or about 52°C, or about 53°C, or about 54°C, or about 55°C, or about 55°C, or about 55.8°C, or about 56°C, or about 57°C, or about 57.5°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 51°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 52°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 53°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 54°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 55°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 55°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 55.8°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 56°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 57°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 57.5°C.

[0245] In some embodiments, compared to the same trimer lacking inter-protopolymer disulfide bonds, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer's melting temperature by about 5°C to about 30°C, or about 5°C to about 25°C, or about 5°C to about 20°C, or about 5°C to about 15°C, or about 5°C to about 12.5°C, or about 5°C to about 10°C, or about 5°C to about 8°C. In some embodiments, compared to the same trimer lacking inter-protopolymer disulfide bonds, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer's melting temperature by about 5°C to about 30°C. In some embodiments, compared to the same trimer lacking inter-protopolymer disulfide bonds, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer's melting temperature by about 5°C to about 25°C. In some embodiments, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by about 5°C to about 20°C compared to the same trimer lacking inter-protopolymer disulfide bonds. In some embodiments, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by about 5°C to about 15°C compared to the same trimer lacking inter-protopolymer disulfide bonds. In some embodiments, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by about 5°C to about 12.5°C compared to the same trimer lacking inter-protopolymer disulfide bonds. In some embodiments, the presence of at least one inter-protopolymer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by about 5°C to about 10°C compared to the same trimer lacking inter-protopolymer disulfide bonds. In some implementations, having at least one interprotomer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by about 5°C to about 8°C compared to the same trimer lacking interprotomer disulfide bonds.

[0246] In one embodiment, the S protein trimer comprises a sequence selected from the following: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 132, and SEQ ID NO: 133. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 4. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 5. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 78. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 79. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 80. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 81. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 82. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 83. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 84. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 108. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 109. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 110. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 117. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 118. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 123. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 124. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 129. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 130. In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 132.In one embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 133.

[0247] Cut off

[0248] In one embodiment, the C-terminus is truncated between residues corresponding to positions 1147 to 1207 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated between residues corresponding to positions 1147 to 1200 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated between residues corresponding to positions 1147 to 1193 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated between residues corresponding to positions 1147 to 1207 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated between residues corresponding to positions 1162 to 1200 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated between residues corresponding to positions 1162 to 1193 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated between residues at positions 1165 to 1193 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.

[0249] In one embodiment, the C-terminus is truncated after residues corresponding to positions 1147, 1157, 1165, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1201, or 1204 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue position 1147 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue position 1157 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after a residue selected from the residues corresponding to positions 1162, 1165, 1192, or 1199 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after a residue selected from the residues corresponding to positions 1165, 1192, or 1199 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after a residue selected from the residues corresponding to positions 1162, 1165, or 1192 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after a residue selected from the residues corresponding to positions 1165 or 1192 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after a residue corresponding to position 1192 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue position 1195 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue position 1196 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue position 1199 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue position 1201 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue position 1204 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0250] In one embodiment, the C-terminus is truncated after residues at positions 1147, 1157, 1165, 1192, 1195, 1196, 1199, 1201, or 1204 of SEQ ID NO: 1 or SEQ ID NO: 2 and before residue position 1208 of SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue position 1147 of SEQ ID NO: 1 or SEQ ID NO: 2 and before residue position 1208 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue position 1157 of SEQ ID NO: 1 or SEQ ID NO: 2 and before residue position 1208 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue 1165 of SEQ ID NO: 1 or SEQ ID NO: 2 and before residue 1208 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue 1192 of SEQ ID NO: 1 or SEQ ID NO: 2 and before residue 1208 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue 1195 of SEQ ID NO: 1 or SEQ ID NO: 2 and before residue 1208 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue 1196 of SEQ ID NO: 1 or SEQ ID NO: 2 and before residue 1208 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue 1199 of SEQ ID NO: 1 or SEQ ID NO: 2 and before residue 1208 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue 1201 of SEQ ID NO: 1 or SEQ ID NO: 2 and before residue 1208 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue 1204 of SEQ ID NO: 1 or SEQ ID NO: 2 and before residue 1208 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.In one embodiment, the C-terminus is truncated after residue 1162 of SEQ ID NO: 1 or SEQ ID NO: 2 and before residue 1200 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue 1165 of SEQ ID NO: 1 or SEQ ID NO: 2 and before residue 1200 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the C-terminus is truncated after residue 1165 of SEQ ID NO: 1 or SEQ ID NO: 2 and before residue 1193 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0251] In one embodiment, the monomer of the S protein trimer comprises residues corresponding to positions 16 to 1147, or residues 16 to 1157, or residues 16 to 1165, or residues 16 to 1192, or residues 16 to 1199, or residues 16 to 1200, or residues 16 to 1201, or residues 16 to 1204 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the monomer of the S protein trimer comprises residues corresponding to positions 16 to 1147 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the monomer of the S protein trimer comprises residues corresponding to positions 16 to 1157 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the monomer of the S protein trimer comprises residues corresponding to positions 16 to 1165 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the monomer of the S protein trimer comprises residues corresponding to positions 16 to 1162 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the monomer of the S protein trimer comprises residues corresponding to positions 16 to 1192 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the monomer of the S protein trimer comprises residues corresponding to positions 16 to 1199 of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the monomer of the S protein trimer comprises residues from position 16 to 1204 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.

[0252] In one implementation, when expressed in a recombinant expression system, the trimer is produced at a higher level compared to the truncated trimer of the same S protein expressed in the same recombinant expression system.

[0253] In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by about 10% to about 60% compared to the level of the same S protein trimer lacking truncation. In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by about 10% to about 50% compared to the level of the same S protein trimer lacking truncation. In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by about 10% to about 40% compared to the level of the same S protein trimer lacking truncation. In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by about 17% to about 36% compared to the level of the same S protein trimer lacking truncation.

[0254] In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by about 2 to about 10 times compared to the level of the same S protein trimer without truncation. In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by about 3 to about 9 times compared to the level of the same S protein trimer without truncation. In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by at least about 2 times compared to the level of the same S protein trimer without truncation. In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by at least about 3 times compared to the level of the same S protein trimer without truncation. In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by at least about 4 times compared to the level of the same S protein trimer without truncation. In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by at least about 5 times compared to the level of the same S protein trimer without truncation. In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by at least about 6-fold compared to the level of the same S protein trimer lacking truncation. In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by at least about 7-fold compared to the level of the same S protein trimer lacking truncation. In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by at least about 8-fold compared to the level of the same S protein trimer lacking truncation. In one embodiment, when expressed in a recombinant expression system, the level of the S protein trimer is increased by at least about 8.75% compared to the level of the same S protein trimer lacking truncation.

[0255] In one embodiment, the S protein trimer is generated at a level greater than about 300 μg / 50 mL. In one embodiment, the S protein trimer is generated at a level from about 300 μg / 50 mL to about 2000 μg / 50 mL. In one embodiment, the S protein trimer is generated at a level from about 300 μg / 50 mL to about 1900 μg / 50 mL. In one embodiment, the S protein trimer is generated at a level from about 300 μg / 50 mL to about 1100 μg / 50 mL.

[0256] In one embodiment, the monomer of the S protein trimer comprises residues corresponding to positions 16 to 1192 of SEQ ID NO: 1 or SEQ ID NO: 2, and a non-endogenous protomer disulfide bond is formed between residues corresponding to positions 571 and 967 of SEQ ID NO: 1 or SEQ ID NO: 2 (D17). In another embodiment, the monomer of the S protein trimer comprises residues corresponding to positions 16 to 1192 of SEQ ID NO: 1 or SEQ ID NO: 2, and a non-endogenous protomer disulfide bond is formed between residues corresponding to positions 570 and 967 of SEQ ID NO: 2 (D17) or SEQ ID NO: 1 or SEQ ID NO: 2 (I1). In one embodiment, the monomer of the S protein trimer comprises residues corresponding to positions 16 to 1192 of SEQ ID NO: 1 or SEQ ID NO: 2, and non-endogenous interprotopolymer disulfide bonds are formed between residues corresponding to positions 571 and 967 (D17) of SEQ ID NO: 1 or SEQ ID NO: 2 or A570 and S967 (I1) of SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the melting temperature of the S protein trimer is from about 50°C to about 55°C.

[0257] In one embodiment, the monomer of the S protein trimer comprises a sequence selected from the following: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 6. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 7. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 8. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 9. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 10. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 11. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 12. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 13.

[0258] In one embodiment, the monomer of the S protein trimer comprises sequences selected from the following: SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49.

[0259] In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 25. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 29. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 31. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 47. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 48. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 49.

[0260] In one embodiment, the monomer of the S protein trimer comprises a sequence selected from the following: SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 102, and SEQ ID NO: 104. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 44. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 45. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 46. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 56. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 57. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 58. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 62. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 63. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 64. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 65. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 70. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 71. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 72. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 102. In one embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 104.

[0261] In one embodiment, the monomer of the S protein trimer comprises a sequence selected from the following: SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO: 124. In one embodiment, the monomer of the S protein trimer comprises the sequence SEQ ID NO: 106. In one embodiment, the monomer of the S protein trimer comprises the sequence SEQ ID NO: 107. In one embodiment, the monomer of the S protein trimer comprises the sequence SEQ ID NO: 108. In one embodiment, the monomer of the S protein trimer comprises the sequence SEQ ID NO: 109. In one embodiment, the monomer of the S protein trimer comprises the sequence SEQ ID NO: 110. In one embodiment, the monomer of the S protein trimer comprises the sequence SEQ ID NO: 116. In one embodiment, the monomer of the S protein trimer comprises the sequence SEQ ID NO: 117. In one embodiment, the monomer of the S protein trimer comprises the sequence SEQ ID NO: 118. In one embodiment, the monomer of the S protein trimer comprises the sequence SEQ ID NO: 122. In one embodiment, the monomer of the S protein trimer comprises the sequence SEQ ID NO: 123. In one embodiment, the monomer of the S protein trimer comprises the sequence SEQ ID NO: 124.

[0262] Structural modifications to reduce alanine cavity size

[0263] In one embodiment, the S protein trimer as described herein further comprises a structural modification that reduces the size of the alanine cavity in the coiled-coil region of the S protein trimer, and wherein said S protein trimer. In one aspect, the structural modification stabilizes the S protein trimer. As used herein, “stabilized” means improving one or more of the following: thermal stability, longevity, immunogenicity and manufacturing stability, yield or uniformity of the S protein trimer, and denaturing stability. In one embodiment, in vitro stability and / or in vivo stability are improved. In one embodiment, in vivo stability is improved (when administered to a subject or after assembly in a subject, e.g., after translation from an mRNA vaccine). In one embodiment, stability is improved in vitro (e.g., during manufacturing).

[0264] In one embodiment, structural modification stabilizes the S protein trimer by reducing the size of the alanine lumen in the coiled helix. In one embodiment, the alanine lumen is partially or completely filled. In one embodiment, the conformation of the alanine lumen is altered. In one embodiment, the size of the alanine lumen is reduced by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100%. In one embodiment, the size of the alanine lumen is reduced by at least 5%. In one embodiment, the size of the alanine lumen is reduced by at least 10%. In one embodiment, the size of the alanine lumen is reduced by at least 20%. In one embodiment, the size of the alanine lumen is reduced by at least 30%. In one embodiment, the size of the alanine lumen is reduced by at least 40%. In one embodiment, the size of the alanine lumen is reduced by at least 50%. In one embodiment, the size of the alanine lumen is reduced by at least 60%. In one embodiment, the size of the alanine lumen is reduced by at least 70%. In one embodiment, the size of the alanine lumen is reduced by at least 80%. In one embodiment, the size of the alanine lumen is reduced by at least 90%. In one embodiment, the size of the alanine lumen is reduced by 100%.

[0265] In one embodiment, the size of the alanine cavity is reduced by about 10% to 100%, or about 10% to about 90%, or about 20% to about 90%, or about 20% to about 80%, or about 30% to about 80%, or about 40% to about 80%, or about 50% to about 80%. In one embodiment, the size of the alanine cavity is reduced by about 10% to 100%. In one embodiment, the size of the alanine cavity is reduced by about 10% to 90%. In one embodiment, the size of the alanine cavity is reduced by about 20% to 90%. In one embodiment, the size of the alanine cavity is reduced by about 20% to 80%. In one embodiment, the size of the alanine cavity is reduced by about 30% to 80%. In one embodiment, the size of the alanine cavity is reduced by about 40% to 80%. In one embodiment, the size of the alanine cavity is reduced by about 50% to 80%.

[0266] In one embodiment, the structural modification improves the stability of the S protein trimer compared to the S protein trimer without structural modification. In one embodiment, the S protein trimer is stable in the absence of at least some or all of the endogenous trimerizing sequences (no trimerizing sequence is required for stability). In one embodiment, the S protein trimer is stable in the absence of non-endogenous trimerizing sequences (no trimerizing sequence is required for stability). In one embodiment, the S protein trimer is stable in the absence of both at least some or all of the endogenous trimerizing sequences (no trimerizing sequence is required for stability) and non-endogenous trimerizing sequences.

[0267] In one embodiment, the structural modification increases the degradation temperature of the S protein trimer compared to the unmodified S protein trimer. In one embodiment, degradation, or a variation thereof, refers to exposing hydrophobic residues at the core of the S protein trimer.

[0268] In one embodiment, the structural modification increases the melting temperature of the S protein trimer compared to the unmodified S protein trimer. In one embodiment, the structural modification improves the thermal stability of the S protein trimer. In one embodiment, the structural modification increases the melting temperature of the S protein trimer by about 5°C to about 25°C. In one embodiment, the structural modification increases the melting temperature of the S protein trimer by about 5°C to about 23°C. In one embodiment, the structural modification increases the melting temperature of the S protein trimer by about 5°C to about 23°C. In one embodiment, the structural modification increases the melting temperature of the S protein trimer by about 10°C to about 23°C. In one embodiment, the structural modification increases the melting temperature of the S protein trimer by about 10°C to about 20°C. In one embodiment, the structural modification increases the melting temperature of the S protein trimer by about 5°C to about 15°C. In one embodiment, the structural modification increases the melting temperature of the S protein trimer by about 5°C to about 10°C.

[0269] In one embodiment, structural modification improves the stability of the S protein trimer against denaturing conditions. Denaturing conditions include, for example, boiling in the presence of a detergent (e.g., sodium dodecyl sulfate) or treatment at room temperature with and without 2-β-mercaptoethanol using a detergent (e.g., sodium dodecyl sulfate).

[0270] In one embodiment, this specification provides a method for improving the stability and / or expression of the coronavirus S antigen.

[0271] In one embodiment, the CoV vaccine antigen is soluble. In one embodiment, the CoV vaccine antigen as described herein does not contain a C-terminal trimer-stabilization clamp sequence, such as an FHA sequence. In one embodiment, the CoV vaccine antigen is stabilized in a pre-fusion S protein trimer conformation. In one embodiment, the ACE2 receptor-binding domain (RBD) of the S protein trimer is in a down (non-ACE2-binding-ready) orientation. In one embodiment, when the RBD is in a down orientation, in addition to neutralizing antibodies targeting an up-conformation RBD, neutralizing antibodies that recognize the down-conformation S-trimer are also generated.

[0272] In one embodiment, the CoV vaccine antigen lacks a trimerized sequence. In one embodiment, the CoV vaccine antigen lacks a transmembrane domain. In one embodiment, the CoV vaccine antigen lacks a folded subsequence / domain.

[0273] In one implementation, when the RBD is oriented downwards, other non-RBD epitopes are in favorable positions to generate additional non-RBD neutralizing antibodies.

[0274] In one embodiment, the structural modification is performed in the coiled helical region. In another embodiment, the structural modification stabilizes the coiled helical region.

[0275] In one implementation, the structural modifications in the coiled helix of S2 have an allosteric effect on the immunogenicity of S1, which enhances the immune response against CoV variants as described herein.

[0276] In one embodiment, the CoV vaccine antigen is suitable for intradermal administration. In one embodiment, the CoV vaccine antigen is suitable for oral administration. In one embodiment, the CoV vaccine antigen is suitable for pulmonary administration. In one embodiment, the CoV vaccine antigen is suitable for nasal administration.

[0277] "Alanine cavity" or "cavity" herein refers to the region and reduced interactions observed between monomers in the trimeric structure within the coiled helix of the SARS-CoV S protein due to the absence of amino acids with nonpolar side chains larger than those of alanine or aromatic residues. In one embodiment, the alanine cavity comprises A1016 and A1020 as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 3.

[0278] Structural modifications to the alanine cavity are achieved using one or more of the following: amino acid substitution of disulfide bonds, hydrogen bonds, π-π stacking, salt bridges, van der Waals interactions, substitution or addition of hydrophobic residues, or proline stabilization within the S protein. In one embodiment, the structural modification of the alanine cavity is achieved by amino acid substitution of one or more amino acids forming the alanine cavity. In one embodiment, the structural modification is the substitution of one or more amino acids for more hydrophobic amino acids. In one embodiment, the structural modification is the substitution of one or more amino acids in the coiled-coil region for more hydrophobic amino acids. In one embodiment, one or two or three S protein monomers in the S protein trimer contain one or more amino acids in the coiled-coil region that have been replaced by more hydrophobic amino acids. In one embodiment, one S protein monomer in the S protein trimer contains one or more amino acids in the coiled-coil region that have been replaced by more hydrophobic amino acids. In one embodiment, two S protein monomers in the S protein trimer contain one or more amino acids in the coiled-coil region that have been replaced by more hydrophobic amino acids. In one embodiment, the three S protein monomers in the S protein trimer contain one or more amino acids in the coiled-coil region that are replaced by more hydrophobic amino acids.

[0279] In one embodiment, the structural modification creates an artificial hydrophobic core within the coiled-coil region. In one embodiment, the structural modification creates an artificial hydrophobic core comprising residues containing an alanine cavity. In one embodiment, the structural modification creates an artificial hydrophobic core within an alanine cavity. In one embodiment, the amino acids at positions 1016 and 1020 contribute to the formation of the artificial hydrophobic core.

[0280] In one embodiment, an artificial hydrophobic core is created by replacing amino acids in the coiled-coil region with more hydrophobic amino acids. In another embodiment, polar residues are replaced with larger hydrophobic residues.

[0281] The term "more hydrophobic amino acid" as used in this article refers to an amino acid that is more hydrophobic than the amino acid that is being replaced and is present in the coronavirus strain. For example, if the modified / replaced amino acid is alanine, it can be replaced by more hydrophobic amino acids such as isoleucine, leucine, methionine, valine, phenylalanine, tyrosine, and tryptophan.

[0282] The hydrophobicity index is a measure of the relative hydrophobicity or solubility of an amino acid in water, and is described, for example, by Sereda et al., 1994 and Monera et al., 1995. The table below provides the hydrophobicity of different amino acids at pH 2 and pH 7, normalized so that the most hydrophobic residue has a value of 100 relative to glycine (0 value).

[0283] Table 1: Hydrophobicity of amino acids.

[0284]

[0285]

[0286] In one embodiment, the more hydrophobic amino acid is a hydrophobic amino acid. In one embodiment, the hydrophobic amino acid is an aliphatic hydrophobic amino acid. In one embodiment, the hydrophobic amino acid is an aromatic hydrophobic amino acid.

[0287] In one embodiment, at least one amino acid in the coiled-coil region of the S protein monomer in the S protein trimer is replaced by a more hydrophobic amino acid. In one embodiment, at least one S protein monomer in the S protein trimer contains a substitution. In one embodiment, at least two S protein monomers in the S protein trimer contain substitutions. In one embodiment, three S protein monomers in the S protein trimer contain substitutions.

[0288] In one embodiment, at least two amino acids in the coiled-coil region of the S protein monomer in the S protein trimer are replaced by more hydrophobic amino acids. In one embodiment, at least one S protein monomer in the S protein trimer contains a substitution. In one embodiment, at least two S protein monomers in the S protein trimer contain substitutions. In one embodiment, three S protein monomers in the S protein trimer contain substitutions.

[0289] In one embodiment, at least one or at least two amino acids are located at the a and / or d positions of the heptapeptide repeat motif in the coiled-coil region of the S protein monomer. The positions of the a and d positions in the heptapeptide repeat motif are specified in PCT / AU2022 / 050429 and PCT / AU2022 / 050880. Figure 1As shown in B. For SARS-CoV-2, positions a and d correspond to amino acids 988, 991, 995, 998, 1002, 1005, 1009, 1013, 1016, 1020, 1023, 1027, and 1031 of SEQ ID NO:1. In one embodiment, a substitution with a more hydrophobic amino acid occurs at position 1016. In one embodiment, a substitution with a more hydrophobic amino acid occurs at position 1020. In one embodiment, substitutions with more hydrophobic amino acids occur at positions 1016 and 1020. In one embodiment, A1016 or A1020 is replaced by leucine, valine, isoleucine, or phenylalanine. In one embodiment, A1016 is replaced by leucine (A1016L), valine (A1016V), or isoleucine (A1016I). In one embodiment, A1020 is replaced with isoleucine (A1020I). In one embodiment, A1016 is replaced with leucine (A1016L) or valine (A1016V), and A1020 is replaced with isoleucine (A1020I). In one embodiment, A1016 is replaced with leucine (A1016L or 16L). In one embodiment, A1016 is replaced with valine (A1016V). In one embodiment, A1020 is replaced with isoleucine (A1020I). In one embodiment, A1020 is not replaced with tryptophan (W). In one embodiment, A1016 is replaced with valine, and A1020 is replaced with isoleucine (referred to herein as "A1016V / A1020I" or "1016 / 20VI" or "VI"). In one embodiment, A1020 is not replaced with tryptophan (A1020W).

[0290] In one embodiment, the more hydrophobic amino acid comprises one or more of the following properties: i) greater hydrophobicity than alanine; ii) a hydrophobic amino acid greater than alanine; iii) hydrophobicity greater than 47 at pH 2; iv) hydrophobicity greater than 41 at pH 7; and iv) selected from: isoleucine, leucine, methionine, valine, phenylalanine, tyrosine, and tryptophan. In one embodiment, the amino acid is selected from: isoleucine, leucine, and valine. In one embodiment, the amino acid is isoleucine. In one embodiment, the amino acid is leucine. In one embodiment, the amino acid is valine. In one embodiment, the amino acid is methionine. In one embodiment, the amino acid is phenylalanine. In one embodiment, the amino acid is tyrosine. In one embodiment, the amino acid is tryptophan. In one embodiment, the hydrophobic amino acid is not tryptophan.

[0291] In one embodiment, the more hydrophobic amino acid has a hydrophobicity greater than that of alanine. In one embodiment, the more hydrophobic amino acid has a hydrophobicity greater than that of alanine. In one embodiment, the more hydrophobic amino acid has a hydrophobicity greater than 47 at pH 2. In one embodiment, the more hydrophobic amino acid has a hydrophobicity greater than 41 at pH 7. In one embodiment, the more hydrophobic amino acid is selected from: isoleucine, leucine, methionine, valine, phenylalanine, tyrosine, and tryptophan. In one embodiment, the more hydrophobic amino acid is isoleucine. In one embodiment, the more hydrophobic amino acid is leucine. In one embodiment, the more hydrophobic amino acid is methionine. In one embodiment, the more hydrophobic amino acid is valine. In one embodiment, the more hydrophobic amino acid is phenylalanine. In one embodiment, the more hydrophobic amino acid is tyrosine. In one embodiment, the more hydrophobic amino acid is tryptophan.

[0292] To avoid any doubt, while optimal hydrophobicity can be achieved by amino acid substitutions in the alanine cavity (including A1016 and A1020), further conserved amino acid mutations can be made in this region without affecting the desired performance of the spike protein as described herein. Conserved amino acid substitutions are known in the art.

[0293] In one embodiment, the S protein trimer as described herein does not contain structural modifications that reduce the size of the alanine cavity in the coiled-coil region of the S protein trimer, and wherein said S protein trimer. In one embodiment, the S protein trimer does not contain VI modification.

[0294] In one embodiment, the S protein monomer of the S protein trimer does not contain one or more of the following substitutions: T887W, A1020W, T887W and A1020W, and P1069F. In one embodiment, the S protein monomer of the S protein trimer does not contain a substitution for T887W. In one embodiment, the S protein monomer of the S protein trimer does not contain a substitution for A1020W. In one embodiment, the S protein monomer of the S protein trimer does not contain substitutions for both T887W and A1020W. In one embodiment, the S protein monomer of the S protein trimer does not contain a substitution for P1069F.

[0295] Immunogenicity and antigenicity

[0296] The S protein is a major protein used as a target antigen in COVID-19 vaccines. Theoretically, antibodies can target the S protein to inhibit viral infection at multiple stages during viral entry. The RBD is a major target for neutralizing antibodies (NAbs) used to interfere with viral receptor binding. To date, most potent NAbs against SARS-CoV-2 target the RBD. Conserved neutralizing sites in S2 include the fusion peptide and the stem region. Furthermore, NAbs targeting the N-terminal domain have been reported in both SARS-CoV-2 and MERS-CoV infections, making them another potential target for inclusion in vaccines. The S2 subunit is also a potential target for neutralizing antibodies that interfere with the structural rearrangement of the S protein and the insertion of fusion proteins required for virus-host membrane fusion.

[0297] As used herein, “neutralizing antibody” or “NAb” is a class of antibodies that bind to antigens, pathogens, or toxins and neutralize their functional activity. NAbs are referred to as functional antibodies because they possess functional antiviral activity. In one embodiment, a neutralizing antibody response inhibits binding to the ACE-2 receptor. In one embodiment, a neutralizing antibody inhibits the binding of the RBD to the ACE-2 receptor. In one embodiment, a neutralizing antibody binds to a non-RBD epitope. As used herein, “neutralizing antibody response” refers to the production of NAbs in a subject following exposure to an antigen.

[0298] The ability of a vaccine to elicit an effective immune response against NAbs or against heterologous strains or emerging variants of interest is one of the key factors influencing the successful rollout of a vaccine program against SARS-CoV-2.

[0299] The ability of a vaccine to elicit an effective immune response against NAbs or against homologous and heterologous strains or emerging variants of interest is one of the key factors influencing the successful rollout of a vaccine program against SARS-CoV-2. This application enables the generation and use of coronavirus S antigen mutants as described herein, which elicit enhanced immunogenicity and / or antigenicity against a wider range of variants, including ancestral and naturally occurring and emerging variants of interest.

[0300] In one embodiment, the vaccine antigen described herein is a pan-coronavirus vaccine antigen. As used herein, "pan-coronavirus vaccine antigen" is an antigen that generates a neutralizing antibody response against more than one SARS-CoV2, sarbecovirus, and / or more than one beta coronavirus. In one embodiment, the pan-coronavirus vaccine antigen generates a neutralizing antibody response against more than one SARS-CoV2. In one embodiment, the pan-coronavirus vaccine generates a neutralizing antibody response against more than one sarbecovirus. In one embodiment, the pan-coronavirus vaccine generates a neutralizing antibody response against at least two sarbecoviruses. In one embodiment, the pan-coronavirus vaccine generates a neutralizing antibody response against clade 1b sarbecovirus and clade 1a sarbecovirus. In one embodiment, the pan-coronavirus vaccine generates a neutralizing antibody response against clade 1b sarbecovirus and clade 3 sarbecovirus. In one embodiment, the pan-coronavirus vaccine generates a neutralizing antibody response against two or more of clade 1b sarbecovirus, clade 1a sarbecovirus, and clade 3 sarbecovirus. Members of the sarbecovirus clades are shown, for example... Figure 57 The pancoronavirus vaccine is described, for example, in Khaledian et al. (2022) and Sallard et al. (2021). In one embodiment, the pancoronavirus vaccine generates a neutralizing antibody response against more than one beta coronavirus. In another embodiment, the pancoronavirus vaccine generates a neutralizing antibody response against at least two beta coronaviruses.

[0301] In one embodiment, when delivered to a subject, the vaccine antigen described herein has altered immunogenicity compared to vaccine antigens lacking i) intermeric disulfide bonds as described herein, ii) C-terminal truncation in the stem region as described herein, or both i) and ii). In one embodiment, when delivered to a subject, the vaccine antigen described herein has altered immunogenicity compared to vaccine antigens lacking intermeric disulfide bonds as described herein. In one embodiment, when delivered to a subject, the vaccine antigen described herein has altered immunogenicity compared to vaccine antigens lacking C-terminal truncation in the stem region as described herein. In one embodiment, when delivered to a subject, the vaccine antigen described herein has altered immunogenicity compared to vaccine antigens lacking both intermeric disulfide bonds as described herein and C-terminal truncation in the stem region as described herein.

[0302] As used herein, “immunogenicity” refers to the ability of a substance to induce cellular and humoral immune responses. In one embodiment, altered immunogenicity is enhanced immunogenicity. In one embodiment, the humoral immune response comprises neutralizing antibodies. Immunogenicity can be measured using any method known to those skilled in the art, including measuring antibody titers.

[0303] In one embodiment, the neutralizing antibody response is an RBD neutralizing antibody response. In one embodiment, the neutralizing antibody response comprises an antibody targeting one or more epitopes comprising part or all of the RBM. In one embodiment, the neutralizing antibody response is not targeted at the RBM.

[0304] In one embodiment, the neutralizing antibody response is a non-RBD neutralizing antibody response. In one embodiment, the non-RBD neutralizing antibody response comprises a neutralizing antibody response against one or more epitopes comprising part or all of the stem region. In one embodiment, the non-RBD neutralizing antibody response comprises a neutralizing antibody response against one or more epitopes comprising part or all of the NTD. In one embodiment, the non-RBD neutralizing antibody response comprises a neutralizing antibody response against one or more epitopes comprising all or part of the RBD flanking region.

[0305] In one embodiment, when delivered to a subject, the vaccine antigen described herein has altered antigenicity compared to vaccine antigens lacking i) intermeric disulfide bonds as described herein, ii) C-terminal truncation in the stem region as described herein, or both i) and ii). In one embodiment, when delivered to a subject, the vaccine antigen described herein has altered antigenicity compared to vaccine antigens lacking intermeric disulfide bonds as described herein. In one embodiment, when delivered to a subject, the vaccine antigen described herein has altered antigenicity compared to vaccine antigens lacking C-terminal truncation in the stem region as described herein. In one embodiment, when delivered to a subject, the vaccine antigen described herein has altered antigenicity compared to vaccine antigens lacking both intermeric disulfide bonds as described herein and C-terminal truncation in the stem region as described herein.

[0306] As used herein, “antigenicity” refers to the ability of a particular substance to be recognized by antibodies generated as a result of a specific immune response. In one embodiment, altered antigenicity is enhanced immunogenicity.

[0307] In one embodiment, the S protein trimer can be bound by an antibody that binds to the N-terminal domain (NTD). In one embodiment, the antibody is selected from C1520 and antibodies that bind to epitopes bound by C1520. In one embodiment, the antibody is C1520. In one embodiment, the antibody that binds to the NTD is an antibody that binds to epitopes bound by C1520. In one embodiment, the antibody binds to an epitope comprising one or more amino acids bound by C1520. As used herein, "epitope bound by C1520" comprises residues corresponding to K97-R102 of SEQ ID NO: 1 and SEQ ID NO: 2; glycans at N122 and N149; K150-R158; Q173; D178-N188; and H245.

[0308] In one embodiment, the S protein trimer can be bound by any antibody that binds to the RBD. In one embodiment, the antibody is selected from: S2H92, SP177, Omi-18, Omi-42, S309, CR3022, antibodies binding to epitopes bound by S2H92, antibodies binding to epitopes bound by SP1-77, antibodies binding to epitopes bound by Omi-18, antibodies binding to epitopes bound by Omi-42, antibodies binding to epitopes bound by S309, and antibodies binding to epitopes bound by CR3022. In one embodiment, the antibody binding comprises an epitope containing one or more amino acids bound by S2H92. In one embodiment, the antibody binding comprises an epitope containing one or more amino acids bound by SP1-77. In one embodiment, the antibody binding comprises an epitope containing one or more amino acids bound by Omi-18. In one embodiment, the antibody binding comprises an epitope containing one or more amino acids bound by Omi-42. In one embodiment, the antibody binding comprises an epitope containing one or more amino acids bound by S309. In one embodiment, the antibody binding comprises an epitope containing one or more amino acids bound by CR3022. As used herein, "epitope bound by S2H92" comprises residues corresponding to W353, R355, R357, T393, N394, Y396, P426-T430, K462-F464, R466, and S514-P521 of SEQ ID NO: 1 and SEQ ID NO: 2. As used herein, "epitope bound by SP177" comprises residues corresponding to N343, T345, R346, K / N440, and L441-V445 of SEQ ID NO: 1 and SEQ ID NO: 2. The term "epitope bound by Omi-18" as used herein includes residues corresponding to R403, R408, Q409, Q414-N417, D420, Y421, Y453-N460, Y473-N477, N487, R493, Y501, G502, and H505 of SEQ ID NO: 1 and SEQ ID NO: 2. The term "epitope bound by Omi-42" as used herein includes residues corresponding to R403, D405, R408, Q409, T415-Y421, Y453, L455, F456, K458, and Y473-N477 of SEQ ID NO: 1 and SEQ ID NO: 2.The term "S309-bound epitope" as used herein includes residues corresponding to T333, N334, L335, P337, G339, E340, V341, N343 glycan, A344, T345, K346, E354, K356, R357, I358, S359, N360, C361, N440, I441, and K444 in SEQ ID NO: 1 and SEQ ID NO: 2 (Pinto et al., 2020).

[0309] The “episodes bound by CR3022” used in this article include residues corresponding to RBD such as Y369, N370, A372, F374, T376, F377, K378, Y380, V382, P384, T385, K386, D389, L390, F392, D428, F429, T430, F515, L517, and H519 (Yuan et al., 2020).

[0310] In one embodiment, the S protein trimer can be bound by any antibody that binds to the RBD flanking region. In one embodiment, the antibody is selected from: S2H97, SP1-77, antibodies binding to epitopes bound by S2H92, and antibodies binding to epitopes bound by SP1-77. In one embodiment, the antibody binding comprises an epitope containing one or more amino acids bound by S2H92. In one embodiment, the antibody binding comprises an epitope containing one or more amino acids bound by SP1-77.

[0311] In one embodiment, the S protein trimer can be bound by any antibody that binds to RBM. In one embodiment, the antibody is selected from: Omi-18, Omi-42, SA55, an antibody binding to an epitope bound by Omi-18, an antibody binding to an epitope bound by Omi-42, and an antibody binding to an epitope bound by SA55. In one embodiment, the antibody binding to RBM is Omi-18. In one embodiment, the antibody binding to RBM is Omi-42. In one embodiment, the antibody binding to RBM is SA55. In one embodiment, the antibody binding to RBM is an antibody binding to an epitope bound by Omi-18. In one embodiment, the antibody binding to RBM is an antibody binding to an epitope bound by Omi-42. In one embodiment, the antibody binding to RBM is an antibody binding to an epitope bound by SA55. In one embodiment, the antibody binding to an epitope comprising one or more amino acids bound by Omi-18. In one embodiment, the antibody binding to an epitope comprising one or more amino acids bound by Omi-42. In one embodiment, the antibody binding comprises an epitope containing one or more amino acids that are bound by SA55.

[0312] In one embodiment, the S protein trimer is capable of being bound by any antibody that binds to the stem. In one embodiment, the antibody is selected from: CV3-25, S2P6, CC40.8, CC95-108, and CC99-103. In one embodiment, the antibody is CV3-25. In one embodiment, the antibody binds an epitope bound by CV3-25. In one embodiment, the antibody binds an epitope comprising one or more amino acids bound by CV3-25. As used herein, "eptope bound by CV3-25" comprises residues corresponding to K1149-D1165 of SEQ ID NO: 1 and SEQ ID NO: 2. In one embodiment, the antibody is S2P6. As used herein, "eptope bound by S2P6" comprises residues corresponding to F1148-F1156 of SEQ ID NO: 1 and SEQ ID NO: 2. In one embodiment, the antibody is CC40.8. As used herein, "epitope bound by CC40.8" comprises residues corresponding to Q1142-H1159 of SEQ ID NO: 1 and SEQ ID NO: 2. In one embodiment, the antibody is CC95-108. In one embodiment, the antibody binds an epitope bound by CC95-108. In one embodiment, the antibody binds an epitope comprising one or more amino acids bound by CC95-108. As used herein, "epitope bound by CC95-108" comprises residues corresponding to F1148-N1158 of SEQ ID NO: 1 and SEQ ID NO: 2. In one embodiment, the antibody is CC99-103. In one embodiment, the antibody binds an epitope bound by CC99-103. In one embodiment, the antibody binds an epitope comprising one or more amino acids bound by CC99-103. The term “epitope bound by CC99-103” as used herein includes residues of F1148-N1158 corresponding to SEQ ID NO:1 and SEQ ID NO:2.

[0313] In one embodiment, the S protein trimer can be bound by any antibody that binds the fusion peptide. In one embodiment, the antibody binding the fusion peptide is COV44-79. In one embodiment, the antibody binds an epitope that is bound by COV44-79. In one embodiment, the antibody binds an epitope comprising one or more amino acids bound by COV44-79. As used herein, "epitope bound by COV44-79" comprises residues S810-D830 corresponding to SEQ ID NO: 1 and SEQ ID NO: 2.

[0314] Additional modifications / additional stabilization modifications

[0315] In one implementation, the CoV vaccine antigen as described herein includes one or more additional modifications to enhance one or more of the stability, immunogenicity, expression, and purification of the S protein trimer.

[0316] In one implementation, the ribonucleic acid encoding the CoV vaccine as described herein includes one or more additional modifications to enhance one or more of the stability, immunogenicity, expression, and purification of the S protein trimer.

[0317] In one embodiment, the additional modification is selected from proline stabilization, furin cleavage site, trimerization sequence, repeat sequence or spacer sequence, or nucleotide sequence encoding them.

[0318] In one embodiment, the proline stabilization modification is 986P and / or 987P. The presence of both 986P and 987P in the S protein trimer is referred to as a "2P" modification. In one embodiment, the proline stabilization modification is 2P and F817P. In one embodiment, the proline stabilization modification is 2P and A892P. In one embodiment, the proline stabilization modification is 2P and A889P. In one embodiment, the proline stabilization modification is 2P and A942P. In one embodiment, the proline stabilization is a 6P modification as described herein.

[0319] In one embodiment, an additional modification is the insertion of a furin cleavage site. In one embodiment, a mutant PG682SAS is introduced to insert into the furin cleavage site (e.g., PG682SAS replaces RR682RAR in Delta and PG682SAS replaces HR682RAR in Omeprone). In one embodiment, an additional modification is the addition of FHA. In one embodiment, an additional modification is the addition of a purification tag.

[0320] In one embodiment, the CoV vaccine antigen described herein does not contain one or more of the following modification pairs: I712C and T1077C, I714C and Y1110C, P715C and P1069C, G889C and L1034C, I909C and Y1047C, Q965C and S1003C, F970C and G999C, A972C and R995C, A890C and V1040C, T874C and S1055C, and N914C and S1123C. In one embodiment, the CoV vaccine antigen described herein does not contain modifications of I712C and T1077C. In one embodiment, the CoV vaccine antigen described herein does not contain modifications of I714C and Y1110C. In one embodiment, the CoV vaccine antigen described herein does not contain modifications of P715C and P1069C. In one embodiment, the CoV vaccine antigen described herein does not contain modifications of G889C and L1034C. In one embodiment, the CoV vaccine antigen described herein does not contain modifications of I909C and Y1047C. In one embodiment, the CoV vaccine antigen described herein does not contain modifications of Q965C and S1003C. In one embodiment, the CoV vaccine antigen described herein does not contain modifications of F970C and G999C. In one embodiment, the CoV vaccine antigen described herein does not contain modifications of A972C and R995C. In one embodiment, the CoV vaccine antigen described herein does not contain modifications of A890C and V1040C. In one embodiment, the CoV vaccine antigen described herein does not contain modifications of T874C and S1055C. In one embodiment, the CoV vaccine antigen described herein does not contain modifications of N914C and S1123C.

[0321] Antigen combination

[0322] In one embodiment, the modified S antigen elicits an immune response against the strain from which it originates and one or more other strains circulating in the community. In another embodiment, an antigen or a vaccine containing the antigen or its coding sequence delivers one or more target antigens to the subject and induces an effective and multifunctional immune response against homologous or heterologous strains, including, for example, T-cell and antibody responses. In one embodiment, the coronavirus antigen from one or more strains is selected from one, two, three, or four of the spike protein, nucleocapsid protein, membrane protein, and envelope protein. In one embodiment, two, three, or four amino acid and / or nucleotide sequences encoding the SARS-CoV protein N, M, E, and S are used. In an illustrative embodiment, N, M, E, and S are employed. In one embodiment, one or two or more different variants of SARS-CoV are combined. In one embodiment, one or two or more different variants of SARS-CoV-2 are combined. In one embodiment, multiple variants and multiple antigens are employed. In one embodiment, the antigen or a vaccine containing the antigen or its coding sequence is administered together with one or more B-cell and / or T-cell epitopes.

[0323] This article considers cell lines capable of expressing the modified S antigen disclosed herein, as well as one or more N, M, E antigens or their coding sequences.

[0324] Methods for generating coronavirus vaccine antigens

[0325] The antigens described herein can be generated via recombinant or synthetic methods known in the art. In one embodiment, the antigen is generated from deoxyribonucleic acid encoding a coronavirus vaccine antigen as described herein. In another embodiment, the antigen is generated from a vector containing deoxyribonucleic acid encoding a coronavirus vaccine antigen as described herein.

[0326] In one aspect, the present invention provides a host cell comprising deoxyribonucleic acid as described herein or a vector as described herein.

[0327] In one aspect, the present invention provides a method for generating coronavirus (CoV) vaccine antigens as described herein, comprising culturing host cells as described herein in a culture medium to generate vaccine antigens. In one embodiment, the method further comprises isolating the vaccine antigens from the cells and / or cell culture medium.

[0328] In one aspect, the present invention provides a vaccine comprising: a coronavirus (CoV) vaccine antigen as described herein, or a protein nanoparticle as described herein, or a virus-like particle as described herein, or a deoxyribonucleic acid as described herein, or a vector as described herein. In one embodiment, the vaccine is selected from: inactivated vaccines, live attenuated vaccines, and protein subunit vaccines. In one embodiment, the vaccine further comprises at least one additional CoV vaccine antigen as described herein.

[0329] In one embodiment, nanoparticles are provided comprising an antigen as described herein fused to a polyhedral protein targeting peptide from CPV or other suitable viruses. Other nanoparticles are known in the art and include SOR particles, luminazine synthase particles, and pyruvate dehydrogenase particles.

[0330] Antigens can be conjugated to carriers or nanoparticles to enhance immunogenicity. Suitable carriers are known in the art.

[0331] Virus-like particles (VLPs) provide antigens with some of the structural complexity / advantages of viral surface proteins and can be derived from any suitable virus. As used herein, "virus-like particles" refers to vaccines that contain viral surface proteins but lack the viral genome and one or more structural proteins. Human hepatotropic DNA virus HBV is a good example. Antigen-containing VLPs can, for example, spontaneously form after protein recombination expression and can be characterized using conventional techniques.

[0332] In one aspect, the present invention provides a method for improving the yield of S protein trimer, comprising modifying CoV S protein trimer to include a stem region C-terminus truncated.

[0333] In one aspect, the present invention provides a method for stabilizing a CoV S protein trimer in a pre-fusion conformation, comprising modifying the CoV S protein trimer to include at least one non-endogenous protomer disulfide bond.

[0334] In one aspect, the present invention provides a method for increasing the melting temperature of the CoV S protein trimer, comprising modifying the CoV S protein trimer to include a stem region truncated at the C-terminus.

[0335] In one aspect, the present invention provides a method for increasing the melting temperature of a CoV S protein trimer that is stable in a pre-fusion conformation, comprising modifying the CoV S protein trimer to include a C-terminal truncation of the stem region.

[0336] In one aspect, the present invention provides a method for enhancing a neutralizing antibody response, comprising modifying a CoV S protein trimer to include at least one interprotomeric disulfide bond and / or modifying the CoV S protein trimer to include a C-terminal truncation of the stem region.

[0337] In some implementations, ribonucleic acid encoding the antigen is applied to the subject, and the antigen is generated in the subject.

[0338] RNA encoding coronavirus vaccines and its production methods

[0339] RNA, as described herein, can be modified to stabilize its sequence, by capping, and by polyadenylation. RNA can be delivered in vectors as described herein. RNA can be delivered as plasmids to express antigens and induce immune responses. RNA can be modified to enhance delivery via lipid nanoparticles. RNA can be modified to improve the stability of RNA molecules. RNA-based methods can include amplified or non-self-amplifying constructs. In one embodiment, the RNA is messenger RNA (mRNA).

[0340] In some embodiments, RNA encoding an antigen as described herein is administered. In some embodiments, the RNA encodes an antigen lacking a coronavirus transmembrane domain as described herein. In some embodiments, the RNA encodes an antigen containing a trimerizing domain as described herein. In some embodiments, the RNA encodes an antigen lacking a trimerizing domain as described herein.

[0341] In some embodiments, the RNA to be administered via transient in vivo transfection is chemically modified RNA, wherein a portion (e.g., 10%, 30%, 50%, or 100%) of at least one type of nucleotide (e.g., cytosine) is chemically modified to improve its stability in vivo. For example, in some cases, the modified cytosine is 5-methylcytosine. Such RNA is particularly useful for in vivo delivery / transfection to cells, especially when combined with transfection / delivery agents. In some cases, the chemically modified RNA is a RNA in which most (e.g., all) of the cytosines is 5-methylcytosine and most (e.g., all) of the uracils is pseudouracil. In some embodiments, non-natural cysteines are modified to generate disulfide bonds (e.g., through recombinant genetic techniques). The synthesis and use of such modified RNA are described, for example, in WO 2011 / 130624. Methods for in vivo transfection of RNA polynucleotides are known in the art.

[0342] The term "RNA" refers to a molecule that contains ribonucleotide residues and is preferably composed entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-furanose group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA by adding, deleting, substituting, and / or altering one or more nucleotides. Such alterations may include the addition of non-nucleotide substances, such as addition to the ends or interior of the RNA, for example, addition at one or more nucleotides in the RNA. The nucleotides in the RNA molecule may also contain non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0343] Therefore, in one embodiment, the G / C content of the coding region is adjusted, particularly increased, compared to the G / C content of the coding region of its specific wild-type coding sequence (i.e., unmodified mRNA). The coding amino acid sequence of the mRNA is preferably unmodified compared to the coding amino acid sequence of the specific wild-type mRNA.

[0344] Optimized mRNA-based compositions may include 5' and 3' untranslated regions (5'-UTR, 3'-UTR) and an open reading frame encoding an S protein, as known in the art for optimizing translation efficiency and intracellular stability. In one embodiment, the removal of the uncapped 5'-triphosphate can be achieved by treating the RNA with a phosphatase. The RNA may have modified ribonucleotides to improve its stability and / or reduce cytotoxicity. For example, in one embodiment, cytidine is partially or completely replaced with 5-methylcytidine in the RNA. As an alternative or supplement, uridine is partially or completely replaced with pseudouridine, preferably completely. These modifications can also reduce indiscriminate immune inactivation that can inhibit RNA translation. In one embodiment, the term "modification" refers to providing RNA having a 5'-cap or a 5'-cap analogue. The term "5'-cap" refers to a cap structure found at the 5' end of an mRNA molecule and is typically composed of a guanosine nucleotide linked to the mRNA via an unusual 5' to 5' triphosphate bond. In one embodiment, the guanosine is methylated at position 7. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, preferably a 7-methylguanosine cap. The term "5'-cap" also includes 5'-cap analogs that resemble RNA cap structures and are modified to have the ability to stabilize RNA and / or enhance RNA translation.

[0345] Further modifications to the RNA can be made by extending or truncating naturally occurring UTRs, such as the tail of the X region, or by altering the 5'- or 3'-untranslated region (UTR), for example, by introducing a UTR unrelated to the coding region of the RNA, such as replacing an existing 3'-UTR with one or more, preferably two copies, of a 3'-UTR derived from a globin gene, or by inserting one or more, preferably two copies, of a 3'-UTR derived from a globin gene, such as α2-globin, α1-globin, or β-globin. RNA with an unmasked poly-A sequence is translated more efficiently than RNA with a masked poly-A sequence.

[0346] The terms "poly(A) tail" or "poly(A) sequence" refer to a sequence of adenosine (A) residues that can be located at the 3' end of an RNA molecule, and "unmasked poly-A sequence" means that the 3' end of the poly-A sequence of the RNA molecule ends with the letter A, and that no nucleotide other than A immediately follows at the 3' end (i.e., downstream) of the poly-A sequence. Furthermore, long poly-A sequences of approximately 120 base pairs result in optimal transcript stability and translation efficiency for RNA.

[0347] Therefore, to improve RNA stability and / or expression, it can be modified to bind with a heterologous poly-A sequence, the length of which is preferably 10 to 500, more preferably 30 to 300, even more preferably 65 to 200, and especially 100 to 150 adenosine residues. In a particularly preferred embodiment, the poly-A sequence is about 120 adenosine residues long. To further improve the stability and / or expression of the RNA used according to the invention, the poly-A sequence can be left unmasked.

[0348] Furthermore, incorporating a 3'-untranslated region (UTR) into the 3'-untranslated region of an RNA molecule can lead to enhanced translation efficiency. A synergistic effect can be achieved by incorporating two or more such 3'-untranslated regions. The 3'-untranslated regions can be autologous or heterologous to the RNA in which they are introduced. In one specific embodiment, the 3'-untranslated region is derived from the human β-globin gene.

[0349] The combination of the above modifications—optionally incorporating a poly-A sequence, leaving the poly-A sequence unmasked, and incorporating one or more 3'-untranslated regions—has a synergistic effect on improving RNA stability and translation efficiency.

[0350] To enhance RNA expression, modifications can be made within the coding region to increase GC content, thereby improving mRNA stability, and to optimize codons, thus enhancing translation in cells. Modified mRNA can be enzymatically synthesized and packaged into nanoparticles, such as lipid nanoparticles, and administered, for example, intramuscularly. RNA approaches using self-replicating RNA or protamine complexes have also been shown to generate immune responses against viral infections.

[0351] RNA molecules can be embedded in microcapsules prepared by, for example, cohesive drug delivery systems (e.g., liposomes, microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, such techniques are known in the art and are disclosed in Remington, the Science and Practice of Pharmacy, 20th edition, Remington, J. (2000).

[0352] Various methods are known for systemic administration of RNA as nanoparticles or colloidal systems. In non-viral methods, cationic liposomes are used to induce RNA aggregation and to promote cellular uptake. Cationic liposomes typically consist of cationic lipids (such as DOTAP) and one or more accessory lipids (such as DOPE). A so-called "lipid complex" can be formed from cationic (positively charged) liposomes and anionic (negatively charged) RNA. In the simplest case, the lipid complex is spontaneously formed by mixing RNA with liposomes using some mixing scheme; however, many other schemes can be applied. In one embodiment, a nanoparticle RNA formulation, such as an RNA-lipid complex, with a defined particle size is produced, wherein the net charge of the particles is close to zero or negative. For example, as disclosed in WO2013 / 143683, an electrically neutral or negatively charged lipid complex derived from RNA and liposomes leads to high RNA expression in spleen or immune cells after systemic administration. In one embodiment, the nanoparticles comprise at least one lipid. In another embodiment, the nanoparticles comprise at least one cationic lipid. The cationic lipids can be monocationic or polycationic. Any cationic amphiphilic molecule, such as a molecule containing at least one hydrophilic and lipophilic moiety, is a cationic lipid within the meaning of this invention. In one embodiment, the positive charge is contributed by at least one cationic lipid, and the negative charge is contributed by RNA. In one embodiment, the nanoparticle comprises at least one accessory lipid. The accessory lipid can be a neutral or anionic lipid. The accessory lipid can be a natural lipid, such as a phospholipid or an analogue of a natural lipid, or a fully synthetic lipid, or a lipid-like molecule that does not resemble a natural lipid. In one embodiment, the cationic lipid and / or accessory lipid are bilayer-forming lipids.

[0353] In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) or its analogues or derivatives and / or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or its analogues or derivatives.

[0354] In one embodiment, the at least one auxiliary lipid comprises 1,2-bis-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine (DOPE) or an analogue or derivative thereof, cholesterol (Choi) or an analogue or derivative thereof, and / or 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC) or an analogue or derivative thereof.

[0355] In one embodiment, the molar ratio of at least one cationic lipid to at least one auxiliary lipid is 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1:1. In one embodiment, in this ratio, the molar amount of the cationic lipid is obtained by multiplying the molar amount of the cationic lipid by the number of positive charges in the cationic lipid. In the nanoparticles described herein, lipids can form complexes with RNA and / or encapsulate RNA. In one embodiment, the nanoparticles comprise lipid complexes or liposomes. In one embodiment, the lipids are contained in vesicles encapsulating the RNA. The vesicles can be multilayered vesicles, monolayered vesicles, or mixtures thereof. The vesicles can be liposomes.

[0356] Lipid nanoparticles (LNPs) are generally referred to as nanoscale particles composed of combinations of different lipids (the aqueous volume is encapsulated by an amphiphilic lipid bilayer, such as a single or multiple layer). Many different types of lipids can be contained in LNPs. In some embodiments, the lipids can be one or more of ionizable lipids, phospholipids, structural lipids, neutral lipids, and PEG lipids. For example, mRNA is encapsulated in an LNP. In another instance, mRNA binds to an LNP. For example, mRNA is absorbed onto an LNP.

[0357] Methods for preparing LNPs are known to those skilled in the art and are described, for example, in Huang et al., 2021 and Schoenmaker et al., 2021. The term "ionizable lipid" or variations thereof as used herein shall refer to lipids having at least one protonable or deprotonable group. For example, lipids are positively charged at a pH of or below physiological pH (e.g., pH 7.4) and neutrally charged at a second pH (e.g., at or above physiological pH). In one embodiment, the lipid nanoparticles comprise ionizable lipids as described in Table 1 of Schoenmaker et al., 2021.

[0358] Suitable ionizable lipids may have hydrophilic head groups that are anionic, cationic, or zwitterionic. Exemplary phospholipids (anionic or zwitterionic) used in this disclosure include, for example, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. In one instance, the lipid is a cationic lipid. Exemplary cationic lipids include, but are not limited to, dioleoyltrimethylammonium propane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-diolenoyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), and 2,5-bis((9Z,12Z)-octadec-9,12-dien-1-yloxy)benzyl-4-(dimethylamino)butyrate (LKY750). In one example, the phospholipid is 2,5-bis((9z,12z)-octadecano-9,12-dien-1-yloxy)benzyl-4-(dimethylamino)butyrate (LKY750). Exemplary zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids, such as dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and dodecylphosphocholine. The lipids may be saturated or unsaturated. In one embodiment, the lipid nanoparticles do not contain cationic lipids.

[0359] Those skilled in the art will understand that PEGylated lipids refer to lipids that have been modified with polyethylene glycol. Exemplary PEGylated lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. For example, PEG lipids include PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0360] Suitable neutral or zwitterionic lipids for use in this disclosure will be apparent to those skilled in the art, and include, for example, 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0Diether 1,2-Oleoyl-2-cholestanoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinanoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidanoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) Lipids include PE, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. Lipids can be saturated or unsaturated.

[0361] Exemplary structural lipids include, but are not limited to, cholesterol, coccosterol, sitosterol, campesterol, stigmasterol, brassosterol, ergosterol, tomatine, tomatine, ursolic acid, and α-tocopherol. In one embodiment, the structural lipid is a sterol. In one embodiment, the structural lipid is cholesterol. In one embodiment, the structural lipid is campesterol.

[0362] This invention covers vaccines in liposome form. The term "liposome" as used herein refers to a single-layer or multi-layer lipid structure encapsulating an aqueous interior. Lipids capable of forming liposomes include all substances having fatty or fatty-like properties. Dynamic laser scattering is a method well-known to those skilled in the art for measuring liposome size. Extensive descriptions of adjuvants can be found in Cox and Coulter, "Advances in Adjuvant Technology and Application", Animal ParasiteControl Utilizing Biotechnology, Chapter 4, edited by Young, WK, CRC Press, 1992, and in Cox and Coulter, Vaccine 15(3): 248-256, 1997. In one aspect, the invention provides host cells comprising ribonucleic acid as described herein or vectors as described herein.

[0363] In one aspect, the present invention provides a method for producing a coronavirus vaccine, comprising culturing host cells as described herein in a culture medium to produce the ribonucleic acid described herein. In one embodiment, the ribonucleic acid as described herein is isolated and formulated into a pharmaceutical composition.

[0364] Cell culture

[0365] Those skilled in the art will understand that CoV vaccine antigens, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or vectors as described herein can all be generated in cell culture. In one instance, the cells are prokaryotic or eukaryotic. In one instance, the cells are derived from mammals, birds, bacteria, or arthropods. In one instance, the cells are mammalian. In one instance, the cells are derived from a continuous cell line. In one instance, the cells are derived from a primary cell line. In one instance, the cells are derived from an immortalized cell line. In one instance, the cells are adherent cells. In one instance, the cells are non-adherent cells (suspension cells). In one instance, the cells are immune cells.

[0366] In one instance, the mammalian cell is a HEK cell, a CHO cell, or a HeLa cell. In another instance, the cell is a HeLa cell.

[0367] Cells can be cultured in any cell culture medium that allows for in vitro cell expansion. Such media and processes are known to those skilled in the art. Exemplary media for culturing the cell populations of the present invention include, but are not limited to: Iscove medium, UltraCHO, CD hybridoma serum-free medium, episerf medium, MediV SF103 (serum-free medium), Dulbecco's modified eagle medium (DMEM), Eagles Modified Eagle Medium (EMEM), Glasgow's modified eagle medium (GMEM), SMIP-8, modified eagle medium (MEM), VP-SFM, DMEM-based SFM, DMEM / F12, DMEM / Ham's F12, VPSFM / William's medium E, ExCell 525 (SFM), adenovirus expression medium (AEM), and Excell 65629. Those skilled in the art will understand that such a culture medium may be supplemented with additional growth factors, such as, but not limited to, amino acids, hormones, vitamins, and minerals. Optionally, such a culture medium may be supplemented with serum, such as fetal bovine serum.

[0368] In one example, cells are cultured using a batch cell culture process. In another example, cells are cultured using a perfusion cell culture process. In one example, cells are cultured in inoculation and production media. In one example, cells are cultured in a stirred tank bioreactor. In one example, the bioreactor volume is from about 1 L to about 2500 L.

[0369] carrier

[0370] Antigens can be delivered in the form of viral or non-viral DNA vectors. As used herein, the term "vector" includes any delivery portion that inserts at least the antigen-coding sequence, including plasmid vectors, granular vectors, phage vectors such as λ phage, virus-like particles, viral vectors such as adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus (HSV), measles virus, CMV, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus, and piconemavirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or PI artificial chromosome (PAC). Vectors include expression vectors and cloning vectors.

[0371] Expression vectors comprise plasmids and viral vectors, and generally contain the desired coding sequence and suitable DNA sequences necessary for expressing the operatively linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to modify and amplify specific desired DNA fragments and may lack the functional sequences required for expressing the desired DNA fragment.

[0372] In one implementation, the vector is a viral vector or a non-viral vector. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses and their attenuated forms, each with its own advantages and disadvantages as known in the art. Viral vectors specifically include, but are not limited to, adenovirus vectors and poxvirus vectors. Typically, for viral vectors, an application rate of approximately 5 × 10⁻⁶ is used. 7 Up to 5 × 10 12 Each virus particle typically consists of approximately 5 × 10⁶ particles. 9 Up to 5 × 10 10 Each virus particle. The vector can be replicating or non-replicating.

[0373] Components, delivery route and dosage

[0374] Those skilled in the art will understand that coronavirus vaccine (CoV) antigens, deoxyribonucleic acid encoding CoV antigens, ribonucleic acid, or vectors as described herein can be formulated into pharmaceutical compositions. In one embodiment, the pharmaceutical composition is a vaccine composition.

[0375] Such compositions may include one or more pharmaceutically acceptable carriers. EW Martin's *Remington's Pharmaceutical Sciences*, Mack Publishing Co., Easton, Pa., 19th edition, 1995, describes compositions and formulations suitable for drug delivery of the disclosed immunogens. Generally, the nature of the carrier will depend on the specific administration method employed. For example, parenteral formulations typically contain injectable fluids, including pharmaceutically and physiologically acceptable carriers (e.g., water, physiological saline, balanced salt solutions, dextran aqueous solutions, glycerol, etc.) as loads. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional nontoxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered (e.g., immunogenic compositions) may also contain small amounts of nontoxic excipients, such as wetting agents or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitol monolaurate. In some specific embodiments, the carrier suitable for administration to a subject may be sterile, and / or suspended, or otherwise contained in a unit dosage form of a composition containing one or more measurable doses suitable for inducing a desired immune response. It may also be accompanied by a drug for therapeutic purposes. For example, the unit dosage form may be in a sealed vial containing sterile contents or in a syringe for injection into the subject, or may be lyophilized for subsequent dissolution and administration, or in a solid or controlled-release dosage form.

[0376] In one embodiment, the composition comprises a vaccine antigen as described herein. In one embodiment, the composition comprises a carrier as described herein. In one embodiment, the carrier comprises deoxyribonucleic acid as described herein. In one embodiment, the composition comprises ribonucleic acid as described herein. In one embodiment, the composition comprises lipid nanoparticles as described herein. In one embodiment, the lipid nanoparticles encapsulate ribonucleic acid as described herein.

[0377] In one embodiment, the composition may contain one or more other epitopes for inducing an immune response, such as B-cell epitopes and / or T-cell epitopes.

[0378] In one embodiment, the composition may contain one or more other RNAs encoding epitopes for inducing an immune response, such as B-cell epitopes and / or T-cell epitopes.

[0379] In one embodiment, the composition is formulated to be compatible with its intended route of administration (e.g., local or systemic). Examples of routes of administration include intradermal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intranasal, sublingual, tonsillar, oral, pulmonary, topical, or other parenteral and mucosal routes.

[0380] In one embodiment, the composition is formulated to be stable at refrigerator temperatures. In one embodiment, the composition is formulated to be suitable for transport and / or storage at refrigerator temperatures. In one embodiment, the refrigerator temperature is about 3°C ​​to about 17°C, or about 4°C to about 10°C, or about 4°C. In one embodiment, the composition is a formulation stable at room temperature. In one embodiment, the room temperature is about 18°C ​​to about 24°C, or about 20°C to about 23°C, or about 23°C. In one embodiment, the composition is formulated to be suitable for non-cold chain transport and / or storage. In one embodiment, the composition is formulated to be suitable for room temperature storage and / or transport. In one embodiment, the composition is formulated to be suitable for transport and / or storage at temperatures above room temperature (e.g., about 25°C to 40°C) (applicable to countries without cold chain and low-temperature storage and transport trains).

[0381] Oral, nasal, and pulmonary administration includes delivery to these sites via inhalation and aerosol. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solutions, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for adjusting tension, such as sodium chloride or dextrose. pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules made of glass or plastic, disposable syringes, or multi-dose vials.

[0382] Suitable compositions for injectable applications include sterile aqueous solutions (where they are water-soluble) or dispersions, non-aqueous solutions, and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and be a fluid present in an injectable manner. It should be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For example, appropriate flowability can be maintained by using coatings (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants. Antimicrobial activity can be achieved using a variety of antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). Isotonic agents (e.g., sugars, polyols (e.g., mannitol, sorbitol), sodium chloride) may also be included in the composition. Extended absorption of the injectable composition can be achieved by including a delay-absorption agent (e.g., aluminum monostearate or gelatin) in the composition.

[0383] Sterile injectable solutions can be prepared by incorporating the required amount of one or a combination of the components listed above (as needed) into a suitable solvent or buffer, followed by filtration sterilization. Generally, dispersions are prepared as sterile carriers containing a basic dispersion medium and any other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, suitable preparation methods include vacuum drying and freeze-drying, which produce powders containing the active ingredient plus any additional desired components from its previously sterile filtered solution.

[0384] Oral compositions generally include an inert diluent or an edible carrier. For oral therapeutic application, excipients are suitable for sprays, tablets, lozenges, or capsules, such as gelatin capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or excipients may be included as part of the composition. Sprays, tablets, pills, capsules, lozenges, etc., may contain any of the following components or compounds with similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginate, PRIMOGEL, or corn starch; lubricants such as magnesium stearate or sterotes; flow aids such as colloidal silica; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring agents.

[0385] Formulations suitable for administration by nasal inhalation include, when the carrier is solid, coarse powders with particle sizes, for example, from about 1 to about 500 micrometers, which are administered by means of a nebulizer, atomizer, inhaler, or snuffing. Suitable formulations where the carrier is a liquid for administration by a nebulizer include aqueous or oily solutions of the pharmaceutical agent. For administration by inhalation, the pharmaceutical agent may also be delivered in the form of drops or aerosol sprays from a pressurized vessel or dispenser or nebulizer containing a suitable propellant (e.g., a gas such as carbon dioxide). Such methods include those described in US 6,468,798.

[0386] Formulations suitable for oral inhalation include, when the carrier is solid, coarse powder with a particle size of, for example, about 20 to about 500 micrometers, which is administered by oral inhalation from a container that holds the powder near the mouth, or when the carrier is a liquid for administration by a nebulizer, the formulation may include an aqueous or oily solution of the agent.

[0387] Systemic administration can also be performed via mucosal or transdermal routes. For mucosal or transdermal administration, a penetrant suitable for the target penetration barrier is used in the formulation. Such penetrants are generally known in the art, and for mucosal administration, include, for example, detergents, bile salts, and fusidic acid derivatives. Mucosal administration can be accomplished using nasal sprays, drops, or suppositories.

[0388] Intradermal delivery of vaccines via needle- or needle-free methods offers advantages in ease of administration and considers methods that effectively target immune-active cells. Liquid formulations can be provided in pre-filled or non-pre-filled syringes, or require disposable jet injectors, hollow microneedles mounted on the syringe, and needles suitable for intradermal delivery. Pre-filled syringes with a single ID needle are commercially available. Alternatively, solid or biodegradable microneedles coated or impregnated with the vaccine (e.g., patches or other microneedle / prick devices), or solid or biodegradable microneedles composed of the vaccine, can be used. These are inserted into the dermis of the skin, where the vaccine coating dissolves, or the microneedles themselves dissolve at the appropriate location. The formulation can be provided as a liquid or semi-liquid formulation, or as a solid or powder formulation. Jet injectors operate by generating a high-pressure stream that propels the liquid vaccine formulation into deeper layers of the skin. However, methods for delivering vaccines in solid form have also proven promising. One such method is the ballistic method, in which solid vaccine particles or vaccine-coated gold particles are accelerated and fired at the skin using a needle-free device, causing the particles to deposit in the epidermis and dermis.

[0389] Intramuscular administration can be performed by any intramuscular method known to those skilled in the art, including, for example, intramuscular injection.

[0390] The composition can also be prepared as a suppository (e.g., with a conventional suppository base such as cocoa butter and other glycerides) or a retention enema for rectal delivery.

[0391] The composition may include adjuvants. When administered as a mixture with one or more adjuvants, it may enhance the immune response to the antigen. Immune adjuvants typically function in one or more ways: (1) immunomodulation; (2) enhanced presentation; (3) CTL generation; (4) targeting; and / or (5) reservoir formation.

[0392] Illustrative adjuvants, which may or may not be included, include: particulate or non-particulate adjuvants, complete Freund's adjuvant (CFA), aluminum salt-based adjuvants, emulsion-based adjuvants, TLR agonists, ISCMS, LPS derivatives (e.g., MPL and its derivatives such as 3D-MPL, GLA, and AGP)), mycobacterial proteins (e.g., muramyl dipeptides or tripeptides), specific saponins from the saponaria tree (Quillaja saponaria) (e.g., QS21, QS7, and ISCOPREP™ saponins), ISCMATRIX™ adjuvants, and peptides (e.g., thymosin α1). In addition to saponin components, adjuvants may also contain sterols such as β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol. In some embodiments, the adjuvant is present in the form of an oil-in-water emulsion, such as containing squalene, α-tocopherol, and a surfactant, or in the form of liposomes. AddaVax, a squalene-based oil-in-water nanoemulsion formulated according to MF-59, has been found to be suitable for use in influenza vaccines. Adjuvants AS03, MF59, and CpG 1018 have been used in licensed vaccines. Other suitable adjuvants include lecithin and caromer homopolymers, matrix M, ASO1, and ALFQ. CpG motifs and co-stimulatory molecules, including TLR agonists, B7, OX-40L, and G-CSF, have been considered. Adjuvants are discussed in Liang et al., 2020.

[0393] In one embodiment, the composition comprises one or more adjuvants selected from the following: aluminum salt-based adjuvants, emulsion adjuvants, or TLR agonists. Some examples of such adjuvants are described, for example, in Liang et al., 2020.

[0394] The subject may receive one, two, or three doses of the composition at predetermined time intervals.

[0395] Antibodies generated against target antigens can be used for treatment or screening. Antibodies include immunoglobulins, antigen-binding fragments, or derivatives thereof that specifically bind to and recognize antigens or antigenic fragments thereof or dimers or multimers of antigens. The term “antibody” is used in the broadest sense herein and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific (and bispecific) antibodies, and antibody fragments. Some examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments, which are generated by modifying whole antibodies or synthesized de novo using recombinant DNA methods (see Kontermann and Dubel (eds.), Antibody Engineering, Vol. 1–2, Ed., Springer, 2010).

[0396] The term epitope refers to a specific peptide sequence on a molecule that possesses antigenicity, thereby triggering a specific immune response. An epitope is an antigenic region that responds to B and / or T cells. Antibodies can bind to specific antigenic epitopes, which can be formed from sequential or non-sequential amino acids.

[0397] Methods of prevention and / or treatment

[0398] In one aspect, the present invention provides a method for preventing and / or treating coronavirus infection in an object.

[0399] As used in this article, the terms "prevention" or "prophylaxis" refer to reducing the likelihood of infection or the occurrence of infection or its symptoms. Prevention does not need to be complete and does not mean that the subject will ultimately not become infected or develop infection or its symptoms.

[0400] As used herein, the term "treatment" or variations thereof refers to at least partially achieving the desired therapeutic outcome. In one embodiment, treatment includes preventing or delaying the onset of one or more symptoms of CoV infection. In another embodiment, treatment includes preventing or reducing the occurrence of one or more symptoms of CoV infection.

[0401] The term "object" and its variations include objects that are susceptible to coronavirus infection or at risk of exposure to coronavirus. Objects may be infected or uninfected and may be asymptomatic or require treatment. In one embodiment, an object is susceptible to SARS-CoV-2 infection or at risk of exposure to SARS-CoV-2 infection. For example, an object can be a mammal, bird, arthropod, chordate, amphibian, or reptile. Exemplary objects include, but are not limited to, humans, primates, livestock (e.g., sheep, cattle, chickens, horses, donkeys, pigs), companion animals (e.g., dogs, cats), laboratory testing animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), captive wild animals (e.g., foxes, deer), zoo animals (e.g., lions, tigers, bears), and reservoir animals (e.g., bats, camels, pangolins). In one embodiment, the object is a mammal. In one embodiment, the object is a human. In one embodiment, the object is a camel. In one embodiment, a human is a fetus, infant, child, early adult, and adult. In one embodiment, an adult is an elderly person. In one implementation, the adult is one or more of the following age groups: 60 years and older, 65 years and older, 70 years and older, 75 years and older, 80 years and older, 85 years and older, and 90 years and older. In one implementation, the subject has a prior history of coronavirus infection. In one implementation, the subject has a prior history of SARS-CoV-2 infection. In one implementation, the subject has received a Level 1 coronavirus treatment regimen as described herein. In one implementation, the subject has received both Level 1 and Level 2 coronavirus treatment regimens. In one implementation, the subject has received Level 1, Level 2, and Level 3 coronavirus treatment regimens. In one implementation, the subject is immunocompromised. In one implementation, the subject has a respiratory illness.

[0402] In one aspect, the present invention provides a method for inducing an immune response against a coronavirus (CoV) in an object, the method comprising delivering a vaccine as described herein to the object.

[0403] In one aspect, the present invention provides a method for enhancing an immune response against a coronavirus (CoV) in an object, the method comprising delivering to the object a vaccine antigen as described herein or a vaccine as described herein.

[0404] In one aspect, the present invention provides a method for preventing or reducing the possibility of coronavirus (CoV) infection in a subject, the method comprising delivering a vaccine antigen as described herein or a vaccine as described herein to the subject.

[0405] In one aspect, the present invention provides a method for preventing or reducing the likelihood or severity of symptoms of coronavirus (CoV) infection in a subject, the method comprising delivering to the subject a vaccine antigen as described herein or a vaccine as described herein.

[0406] In one aspect, the present invention provides a method for reducing the severity and / or duration of coronavirus (CoV) infection in a subject, the method comprising delivering to the subject a vaccine antigen as described herein or a vaccine as described herein.

[0407] In one aspect, the present invention provides a method for preventing or reducing viral shedding in human individuals infected with coronavirus (CoV), the method comprising delivering to the subject a vaccine antigen as described herein or a vaccine as described herein.

[0408] In one implementation, the vaccine is delivered via intramuscular, intradermal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intranasal, sublingual, tonsillar, oral, pulmonary, surface, or other parenteral and mucosal routes.

[0409] In one aspect, the present invention provides a vaccine antigen as described herein or a vaccine as described herein, which is used for one or more of the following: i) inducing an immune response against CoV in a subject; ii) enhancing an immune response against CoV in a subject; iii) preventing or reducing the likelihood of CoV infection in a subject; iv) preventing or reducing the likelihood or severity of CoV symptoms in a subject; v) reducing the severity and / or duration of CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating CoV infection in a subject.

[0410] In one aspect, the present invention provides kits, devices, surfaces, or test strips comprising coronavirus (CoV) vaccine antigens as described herein.

[0411] In one aspect, the present invention provides the use of coronavirus (CoV) vaccine antigens as described herein in the preparation of medicaments for one or more of: i) inducing an immune response against CoV in a subject; ii) enhancing an immune response against CoV in a subject; iii) preventing or reducing the likelihood of CoV infection in a subject; iv) preventing or reducing the likelihood or severity of CoV symptoms in a subject; v) reducing the severity and / or duration of CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating CoV infection in a subject.

[0412] In one aspect, the present invention provides a method for inducing an immune response against a coronavirus (CoV) in a subject, the method comprising delivering to the subject a ribonucleic acid vaccine as described herein or an RNA vaccine as described herein.

[0413] In one aspect, the present invention provides a method for enhancing an immune response against a coronavirus (CoV) in a subject, the method comprising delivering to the subject a ribonucleic acid vaccine as described herein or an RNA vaccine as described herein.

[0414] In one aspect, the present invention provides a method for preventing or reducing the possibility of coronavirus (CoV) infection in a subject, the method comprising delivering to the subject a ribonucleic acid vaccine as described herein or an RNA vaccine as described herein.

[0415] In one aspect, the present invention provides a method for preventing or reducing the likelihood or severity of symptoms of coronavirus (CoV) infection in a subject, the method comprising delivering to the subject a ribonucleic acid vaccine as described herein or an RNA vaccine as described herein.

[0416] In one aspect, the present invention provides a method for reducing the severity and / or duration of coronavirus (CoV) infection in a subject, the method comprising delivering to the subject a vaccine ribonucleic acid vaccine as described herein or an RNA vaccine as described herein.

[0417] In one aspect, the present invention provides a method for preventing or reducing viral shedding in human individuals infected with coronavirus (CoV), the method comprising delivering to the subject a ribonucleic acid vaccine as described herein or an RNA vaccine as described herein.

[0418] In one implementation, the ribonucleic acid or RNA vaccine is delivered via intramuscular, intradermal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intranasal, sublingual, tonsillar, oral, pulmonary, surface, or other parenteral and mucosal routes.

[0419] In one aspect, the present invention provides a ribonucleic acid or a vaccine as described herein, which is used for one or more of the following: i) inducing an immune response against CoV in a subject; ii) enhancing an immune response against CoV in a subject; iii) preventing or reducing the likelihood of CoV infection in a subject; iv) preventing or reducing the likelihood or severity of CoV symptoms in a subject; v) reducing the severity and / or duration of CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating CoV infection in a subject.

[0420] In one aspect, the present invention provides kits, devices, surfaces, or test strips comprising coronavirus (CoV) ribonucleic acid as described herein.

[0421] In one aspect, the present invention provides the use of coronavirus (CoV) ribonucleic acid as described herein in the preparation of a medicament for one or more of: i) inducing an immune response against CoV in a subject; ii) enhancing an immune response against CoV in a subject; iii) preventing or reducing the likelihood of CoV infection in a subject; iv) preventing or reducing the likelihood or severity of CoV symptoms in a subject; v) reducing the severity and / or duration of CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating CoV infection in a subject.

[0422] The phrase “reducing the severity of infection” or similar phrases used in this article include reducing one or more of the following in an individual: viral titer, duration of viral infection, severity or duration of one or more symptoms of coronavirus infection in the subject. The phrase “duration of coronavirus infection” used in this article refers to the time an individual has had CoV infection or symptoms caused by CoV infection.

[0423] In one embodiment, the present invention provides a vaccine as a primary vaccine regimen. As used herein, a "primary vaccine regimen" is a first vaccine regimen administered to a subject to generate an immune response against a specific pathogen. In the context of SARS-CoV-2, a primary vaccine is a first vaccine regimen administered to a subject to generate an immune response against an ancestral strain and / or its variants.

[0424] In one embodiment, the present invention provides a booster vaccine for a primary coronavirus vaccine regimen. In one embodiment, the present invention provides a booster vaccine for situations where a subject has received more than one prior coronavirus vaccine regimen. In one embodiment, the booster works by enhancing the immune response induced by the primary vaccine regimen. In one embodiment, the booster works by enhancing the immune response against VOCs, VOIs, or VHCs against which the protective immune response induced by the primary vaccine regimen is weak, very weak, or nonexistent. In one embodiment, the booster is administered at least 6 months, or at least 12 months, or at least 18 months, or at least 2 years, or at least 3 years, or at least 5 years, or at least 6 years, or at least 7 years after the primary vaccine regimen. In one embodiment, the booster is administered sequentially or in combination with one or more other booster vaccines.

[0425] Combination therapy

[0426] Coronavirus vaccine antigens, vaccines, or ribonucleic acids as described herein can be administered to the recipient in combination with one or more additional ribonucleic acids, vaccine antigens, or vaccines. These additional ribonucleic acids, vaccine antigens, or vaccines can elicit an immune response against infectious pathogens that may be the same as or different from SARS-CoV-2. In one embodiment, the pathogen is selected from influenza, respiratory syncytial virus, SARS-CoV-2, or specific VOCs, VOIs, or VHCs thereof. Administration can be combined (simultaneously) or performed sequentially in any order.

[0427] Reagent kit, device, surface or test strip

[0428] As described herein, coronavirus antigens, deoxyribonucleic acid (DNA), or ribonucleic acid (RNA) are captured on solid or semi-solid surfaces for assay purposes, including epidemiological, diagnostic, purification, drug screening, and vaccine screening applications. Many such applications and methods for immobilizing antigens on surfaces are known in the art and are covered herein.

[0429] Example

[0430] Example 1 - Materials and Methods

[0431] Recombinant spike protein. S2P.BA45.VI-1208.H8 and its C-terminal truncated mutant. The synthetic gene encoding the SARS CoV-2 omicron BA4 / omicron BA5 S extracellular domain (omicron BA4 and omicron BA5 S have the same amino acid sequence and are collectively referred to as BA45 herein) was obtained from GeneART-ThermoFisher Scientific, which corresponds to the S2P protein described by Wrapp et al., 2020. This gene encodes amino acids 16 to 1208 of S with mutations at the furin cleavage site (H681RRAR->P681GSAS), a '2P' mutation (K986P / V987P), and a VI mutation (A1016V / A1020I). The C-terminus of S2P is linked to a Gly-Ser-Gly-Ser linker and an eight-His affinity tag. The synthesized S2P.BA45.VI-1208.H8 gene was ligated downstream of the DNA sequence encoding the tissue plasminogen activator precursor within pcDNA3 (Invitrogen) via NheI. The DNA fragment encoding the C-terminal truncated mutant was then processed via polymerase chain reaction using Phusion DNA polymerase (ThermoFisher), with S2P.BA45.VI-1208.H8 DNA as a template and a forward primer (…). ), and to prepare reverse primers designed to encode the Gly-Ser-His6 sequence and stop codon following the desired C-terminal spike residues:

[0432]

[0433] PCR products encoding C-terminal truncated PCR products were used as replacements Figure 4 The DNA sequence within the pcDNA3-S2P.BA45.VI-1208.H8 expression vector, surrounded by EcoRV and XbaI restriction sites. All sequences were validated using fluorescent Sanger sequencing (BigDyeTerminator v3.1, ABI).

[0434] The S1147 truncated sequence was initially introduced into the pcDNA3-S2P-FHA vector containing the ancestral Hu-1 spike sequence described in Poumbourios et al., 2023, using a synthetic gene encoding amino acid 979-1147-GlySer-His6 followed by a stop codon. The synthetic fragment was surrounded by EcoRV and XbaI restriction sites. The VI mutation was then introduced by overlap extension PCR mutagenesis. The ancestral Hu-1 spike DNA was then replaced with the corresponding omicron BA4 / 5 spike DNA fragment from S2P.BA45.VI-1208.H8.

[0435] Cysteine ​​substitution mutants. Using synthetic genes encoding paired Cys substitution mutations listed in Table 2 (GeneART-ThermoFisher Scientific), cysteine ​​substitution mutations were introduced into the expression vectors S2P.BA45.VI-1147, S2P.BA45.VI-1192, and S2P.BA45.VI-1204. Using synthetic genes encoding suitable mutations (GeneART-ThermoFisher Scientific), reverse mutations of V1016A / I1020A (AA) and P986V / P987K (noP) were introduced into multiple vectors.

[0436] Table 2 shows the nearest neighbor contacts between SARS Cov-2 subunits determined using ligand-protein contact and structural unit contact servers.

[0437]

[0438] The coordinates of the pre-fusion stable spike trimer of ancestral SARS-CoV-2 in the 1RBD-up, 2RBD-down conformation (PDBID: 6VSB) were analyzed to determine the nearest neighbor contact between subunits as a potential target for pairwise Cys substitution mutagenesis. The nearest distance, buried surface area, and C between interacting amino acid pairs are shown. α -C α Distance, the subunit containing the interaction pair, and the mutation code.

[0439] S2P.BA45-1273 expression vector. Synthetic genes encoding residues 1-1273 of S in the Omicron BA.4 / BA.5 variant (sequence identical) were generated by GeneART-Thermo Fisher. These genes contain a KpnI restriction site at the 5' end followed by the TATCGCCACC (SEQ ID NO: 100) sequence (before the ATG start codon) and an XbaI site at the 3' end (after the TAA stop codon). The synthetic genes encode a furin cleavage site mutation (H681RRAR->P681GSAS) and double Pro'2P' substitutions at positions 986 and 987. The synthetic genes were cloned into the KpnI-XbaI site of pCDNA3. D17, I1, L23, DL, and IL mutations were introduced into the vector by subcloning suitable fragments containing mutations from various vectors or synthetic gene products (GeneART). Fluorescent sequencing was used to confirm that the mutations had been transferred to the appropriate vector.

[0440] Expression and purification of recombinant spike protein. The spike expression vector was transfected into Expi293F cells using Expifectamine according to the manufacturer's (Thermo Fisher Scientific) recommendations. Cells were cultured at 34°C for 7 days, after which the transfection supernatant was clarified by centrifugation and filtration through a 0.45 μm nitrocellulose filter. The SARS-CoV-2 glycoprotein was then purified by divalent cation affinity chromatography using TALON resin (Merck), followed by size exclusion chromatography using a Superose 6 Increase 10 / 300 column connected to an AKTApure instrument (Cytiva). All proteins were concentrated using an Amicon centrifugal filter unit. The protein solutions were sterilized by filtration through a 0.45 μm nitrocellulose filter and aliquots were stored at -80°C. The buffer was phosphate-buffered saline at pH 7.4. Protein purity was assessed by SDS-PAGE and SEC. SDS-PAGE was performed using NuPAGE Bis-Tris and Tris-acetate pre-prepared gels and an XCell SureLock Mini-Cell electrophoresis system (Thermo Fisher) according to the manufacturer's recommendations. Pre-stained Precision plus protein standards (Bio-Rad) were used as molecular weight markers.

[0441] Recombinant spike ligand. hACE2-Fc is a recombinant fusion protein containing amino acids 19 through 615 of the extracellular domain of human ACE2 linked to the Fc domain of human IgG1 via a GS linker, and has been previously described (Poumbourios et al., 2023). Recombinant mNAb. As described in Poumbourios et al., 2023, an expression vector (Center et al., 2020) based on pCDNA3 of human IgG1 heavy chain and κ and λ light chains is generated internally, containing variable regions of mNAb S2H97 (Starr et al., 2021), Omi-18 and Omi-42 (Nutalai et al., 2022), SP1-77 (Luo et al., 2022), C1520 (Wang et al., 2022), COV44-79 (Dacon et al., 2022), and CV3-25 (Jennewein et al., 2021) targeting SARS CoV-2.

[0442] Differential scanning fluorescence (DSF) was used to assess the thermal stability of proteins (Niesen et al., 2007). Protein (10 µg) was diluted twice to 25 µL with 5× SYPRO orange protein gel staining agent (Sigma Aldrich). The sample was then heated from 25 °C to 95 °C in 0.5 °C increments, 1 minute per increment. Fluorescence measurements were performed at the end of each increment. The excitation wavelength was 492 nm, and the emission wavelength was 610 nm. Tm was determined as the minimum of the negative first derivative of the melting curve.

[0443] Biolayer interferometry. BLI-based measurements were determined using the OctetRED96 system (ForteBio, Fremont CA). Antibodies were diluted to 10 μg / ml in kinetic buffer and immobilized on an anti-human IgG Fc capture biosensor (AHC, ForteBio). Kinetic assays were performed at 30°C using standard kinetic acquisition rate settings (5.0 Hz, average 20) and a sample plate shaking speed of 1,000 rpm. The kinetic experiments consisted of five steps: (a) baseline (180 s); (b) antibody loading (300 s); (c) second baseline (180 s); (d) antigen association (300 s); and (e) antigen dissociation (300 s). Fitted curves were constructed using a 1:1 binding model using ForteBio Data Analysis 10.0 software and corrected using a dual-reference subtraction method.

[0444] Western blot analysis of the S2P-1273 glycoprotein expressed in 293T cells. 293T cells were transfected with the expression vector using a FUGENE HD (Promega) membrane according to the manufacturer's instructions. Forty-eight hours post-transfection, cells were washed with ice-cold PBS, centrifuged at 10,000 rpm for 90 seconds, and the precipitate was lysed on ice in lysis buffer (1% Triton X100 in PBS containing 1 mM EDTA) for 30 minutes. The lysate was clarified by centrifugation at 10,000 rpm for 10 minutes at 4°C and subjected to SDS-PAGE with or without 3% β-mercaptoethanol. Proteins were transferred to nitrocellulose using the iBLOT2 system (ThermoFisher), and the membrane was blocked with 5% skim milk powder in PBS. The filter was probed with rabbit anti-S1 polyclonal antibody (Sino Biological) and anti-rabbit IRDye800CW (Odyssey). The filter is then scanned in the LI-CORE imager.

[0445] Flow cytometry. 293T cells were transfected using FUGENE 6 (Promega) with S2P.BA45.VI-1273, D17.VI-1273, and I1.VI-1273 expression vectors along with the EGFP expression vector (EGFP-N1) according to the manufacturer's instructions. Forty-eight hours post-transfection, cells were washed with PBS and subsequently isolated using versene solution. Cells were resuspended in 1000 μl PBS and stained for 30 min at room temperature with a 1:1000 dilution of LIVE / DEAD Red Dead Cell (Invitrogen) staining agent. Cells were washed twice by centrifugation at 400 × g for 5 min in ice-cold FACS buffer (5% v / v fetal bovine serum in PBS containing 2 mM EDTA). Cells were resuspended in 800 µl of FACS buffer, added to 96-well U-bottom plates, and incubated for 1 hour at room temperature with 5 µg / ml ACE2-Fc and human mNAb in FACS buffer. Cells were washed twice in ice-cold FACS buffer by centrifugation at 400 ×g for 5 min. Cells were then incubated with Alexa Fluor 647 goat anti-human (H+L) (Invitrogen) in the dark at room temperature for 30 min. Cells were washed twice in ice-cold FACS buffer by centrifugation at 400 ×g for 5 min, resuspended in 100 μl of FACS buffer, and immediately used in a Canto II flow cytometer. Ten thousand events were captured for each antibody-S2P.BA45.VI-1273 variant combination. FlowJo software was used for data analysis. The viable 293T cell population was first gated by forward and side scattering, and single cells were selected for analysis after doublet discrimination. Analyze the fluorescence intensity of EGFP / S2P-1273 glycoprotein double-positive cells.

[0446] S6P-1192 glycoprotein expression vector. Using a synthetic "six-Pro (or 6P) converter" gene fragment generated by GenSCRIPT, six-Pro mutations: F817P, A892P, A899P, A942P, V986P, K987P (Hsieh et al., (2020)) were introduced into the S2P.BA45-1192 expression vector. Two converters were used: the 'six-Pro converter BsrGI-EcoRV' (SEQ ID NO: 143) targeting the S6P.BA45-1192 protein lacking the D17 and I1 mutations. And the 'six-Pro S967C converter BsrGI-EcoRV' (SEQ ID NO: 144) targeting the S6P.BA45-1192 protein containing D17 and I1 mutations. . The converter was attached to the BsrGI and EcoRV restriction sites of the appropriate S2P.BA45-1192 vector.

[0447] For the spikes of omicron BA.2.86 and clade 3 bat sabevirus PRD-0038, the synthetic gene corresponding to amino acids 16 to 1192 of the ancestral Hu-1 reference isolate was obtained from Genscript. The synthetic gene encodes a six-Pro mutation, a furin cleavage site mutation: R681RRAR->P681GSAS for omicron BA.2.86 (PRD-0038 S lacks a furin cleavage site), and in some cases, a VI mutation: A1016V / A1020I (Poumbourios et al., 2023). The C-terminus of the S6P-1192 spike is linked to a Gly-Ser linker and a six-His affinity tag. The synthetic gene is ligated downstream of the DNA sequence encoding the tissue plasminogen activator precursor within pcDNA3 (Invitrogen) via NheI. Cysteine ​​substitution mutations were introduced into the S6P-1192 expression vector using a synthetic gene fragment encoding paired Cys substitution mutations corresponding to D17 (D571C / S967C) and I1 (A570C / S967C).

[0448] The S6P.BA45-1273 glycoprotein expression vector was created by replacing the NotI-EcoRV restriction fragment of the S2P.BA45-1273 expression vector with the corresponding gene fragments encoding the double Cys, furin protease sites, and six Pro mutations from the appropriate S6P.BA45-1192 expression vector.

[0449] Other recombinant monoclonal antibodies, such as those described in Poumbourios et al., 2023, internally generate human IgG1 heavy chain and κ and λ light chain expression vectors based on pCDNA3 (Center et al., 2020), which contain variable regions targeting SARS CoV-2 mNAb SA55 (Cao et al., 2020), S309 (Shang et al. (2020)), CC95-108, CC99-103 (Zhou et al., 2023), CR3022 (terMeulen et al., 2006), S2P6 (Pinto et al., 2020), and CC40.8 (Zhou et al., 2022).

[0450] RBD expression vector. Synthetic genes encoding the receptor-binding domain (RBD; amino acids 332-532) of the ancestral isolates Hu-1, Omicron BA.5, XBB.1.5, and JN.1 were obtained from GeneART-ThermoFisher Scientific or GenSCRIPT and ligated into tissue plasminogen activator precursors in pcDNA3 via NheI. These genes encode C-terminal six His tags and Avitag sequences.

[0451] NTD expression vector. The gene fragment encoding amino acids 16 to 305 corresponding to the S-terminal domain was amplified by polymerase chain reaction. The templates were S2P.16L-FHA, encoding the ancestral Hu-1 sequence (Poumbourios et al., 2023); S6P.BA45.AA-1192 and S6P.BA286.AA-1192. Notably, BA.2.86 and JN.1 NTD are identical. The forward primer was CMVf (SEQ ID NO: 145). The reverse primer is: CoV2 NTD 3'_A (SEQ ID NO: 146) , CoV2 NTD 3'_B (SEQ IDNO: 147) And CoV2 NTD 3'_C (SEQ ID NO: 148) The NTD fragment is joined to the six His and Avitag sequences at the 3' end.

[0452] Maltose-binding protein-stem chimeric protein, MBP-stem (positions 1138-1208). The gene fragment encoding the S-stem region (amino acids 1138-1208) was amplified by polymerase chain reaction. The template was S2P-FHA, which encodes the sequence derived from the ancestral Hu-1 spike (Poumbourios et al., 2023), and the primers were WuStemY1138NotIforw (SEQ ID NO: 149). And WuSTEMQ1208SalIRev (SEQ ID NO: 150) The DNA fragment was ligated to the NotI-SalI site (positions 522-654) of MBP / gp41 (Lay et al., 2004). MBP-stem (positions 1138-1208) expression was induced in *E. coli* BL21 cells at 24 °C for 18 h using 100 mM isopropyl β-d-1-thiogalactopyranoside. Cells were then pelleted, subjected to freeze-thaw cycles (-80 °C), and disrupted by acoustic treatment in 300 mM NaCl / 100 mM Tris.HCl pH8 / 1 mM EDTA / 1 mM phenylmethylsulfonyl fluoride (S buffer). The clarified supernatant was then affinity purified by elution onto amylose agarose using 10 mM maltose in S buffer, followed by Superose 12 SEC.

[0453] SFur BA45 expression vector. The wild-type omicron BA.4 / 5 spike expression vector (SFur BA45) is derived from S2P.BA45-1273 after the wild-type spike sequence was restored using standard molecular biology techniques.

[0454] Biolayer interferometry. BLI-based measurements were determined using an OctetRED96 system (ForteBio, Fremont CA). Antibodies were diluted to 10 μg / ml in kinetic buffer and immobilized on an anti-human IgG Fc capture biosensor (AHC, Sartorius). Kinetic assays were performed at 30°C using standard kinetic acquisition rate settings (5.0 Hz, average 20) with a sample plate shaking speed of 1,000 rpm. The kinetic experiments consisted of five steps: (a) baseline (180 s); (b) antibody loading (300 s); (c) second baseline (180 s); (d) antigen association (300 s); and (e) antigen dissociation (300 s). Fitted curves were constructed using a 1:1 binding model using Octet Analysis Studio 13.0.3.52 software and corrected using a dual-reference subtraction method.

[0455] Immunization and viral challenge in K18hACE2 mice. Sixteen 8- to 10-week-old female B6.Cg-Tg(K18-ACE2)2Priman (K18hACE2) mice were immunized at weeks 0, 3, and 6 via subcutaneous intrascapular injection of 10 μg (100 μL) of S6P.BA45-1192 protein mixed with AddaVax adjuvant (InvivoGen, San Diego, CA) at a 1:1 (v / v) ratio. The negative control group was immunized as described above with a 1:1 (v / v) mixture of PBS and adjuvant. Serological analysis was performed on 10% of total blood volume mandibular blood collected one day before the final booster and seven days before viral challenge. Additional terminal blood collection was performed on four mice from each treatment group at the same time as the final blood collection to ensure sufficient sample collection. Serum was stored at -20°C and heat-inactivated at 56°C for 30 minutes prior to immunological assays. Two weeks after the second booster, all mice were lightly anesthetized with 4% v / v isoflurane and administered 10% isoflurane in 50 µL PBS. 4 Intranasal infection was performed using SARS-CoV-2 Omicron BA.5 (hCoV-19 / Australia / VIC61194 / 2022; GISAID: EPI_ISL_13276063) with TCID50. A group of four mice was treated with 50 µL PBS as a simulated challenge. Four days post-infection, eight mice in each group were cull by CO2 asphyxiation, and lung and nasal turbinate tissues were collected in PBS containing penicillin / streptomycin and amphotericin B. Lung and nasal turbinate tissues were homogenized in 2 mL and 1 mL PBS, respectively. Vero-TMPRSS2 cells were infected to determine the tissue culture infection dose (TCID50 / ml). Titration was performed using Vero-TMPRSS2 cells in 24-well and 96-well plates. Cells were infected in quadruplicate. Cells were incubated at 37°C and 5% CO2 for 5 days. After incubation, wells exhibiting cytopathic effects were recorded, and the viral titer (TCID50 / mL) of each sample was determined by limiting dilution using the Reed and Muench method.

[0456] Real-world virus neutralization assay. The neutralizing activity of serum against real ancestral hCoV-19 / Australia / NSW2715 / 2020, Omicron BA.5, XBB.1.5, and JN.1 SARS-CoV-2 was determined using a rapid high-content SARS-CoV-2 microneutralization assay described by Aggarwal et al. 2022. Briefly, Hoechst-33342-stained HAT-24 cells were seeded in 384-well plates (Corning, CLS3985). Serially diluted heat-inactivated vaccine serum was co-incubated with an equal volume of SARS-CoV-2 virus solution at twice the moderately lethal dose at 37°C for 1 hour. 40 μl of the serum-virus mixture was added to an equal volume of pre-plate-seeded cells, incubated for 20 hours, and subsequently imaged directly on an ImageXpress Pico automated cell imaging system (Molecular Devices). Cell nucleus counts were obtained using CellReporterXpress image acquisition and analysis software (MolecularDevices), and the percentage of virus neutralization was calculated as described in Aggarwal et al. 2022. Neutralization ID 50 It is the final continuous dilution that achieves ≥ 50% neutralization.

[0457] Chemiluminescent immunoassay (ELISA). Nunc Maxisorp 384-well white plates were coated overnight at 4°C with 1 μg / ml of S glycoprotein, NTD protein, RBD protein, stem protein, or synthetic peptide. The plates were washed with PBS and blocked with BSA (10 mg / ml, PBS) for 1 hour at room temperature. The plates were washed again and incubated with serially diluted serum samples or mNAb for 2 hours at room temperature. Antibody binding was detected using horseradish peroxidase-labeled rabbit anti-guinea pig antibodies (Dako, Glostrup, Denmark). The light signal was detected using the SuperSignal ELISA Pico chemiluminescent substrate (ThermoFisher Scientific) and measured immediately for 0.5 seconds using CLARIOstar (BMG Lab Technologies). Relative light units (RLU) were plotted against the reciprocal of relative dilutions in GraphPad Prism 10.1.0, and curve fitting was performed using specific binding with a Hill slope. The binding titer is defined as the reciprocal of the serum dilution that brings RLU to the background (as defined by binding to BSA).

[0458] Flow cytometry. 293T cells were transfected using FUGENE 6 (Promega) with the S6P.BA451273 expression vector and the EGFP expression vector (EGFP-N1) according to the manufacturer's instructions. Forty-eight hours post-transfection, cells were washed with PBS and subsequently isolated using versene solution. Cells were resuspended in 1000 μl PBS and stained for 30 min at room temperature with a 1:1000 dilution of LIVE / DEAD Red Dead Cell (Invitrogen) staining agent. Cells were washed twice by centrifugation at 400 × g for 5 min in ice-cold FACS buffer (5% v / v fetal bovine serum in PBS containing 2 mM EDTA). Cells were resuspended in 800 µl FACS buffer, added to 96-well V-bottom plates, and incubated for 1 h at room temperature with 5 µg / ml ACE2-Fc and human mNAb in FACS buffer. Cells were washed twice in ice-cold FACS buffer by centrifugation at 400 × g for 5 min. Cells were then incubated with Alexa Fluor 647 goat anti-human (H+L) (Invitrogen) in the dark at room temperature for 30 min. Cells were washed twice in ice-cold FACS buffer by centrifugation at 400 × g for 5 min, resuspended in 100 μl FACS buffer, and immediately used in a Canto II flow cytometer. Ten thousand events were captured for each antibody-S2P.BA45.VI-1273 variant combination. FlowJo software was used for data analysis. Viable 293T cell populations were first gated by forward and side scattering, and single cells were selected for analysis after duplex identification. The fluorescence intensity of EGFP / S2P-1273 glycoprotein double-positive cells was analyzed.

[0459] Generation and analysis of pseudotyped viruses. S-pseudotyped HIV luciferase reporter virus was prepared according to the method of Jackson et al., 2020. The plasmids used to generate S-HIV pseudotyped particles were a gift from Professor Doria-Rose of the NIH Vaccine Research Center and included the packaging plasmid pCMVΔR8.2 and the luciferase reporter plasmid pHR'CMV Luc (Naldini et al., 1996), as well as the TMPRSS2 plasmid (Böttcher et al., 2006). Together with the S-expression plasmid, the three plasmids were co-transfected into HEK293T cells. After 18 hours of incubation, the medium was replaced with fresh Dulbecco modified minimum essential medium (DMF10) containing 10% fetal bovine serum, and the cells were cultured for another 2 days. The supernatant containing the pseudotyped virus was filtered through a 0.45 μm membrane filter before use. Three days after seeding 293-ACE2 cells (10,000 cells / well) into poly-L-lysine-coated 96-well plates, the infectivity of filtered supernatant containing pseudomorphic particles was determined. Luteinase activity was measured using a Promega luciferase assay system in a Clariostar microplate reader (BMG Labtech).

[0460] mRNA transfection. Using Liposome MessengerMAX (Thermo Fisher Scientific), mRNAs encoding multiple spike constructs were transfected into Expi293F (soluble spike glycoprotein) or 293T cells (membrane-anchored spike glycoprotein) according to the manufacturer's recommendations.

[0461] Example 2 - Identification of amino acid sequence determinants controlling the thermal stability and yield of the S2P.BA45 trimer within the S2 stem

[0462] The basic S-glycoprotein construct used here to identify the amino acid sequence determinants controlling the biophysical properties of the S-trimer within the S2 stem is S2P.omiBA45.VI-1208.H8 (referred to herein as S2P.BA45.VI.1208), as described in (Poumbourios et al., 2023). This protein is generated by a CMV promoter-driven vector expressing residues 16 through 1208 of the omicron BA.4 S-glycoprotein (omicron BA.4 and omicron BA.5 sequences are identical (Tegally et al., 2022)); a '2P' mutation—a double Pro substitution at positions 986 and 987—which maintains the S-trimer in its pre-fusion conformation; and a furin cleavage site mutation (H681RRAR->P681GSAS) (Wrapp et al., 2020). The A1016V / A1020I 'VI' mutation was added to the CH coil-coil forming sequence of S2 (Poumbourios et al., 2023). A Gly-Ser-Gly-Ser linker and an eight-His or six-His tag were added to the C-terminal residue Q1208 of the extracellular domain. The N-terminal tissue plasminogen activator leader (tPAL) was linked to residue 16 via the Ala-Ser linker to enable secretion (…). Figure 2 B). See amino acid and DNA sequences respectively. Figure 3 and Figure 4 This construct is consistently referred to as S2P.BA45.VI-1208.

[0463] The expected 3D architecture model of S2P.BA45.VI-1208 is in Figure 5 The diagram is shown as follows: 1) Head (residues 16 to 1139), which contains the RBD, fusion peptide, and CH coiled helix introducing the VI mutation; 2) Stem (residues 1140 to 1208), which connects the extracellular domain to the transmembrane domain (TMD). A short coiled helix at the top of the stem is shown in cryo-electron microscopy (cryo-EM) structures (e.g., Wrapp et al., 2020). 3) The location and partial structure of the membrane anchor (Fu et al., 2021) are shown at the bottom. 4) Recombinant soluble spike proteins typically have a TMD replaced by a trimerizing clamp (e.g., T4 foldon) (also shown in Guthe et al., 2004) to stabilize the trimer.

[0464] To identify the amino acid sequence that determines the biochemical characteristics of S2P.BA45.VI, a group of C-terminal stem-truncation mutants were created. Figure 6 The sequence of the stem with key features and truncation point annotations is shown. The new C-terminus is:

[0465] • S1147, which includes a short, coiled spiral at the top of the stem observed in most cryo-electron microscopy (cryo-EM) structures of soluble S2P trimers (e.g., Wrapp et al., 2020); (referred to as S2P.BA45.VI-1147).

[0466] • K1157, which completes the hydrophobic repetition of short coiled spirals located at the top of the stem; (referred to as S2P.BA45.VI-1157).

[0467] • D1165, which includes an N-linked glycosylation site located at N1158; (referred to as S2P.BA45.VI-1165).

[0468] • N1192, which includes a hydrophobic repeat of the N-linked glycosylation site located at N1194; (referred to as S2P.BA45.VI-1192).

[0469] • D1199, which includes an N-linked glycosylation site located at N1194; (referred to as S2P.BA45.VI-1199).

[0470] • L1200, Q1201, and G1204, which are hydrophobically repeated to the C-terminal residue Q1208 of the extracellular domain. (Referred to as S2P.BA45.VI-1200, S2P.BA45.VI-1201, and S2P.BA45.VI-1204, respectively).

[0471] The amino acid and DNA sequences are respectively in Figure 7 and 8 As shown in the image.

[0472] The S2P.BA45.VI protein was expressed in Expi293F cells and partially purified from the culture supernatant by TALON affinity chromatography. Superose 6 size exclusion chromatography (SEC) was used to reveal the size characteristics of the secreted protein. A thyroglobulin (669 kDa) molecular weight standard was used to label the location of the S2P trimer (Wrapp et al., 2020).

[0473] The SEC filing for S2P.BA45.VI-1208 revealed heterogeneity in the protein formulation, with putative trimers comprising <20% of the total affinity-purified protein. Figure 9A). C-terminus truncation to G1204, Q1201, and L1200 resulted in significant putative trimer peaks co-eluting with the 669 kDa label in many low molecular weight compounds. Fractions consistent with the expected size of the S2P.BA45.VI trimer were collected, concentrated, and re-analyzed on a Superose 6 after freeze-thaw cycles (-80°C). Figure 9 B indicates a purity ≥ 90% and the putative trimer is stable. The melting temperature (Tm) of the purified trimer was determined to be 60°C to 61°C by differential scanning fluorometry (DSF). Figure 9 C).

[0474] Further truncation to D1199, N1192, D1165, K1157, and S1147 produced the main peaks co-eluted with the 440 kDa label. Figure 9 A), which was purified to homogeneity ( Figure 9 B). The elution positions of these truncated S2P.BA45.VI substances indicate that their molecular weight is lower than that of S2P.BA45.VI-1208. Protein elution is determined by its hydrodynamic radius or Stokes radius (La Verde et al., 2017). Therefore, the Stokes radii of S2P.BA45.VI-1147, -1157, -1165, and -1192 may be smaller than those of S2P.BA45.VI-1208, -1204, -1201, and -1200 due to the truncation of most of the stem (see B). Figure 5 DSF indicates that the Tm of these shorter S2P.BA45.VI proteins is 38.4℃ to 40℃. Figure 9 C), indicating that its thermal stability is lower than that of the longer-stemmed counterpart with a Tm of 60 to 61 °C. Despite this reduced thermal stability, the putative trimer is stable after freeze (-80 °C)-thaw cycles. The yield and elution position of the purified putative trimer and its Tm are shown in [the table / description]. Figure 9 As shown in D. SDS-PAGE under both reducing and non-reducing conditions showed that all constructs exhibited distinct bands at approximately 160 to 170 kDa, with a purity > 95%. Figure 10 ).

[0475] The graph showing Tm and trimer yield as a function of C-terminal length indicates that L1200 is the key determinant of thermal stability. However, the thermally stable trimer yield is low. Conversely, truncation to N1192, D1165, and K1157 results in high-yield trimers, but with lower thermal stability. Figure 11 ).

[0476] Example 3 – Epitope profile of neutralizing antibody (NAb) with truncated stem S2P.BA45.VI trimer

[0477] Next, the antigenic structure of the S2P.BA45.VI stem truncated protein was detected by biolayer interferometry using ACE2-Fc and human monoclonal NAb (mNAb). Figure 12 The human mNAb studied included C1520 targeting NTDs (Wang et al., 2022), Omi-18 and Omi-42 targeting RBMs (Nutalai et al., 2022), S2H97 (Starr et al., 2021) and SP1-77 (Luo et al., 2022) targeting conserved epitopes within RBDs other than RBMs, COV44-79 targeting fusion peptides (Dacon et al., 2022), and CV3-25 targeting S2 stems (Jennewein et al., 2021 and Li et al., 2022). ACE2-Fc and human mNAb were attached to an anti-human IgG Fc capture biosensor, while the S2P glycoprotein was in the analyte phase. Figure 12 The sensing plots shown are related to a 1:1 bimolecular interaction model, with R values ​​for all but one case. 2 The values ​​are all > 0.93 and Value < 2 (Table 1). Combined with the response at binding equilibrium (R... eq The figure provides a summary of the interactions between the S ligand and the purified S2P.BA45.VI stem-truncation mutant oligomers. Figure 13 All mutants achieved roughly equivalent binding to NAb C1520 targeting the NTD, while proteins terminating at 1157, 1165, and 1192 bound to the RBM-targeting ligand R. eq The values ​​were slightly higher than those of proteins terminating at 1199, 1201, and 1204. A similar trend was observed for RBD / non-RBM specific Nab S2H97 and SP1-77. Poor overall binding to the fusion peptide NAb COV44-79 was observed, indicating that this epitope is difficult to access. Maximum binding was observed between S2P.BA45.VI-1192 and NAb CV3-25, whose epitope covers the stem amino acids K1147 to D1165. Binding kinetic studies (Table 3) showed relatively high affinity interactions between all spike proteins and ligands (except COV44-79), with KD affinity constants ≤ 6.7 × 10⁻⁶. -9M.S2P.BA45.VI-1192 exhibited strong binding to all ligands, and among all spike constructs, it achieved the highest R-value against stem NAb CV3-25, which has pan-SARS-CoV-2 variant neutralizing properties. eq .

[0478] Table 3. Binding kinetics of S spike trimer with ACE2-Fc and human monoclonal Nab.

[0479]

[0480] Example 4 - Structure-directed cysteine ​​substitution mutagenesis to covalently stabilize the S2P.BA45.VI trimer

[0481] A complementary approach was used to stabilize the S2P.BA45.VI extracellular domain trimer by introducing cysteine ​​pairs into the subunit interface to generate intermolecular disulfide bonds. To this end, S1-S1, S1-S2, and S2-S2 interface residues in the SARS-CoV-2 S trimer (PDB ID: 6VSB) were identified using the LPC-CSU server (http: / / oca.weizmann.ac.il / oca-bin / lpccsu) for Cys substitution mutagenesis. Small polar residues (e.g., Ser, Thr, Asn, and Asp) that are not glycosylated sequences or salt bridge components, as well as glycine and alanine, were targets for Cys substitution, as there is reason to believe that such side-chain substitutions are unlikely to affect the overall folding of the spike glycoprotein trimer. Eleven contact residue pairs were identified for Cys substitution mutagenesis of S2P.BA45.VI-1147, the contact residue pairs having C α The atoms are spaced approximately 4.2 to 6.6 Å apart, representing the C atoms of the Cys residues involved in the disulfide bond. α The ideal distance between atoms (Reiter et al., 1995 (Table 3); Figure 14 The amino acid and DNA sequences of the S2P.BA45.VI-1147 Cys mutant are respectively located in... Figure 15 and 16 As shown in the image.

[0482] Example 4 - Biochemical characteristics of the S2P.BA45.VI-1147 Cys substitution mutant

[0483] The S2P.BA45.VI-1147 Cys mutant was expressed in Expi293F cells (50 ml culture) and affinity purified from the culture supernatant by divalent cation affinity chromatography as described above. Superose 6 SEC showed that only 3 of the 11 double Cys mutants were efficiently affinity purified, indicating that most mutations introduce folding defects that inhibit expression and / or secretion. Figure 17 A). Therefore, D17 (D571C / S967C), I1 (A570C / S967C), and L23 (N914C / S1123C) are mainly produced as trimer proteins, with the last one existing almost entirely as a trimer. Figure 17 A and B). D17 and I1 increased the melting temperature of S2P.BA45.VI-1147 from 38.5℃ to 55.5℃ and 51.5℃, respectively. L23 caused a moderate increase in the melting temperature from 38.5℃ to 44℃. Figure 17 C). Importantly, relative to the parental S2P.BA45.VI-1147 construct, the D17, I1, and L23 mutations were associated with 11-fold, 8.75-fold, and 17.5-fold increases in trimer yield, respectively. Figure 17 D).

[0484] Non-reducing and reducing SDS-PAGE were used to determine whether inter-monomer disulfide bonds were formed in the three mutants. Under non-reducing conditions, S2P.BA45.VI-1147 migrated to its expected monomer molecular weight (approximately 150 kDa), while D17 and I1 migrated to positions consistent with higher molecular weights; L23 migrated to an intermediate position (…). Figure 18 Under reducing conditions, all proteins decompose into their monomeric molecular weights. This is because the D17, I1, and L23 disulfide bonds are subject to the C-forces imposed on them by their design. α Limited by distance and geometric constraints, it theoretically forms only in the context of trimer spikes (Table 3 and...). Figure 14 Therefore, it can be deduced that D17 and I1 crosslink each monomer in the trimer, while L23 crosslinks 2 monomers and 1 monomer remains unbound.

[0485] Example 5 - Mutant covalent crosslinking of S2P.BA45.VI-1192 and S2P.BA45.VI-1204 trimers by I1 (A570C / S967C) and D17 (D571C / S967C)

[0486] The D17, I1, and L23 mutations were introduced into S2P.BA45.VI-1192 (yielding D17.VI-1192, I1.VI-1192, and L23.VI-1192, respectively) and S2P.BA45.VI-1204 (yielding D17.VI-1204, I1.VI-1204, and L23.VI-1204, respectively). S2P.BA45.VI-1192 is a putative trimer with a high yield and a low Tm (39.5 °C), while S2P.BA45.VI-1204 is a putative trimer with a low yield and a high Tm (60.5 °C) (see [link to relevant documentation]). Figure 11 Importantly, unlike S2P.BA45.VI-1147, these longer constructs contain highly conserved neutralizing epitopes within the stem, recognized by mNAb CV3-25. This experiment was performed to determine whether these constructs with longer stems possess a suitable trimer geometry that allows for the formation of I1, D17, and L23 disulfide bonds. The protein and DNA sequences of these constructs are respectively... Figure 19 and 20 The spike mutant was expressed in Expi-293F cells and purified from the supernatant by divalent cation affinity chromatography using TALON resin followed by Superose 6 SEC. Figure 21 A shows that the affinity-purified D17.VI-1192 and I1.VI-1192 proteins consisted primarily of a single substance co-eluted with the parental S2P.BA45.VI-1192 and a 440 kDa label. DSF shows that the Tm (55℃ and 51℃, respectively) of the purified D17.VI-1192 and I1.VI-1192 proteins were significantly higher than those of the parental S2P.BA45.VI-1192 (40℃). Figure 21 B). The D17 and I1 disulfide bonds increased the yield of the S2P.BA45.VI-1192 trimer by 2.3-fold and 1.7-fold, respectively. SDS-PAGE under non-reducing conditions showed that the bands for both mutants were >250 kDa, while under reducing conditions, the bands dissociated to the expected monomer molecular weight of approximately 170 kDa, with some residual oligomers remaining in I1.VI-1192. Figure 21 C). Compared to I1 and D17, the L23 (N914C / S1123C) mutation is intolerant in the S2P.BA45.VI-1192 background and secretes very little L23.VI-1192 protein.

[0487] The I1, D17, and L23 mutations performed relatively poorly in the thermostable S2P.BA45.VI-1204, and produced lower putative trimer yields. Figure 21D). The purified D17.VI-1204 and I1.VI-1204 trimers showed almost identical Tm values ​​(5.15 °C and 55 °C, respectively) to their 1192 counterparts. Figure 21 E), and non-reducing SDS-PAGE indicates that intermolecular disulfide bonds have been formed (E), and non-reducing SDS-PAGE shows that intermolecular disulfide bonds have been formed ( Figure 21 F). As for S2P.BA45-1192, the L23 mutation is intolerant in the S2P.BA45.VI-1204 background, and very little L23-1204 protein is secreted.

[0488] These data indicate that: 1) S2P.BA45.VI-1192 and S2P.BA45.VI-1204 acquire a trimer quaternary structure, enabling the formation of I1 and D17 disulfide bonds; 2) I1 and D17 mutations confer thermal stability to the S2P.BA45.VI-1192 trimer and improve its yield; 3) I1 and D17 do not improve the biophysical properties of longer S2P.BA45.VI-1204 proteins; 4) I1 and D17 disulfide bonds show that the Tm determines both the low-thermal-stability and high-thermal-stability S2P.BA45.VI trimers; 5) The L23 mutation is not accommodated in S2P.BA45.VI constructs with C-terminal stem extensions beyond S1147 to N1192 and G1204.

[0489] Example 6 - NAb epitope spectrum of covalently linked D17-1192 and I1-1192 trimers

[0490] The presentation of a wide range of NAb epitopes in the D17-1192 and I1-1192 trimers was investigated in BLI using the following ligands: ACE2-Fc, Omi-42, and Omi-18 for RBM, S2H92 and SP1-77 for RBD (excluding RBM), and CV3-25 for stem. The 3D structures of complexes between RBD and the extracellular domain of ACE2 (PDB ID: 6VW1) (Shang et al., 2020) or with Fabs derived from Omi-18 (PDB ID: 7ZFB), Omi-42 (PDB ID: 7ZR7) (Nutalai et al., 2022), S2H97 (PDB ID: 7M7W) (Starr et al., 2021), and SP1-77 (PDB ID: 7UPX) (Altman et al., 2021), or the 3D structures of complexes between Fabs derived from CV3-25 and the stem region of the trimer (Li et al., 2022). Figure 21 As shown on the left.

[0491] Notably, ACE2 ECD, Omi-18, and Omi-42 exhibited a similar binding pattern to RBM (ACE2 contact residues are shown as black spheres) at the top of the RBD. S2P.BA45.VI-1192 showed robust binding to ACE2-Fc, Omi-18, and Omi-42, while D17-1192 bound poorly to the three RBM-targeting ligands. The data suggest that D17 likely occludes RBM, a major site of immune escape in VOCs, by inducing a 3-RBD downconformation in the spike trimer. I1 partially occludes RBM, blocking its interaction with ACE2, but not RBM-dependent epitopes located at the “shoulder” and “back” of the RBD (Nutalai et al., 2022), as shown by Omi-42 and Omi-18, respectively. Next, two Nab ligands with pan-VOC neutralizing activity (S2H97 and SP1-77) were investigated. Both epitopes are conserved and located “below” the RBM on the opposite flank of the RBD. S2H97 and SP1-77 bind with similar potency to WT and the two mutants, indicating that exposure to these two epitopes is unaffected by the I1 and D17 mutations. Compared to the parental S2P.BA45.VI-1192, the pangeny neutralizing stem Nab (CV3-25) exhibits a significantly reduced shut-off rate when binding to the two mutants.

[0492] Table 4 shows the binding kinetics between D17-1192 and I1-1192 with various S-ligands. In all cases except for the interaction between D17-1192 and ACE2-Fc, Omi-18, and Omi42, the following conditions are observed. Figure 22 The sensing plots shown are related to a 1:1 bimolecular interaction model, where Ri is the largest for all but one case. 2 Value > 0.96 and Value < 1.65. Strong interactions between all spike proteins and ligands are defined by an affinity constant (KD) ≤ 6 × 10⁻⁶. -9 M is used to define the mutation. The significant role of D17 and I1 mutations is the imperceptible dissociation rate of CV3-25, which targets the stem and has neutralizing properties against pan-SARS-CoV-2 variants.

[0493] Table 4. Binding kinetics of S2P.BA45.VI-1192 spike trimer with ACE2-Fc and human monoclonal NAb.

[0494]

[0495] Therefore, D17 and I1 represent potential methods for modulating exposure to the highly immunogenic but also highly variable RBM and for refocusing antibody responses on other conserved epitopes within the RBM. Furthermore, the I1 and D17 disulfide bonds at the top of the head domain may stabilize the CV3-25 NAb-stem interaction at the base of the trimer via an allosteric mechanism. In an immunization setting, the latter property of D17 and I1 could theoretically lead to a sustained interaction between the stem and the CV3-25-like B cell receptor to induce high-affinity NAbs targeting this highly conserved site.

[0496] Example 7 - The VI mutation is not essential for the thermal stability of the S2P.BA45-1192 trimer ± D17 and I1 mutations.

[0497] Previous studies on S2P.BA45-1208 have shown that the VI mutation is associated with improved thermal stability and is essential for maintaining the trimer structure (Poumbourios et al., 2023). To determine whether the VI mutation is also required to maintain the S2P.BA45-1192 trimer, V1016 and I1020 were restored to native Ala residues to obtain S2P.BA45.AA-1192. The protein and DNA sequences of these constructs are as follows: Figure 23 and 24 As shown in the figure. Divalent cation affinity chromatography (TALON) of S2P.BA45.AA-1192 followed by SEC produced a major putative trimer peak co-eluted with a 440 kDa label, which was purified to homogeneity. Figure 25 A). The S2P.BA45.AA-1192 trimer is stable after a freeze-thaw cycle (-80°C). Figure 25 B), and the DSF indicates Tm is 43℃ ​​( Figure 25 C), close to the Tm (41℃) of S2P.BA45.VI-1192.

[0498] The D17 and I1 mutations were introduced into S2P.BA45.AA-1192 to obtain D17.AA-1192 and I1.AA-1192, respectively. The protein and DNA sequences of these constructs are shown in [the original text]. Figure 23 and 24 The results are shown in the figure. Divalent cation affinity chromatography (TALON) of D17.AA-1192 and I1.AA-1192 followed by SEC yielded the major putative trimer peak co-eluted with the 440 kDa labels of both proteins. Figure 25 A). Both proteins were purified to homogeneity. Figure 25 B). The Tm values ​​of the purified D17.AA-1192 and I1.AA-1192 trimers were 55℃ and 52℃, respectively. Figure 25 C), which is almost identical to their VI counterparts. Both proteins are stable after freeze (-80°C)-thaw cycles. SDS-PAGE under non-reducing conditions showed that the bands for both mutants were >250 kDa, and that the bands dissociated into the expected monomeric molecular weight of approximately 170 kDa, with some residual oligomers remaining in I1.AA-1192. Figure 25 D).

[0499] To further investigate the relative contributions of disulfide bonds and VI mutations to the thermal stability of spike trimers, DSF was performed on D17-1192 and I1-1192 ± VI in the presence of increased β-mercaptoethanol concentration. Figure 26 The results show that the Tm of the VI and AA versions of S2P.D17-1192 decreases with increasing β-mercaptoethanol concentration, which is largely consistent with the data. The data again show that VI does not contribute to the stability of the S2P.D17-1192 and S2P.D17-1192 trimers.

[0500] Therefore, preserving the structural stability of the S2P.BA45-1192 trimer does not require a VI mutation, and both the D17 and I1 disulfide bonds can form and improve the thermal stability of S2P.BA45-1192 in the presence or absence of VI. The L23 mutation is compatible with VI-deficient S2P.BA45-1192; however, intermolecular disulfide bonds cannot form.

[0501] Example 8 - NAb epitope spectra of covalently linked I1-1192 and D17-1192 trimers with and without VI

[0502] BLI was used to compare the NAb epitope spectra of covalently linked S2P.I1-1192 and S2P.D17-1192 trimers with and without VI. ACE2-Fc and human mNAb were attached to the anti-human IgG Fc capture biosensor, while the S2P glycoprotein (30 nM) was in the analyte phase. Data showed that the S2P.BA45.VI-1192 and S2P.BA45.VI-1192 trimers had nearly identical binding affinity to the neutralizing ligands studied. Figure 27The D17.VI-1192 and D17.AA-1192 trimers bind poorly to RBM ligands (ACE2-Fc, Omi-18, and Omi-42), but strongly bind to NTD mNAb C1520, broadly neutralizing RBM mNAbs (S2H97 and SP1-77), and stem mNAb CV3-25. While S2P.I1.VI-1192 showed reduced binding to RBM ligands, the VI-AA reversion in S2P.I1.AA-1192 restored strong binding to Omi-18 and Omi-42. As for their D17 counterparts, both S2P.I1.VI-1192 and S2P.I1.AA-1192 strongly bind to C1520, S2H97, SP1-77, and CV3-25. These data indicate that the VI-AA reversion does not affect the NAb epitope profile of D17-1192, suggesting a 3RBD-down conformation. However, the VI-AA reversion in I1-1192 improves NAb binding to RBM, indicating a more open RBM conformation. Other conserved NAb epitopes studied also performed well in the four spike backgrounds. Therefore, five constructs were generated under different levels of RBM exposure: (S2P.BA45.VI-1192=S2P.BA45.VI-1192) > I1.AA-1192 > (I1.VI-1192=D17.AA-1192) > D17.VI-1192. The biochemical and antigenic characteristics of the S2P.BA45-1192 variant are summarized in Table 5.

[0503] Table 5. Summary of the biophysical and antigenic characteristics of the spike constructs

[0504]

[0505] a Determined by preparative SEC elution spectra

[0506] b Total yield of pure trimeric protein from 50 ml suspension culture of Expi293F cells

[0507] c Determined in DSF

[0508] d R @ 300 seconds: Response (nm) at the end of the association phase in BLI at 300 seconds.

[0509] n / a: Not applicable

[0510] Example 9 - Combined mutants of D17, I1 and L23

[0511] D17 and I1 were combined with L23 in S2P.BA45.AA-1192 and S2P.BA45.VI-1192, respectively, to determine whether combinatorial mutations could improve spike yield and thermal stability. The protein and DNA sequences of these constructs are shown in [details missing]. Figure 28 and 29 The results are shown in the figure. Divalent cation affinity chromatography (TALON) followed by SEC showed a difference in expression between DL.VI-1192 and IL.VI-1192, consistent with the incompatibility between L23 and S2P.BA45.VI-1192. Figure 30 A). In contrast, both DL.AA-1192 and LI.AA-1192 produced the main putative trimer peak (A). Figure 30 A), which can be purified to homogeneity ( Figure 30 B). The DL.AA-1192 and IL.AA-92 trimers are stable after freeze (-80°C) - thaw cycles. Figure 30 B), and the DSF indicated Tm of 56℃ and 52.4℃ respectively. Figure 30 C). Non-reducing SDS-PAGE showed that inter-monomer disulfide bonds were formed in the combinatorial mutants, with the same efficiency as D17 and I1 ( Figure 30 D). However, the yields of DL.AA-1192 and IL.AA-1192 trimers were lower than those of their D17 and I1 counterparts, respectively (Table 2).

[0512] Example 10 - The 2P mutation is not essential for the stability of the BA45-1192 trimer, but it is important for the trimer yield.

[0513] The '2P' mutation functions by stabilizing the spike trimer in the pre-fusion conformation by blocking the spontaneous transition of S2 to the fusion-activated hairpin trimer (Wrapp et al., 2020). To investigate the role of 2P in the expression and stability of the BA45.VI-1192 trimer and in the formation of D17 and I1 disulfide bonds, Pro986Pro987 was reverted to the native amino acids Lys and Val in D17.VI-1192, D17.AA-1192, I1.VI-1192, and I1.AA-1192, respectively, yielding SnoP.VI-1192, SnoP.D17.AA-1192, SnoP.I1.VI-1192, and SnoP.I1.AA-1192, respectively. The protein and DNA sequences of these constructs are shown in […]. Figure 31 and 32As shown in the figure. Divalent cation affinity chromatography (TALON) followed by SEC revealed the presence of spike trimers; however, for constructs containing VI, these trimers accounted for approximately 30% of the total secreted spike protein, and for constructs containing AA, they accounted for 53% to 58% of the total secreted spike protein. Figure 33 A). Purified trimer ( Figure 33 The Tm of B) is the same as its S2P-containing counterpart, but it is obtained in a significantly lower yield. Figure 33 C and D; Table 2). SDS-PAGE under non-reducing conditions revealed that the purified SnoP.VI-1192, SnoP.D17.AA-1192, SnoP.I1.VI-1192, and SnoP.I1.AA-1192 trimers migrated to single bands above 250 kDa, while SDS-PAGE under reducing conditions dissociated these bands into monomers with molecular weights of approximately 180 kDa. Figure 34 These data indicate that D17 and I1 disulfide bonds can form in the presence and absence of 2P and VI mutations.

[0514] In summary, the data indicate that VI and 2P mutations are not essential for trimer stability or the formation of D17 and I1 disulfide bonds, but 2P is essential for high trimer yield.

[0515] Example 11 - D17 and I1 mutations guide the formation of intermolecular disulfide bonds in full-length membrane-anchored S2P glycoproteins.

[0516] The ability of D17, I1, L23, DL, and IL disulfide mutants to covalently stabilize the full-length S2P.BA45.VI-1273 trimer containing the native transmembrane domain and cytoplasmic tail region was investigated. CMV-driven expression vectors containing codon-optimized genes encoding residues 1-1273 of the S glycoprotein derived from Omicron BA.4 / BA.5 were prepared. The vectors also included an H681RRAR->P681GSAS mutation at the furin protease site and double Pro "2P" substitutions at positions 986 and 987. The protein and DNA sequences of these constructs were obtained from [data missing]. Figure 35 and 36 As shown in the image.

[0517] To demonstrate the expression of the S2P.BA45-1273 glycoprotein and its cysteine ​​mutant, the DNA vector was transfected into 293T cells. Cells were lysed, and the lysates were subjected to SDS-PAGE and Western blotting with rabbit anti-S1 polyclonal antibody. Under non-reducing conditions, Figure 37The left panel shows the dominant approximately 180 kDa protein bands corresponding to the monomers observed in S2P.BA45-1273 and S2P.BA45.VI-1273, the latter containing the VI mutation. Addition of D17 and I1 to S2P.BA45.VI-1273 produced two major high-molecular-weight substances, likely corresponding to disulfide-linked trimers and dimers (dsl-spikes). These data suggest that the D17 and I1 disulfide bonds can covalently stabilize the full-length Omicron BA.4 / 5.VI oligomers. In contrast, the mutant containing L23 may be poorly expressed due to incompatibility of the L23, VI, and C-terminal stem sequences, as observed with the soluble S2P.BA45-1192 glycoprotein. However, two weak high-molecular-weight bands were observed, which are likely residual trimers and dimers resistant to sodium dodecyl sulfate plus thermal degradation. SDS-PAGE under reducing conditions (…) Figure 37 (See right figure) This results in doublets in all constructs, which migrate to locations close to the monomeric spikes. These doublets may be due to cleavage by contaminated proteases following spike mutation, or due to the presence of other glycoforms. Data indicate that disulfide bond mutations at D17 (D571C / S967C) and I1 (A570 / S967C) covalently stabilize the full-length S2P.BA45.VI-1273 oligomer.

[0518] Example 12 - Effects of D17 and I1 mutations on the NAb epitope profile of full-length membrane-anchored S2P glycoprotein

[0519] Flow cytometry, ACE2-Fc, and human monoclonal antibodies were used to investigate the role of D17 and I1 mutations in the presentation of key neutralizing epitopes against the background of full-length S2P.BA45.VI expressed on the cell surface. 293T cells were co-transfected with various S2P.BA45.VI-1273 expression vectors and EGFP expression vectors, and intact cells were stained with ACE2-Fc, human monoclonal NAb, and AlexaFluor-conjugated anti-human immunoglobulin. Cells were counterstained with LIVE / DEAD staining to exclude dead cells from the analysis. Figure 38 Histogram A shows the binding of S2P.BA45.VI-1273, D17.VI-1273, and I1.VI-1273 glycoproteins to ACE2-Fc and to all tested monoclonal NAbs. The isotype control HCV-specific antibody (HCV1) did not show binding. These data indicate that the S2P.BA45.VI-1273 variant glycoproteins are expressed on the cell surface and present distinct NAb epitopes.

[0520] Confirmed Figure 38 The geometric mean of the fluorescence intensity of the histogram shown in Figure A. Figure 38Data in B indicate that binding of D17.VI-1273 and I1.VI-1273 to ACE2-Fc, Omi-18 and Omi-42 (for RBM), C1520 (for NTD), and SP1-77 (for RBD flanks) is reduced, while binding to S2H97 (for RBD flanks, opposite to the SP1-77 epitope) and CV3-25 (stem) is similar across the three constructs. Therefore, in the context of full-length membrane anchoring, D17 and I1 mutations reduce exposure to bNAb epitopes involved in RBM, NTD, and RBD flanks, while epitopes identified by S2H97 and CV3-25 remain unaffected.

[0521] Example 13 - BA45-1192 spike protein containing hexapro (S6P)

[0522] Hsieh et al. (2020) identified six concurrent proline substitutions (F817P, A892P, A899P, A942P, V986P, K987P) within S2, which conferred higher expression and stability of the ancestral spike trimer containing the foldon trimer clamp. This mutation is termed hexapro (or S6P or 6P mutation). Therefore, the effects of hexapro on the expression, stability, and epitope profiles of the clipless omicronBA45-1192 spike and its disulfide-bonded D17 and I1 derivatives were investigated. Four hexapro derivatives were prepared: S6P.BA45.AA-1192, S6P.BA45.D17.AA-1192, S6P.BA45.I1.AA-1192, and S6P.BA45.I1.VI-1192, each a corresponding S2P protein described in Examples 5 and 7. The protein and DNA sequences of these constructs are respectively in Figure 39 and 40 As shown in the figure. Divalent cation affinity chromatography (TALON) of S6P.BA45.AA-1192 followed by SEC produced the main putative trimer peak co-eluted with the 440 kDa label and its S2P counterpart. Figure 41 A). The purified S6P.BA45.AA-1192 trimer was stable after freeze-thaw cycles (-80°C). Figure 41 B), and the DSF indicates Tm is 43℃ ​​( Figure 41 C). S6P.BA45.AA-1192 and its S2P counterpart are almost identical in biophysical characteristics, except that the former is expressed in higher yields: 86 mg / L and 36 mg / L.

[0523] The D17 and I1 disulfide mutants were converted to hexapro to obtain: S6P.BA45.D17.AA-1192, S6P.BA45.I1.AA-1192, and S6P.BA45.I1.VI-1192; the last one also includes a VI core cavity-filling mutant. Divalent cation affinity chromatography (TALON) of S6P.BA45.D17.AA-1192, S6P.BA45.I1.AA-1192, and S6P.BA45.I1.VI-1192 subsequently produced major putative trimer peaks eluted between 669 kDa and 440 kDa labels. Figure 41 A). The SEC spectrum of S6P.BA45.I1.AA-1192 indicates the presence of another substance that elutes more slowly (lower molecular weight). Figure 41 Fractions between the vertical dashed lines in A were merged and concentrated, and the sample was subjected to SEC after freeze-thaw cycles. A single symmetrical peak in the chromatogram indicates that the S6P protein has been purified to a homogeneous state. Figure 41 B). The Tm of the purified S6P.BA45.D17.AA-1192 trimer was 55℃, while the Tm of S6P.BA45.I1.AA-1192 and S6P.BA45.I1.VI-1192 was 52℃. Figure 41 C), which is almost identical to its S2P counterpart. It is noteworthy that the S2P.BA45.I1.AA-1192 sample contained another substance with poor thermal stability (Tm approximately 43℃). SDS-PAGE under non-reducing conditions indicated bands for the three disulfide bond mutants >250 kDa, while under reducing conditions, these bands dissociated to the expected monomer molecular weight of approximately 170 kDa, consistent with quantitative disulfide bond formation. Figure 41 D). The yields of the trimers S6P.BA45.D17.AA-1192 and S2P.BA45.D17.AA-1192 were almost identical (27 and 30 mg / L, respectively) (Table 6). In contrast, the yield of S6P.BA45.I1.VI-1192 was more than twice that of its S2P counterpart: 45 and 19.2 mg / L, respectively.

[0524] Therefore, compared to the 2P version, the hexapro mutation improved the yields of the parental and I1.VI forms of the omicron BA.4 / 5-1192 trimer, but not the yield of the D17 form. However, in this context, the hexapro mutation did not improve thermostability. The biochemical and antigenic characteristics of the S2P.BA45-1192 and S6P.BA45-1192 variants are summarized in Table 6.

[0525] Table 6. Characteristics of spike trimers.

[0526]

[0527] Example 14 - Epitope spectrum of S6P.BA45-1192 trimer

[0528] BLI was used to study the NAb epitope spectra of the disulfide-linked S6P.BA45-1192 trimer. ACE2-Fc and human mNAb were attached to an anti-human IgG Fc capture biosensor, while the S6P glycoprotein was in the analyte phase. Sensing plots showed that the binding response of the D17.AA and I1.VI mutants to ligands targeting RBM (i.e., ACE2-Fc, Omi-18, Omi-42, and SA55) was reduced compared to the parental S6P trimer. Figure 42 The binding kinetics showed that the opening and closing rates of the ligands targeting the RBM were relatively slow (Table 7). The covalently stable trimer exhibited a significantly reduced dissociation constant (KD) with ACE2-Fc (approximately 100-fold), and the D17 variant showed a KD approximately 10-fold lower than that of Omi-18. In contrast, the parental, D17.AA, and I1.VI S6P trimers showed almost identical binding characteristics to NAb S2H97, SP1-77, and S309 targeting conserved epitopes within the RBM (excluding the RBM) and to C1520 targeting the NTD, with dissociation constants of 10. -9 Up to < 10 -12 The projections of the M. ACE2, Omi-18, Omi-42, and SA55 epitopes onto the "open" and "closed" RBD conformations within the spike trimer indicate that they are masked in the closed conformation. In contrast, the RBD epitopes of S2H97, SP1-77, and S309 are exposed in both conformations. Figure 43 This analysis reveals a mechanism in which the disulfide bond stabilization of the spike trimer via D17 and I1 promotes a closed RBD conformation, thereby restricting access to RBM-dependent epitopes but not to conserved epitopes on the RBD (not involving the RBM).

[0529] CV3-25, CC99-103, and CC95-108 target the stem of S2, which encompasses conserved key neutralizing epitopes in β-coronaviruses (Zhou et al., 2023). These Nabs exhibit significantly reduced dissociation rates and dissociation constants (KD reduction of at least 2 to 3 orders of magnitude) when bound to D17 and I1.VI, indicating that disulfide bond stabilization of the spike trimer enables more stable NAb-stem interactions. Figure 42 ).

[0530] Table 7. Binding kinetics of S6P.BA45-1192 variant trimer with neutralizing ligands

[0531]

[0532] Example 15 - Trimeric S6P.BA45-1192 vaccine protects K18hACE mice from viral attack.

[0533] The protective efficacy and immunogenicity of purified S6P.BA45.AA-1192, S6P.BA45.D17.AA-1192, and S6P.I1.VI-1192 trimers were evaluated in K18hACE2 transgenic mice. Sixteen 6- to 8-week-old female mice in four groups were subcutaneously immunized with the trimer in either a carrier or 10 μg of squalene in 50% (vol / vol) AddaVAX adjuvant (squalene oil-in-water emulsion). Mice received three doses of the carrier or vaccine at 3-week intervals, with blood collected either before the third dose or immediately two weeks after the third dose. Three weeks after the third dose, 12 mice in each group were immunized with 10 μg of the carrier or vaccine. 4 TCID 50 The mice were challenged with Omicron BA.5 virus. Four days after challenge, nasal turbinates (NT) and lungs were collected from 8 mice in each group, and lungs from 4 mice in each group were fixed with 10% neutral buffered formalin for pathological examination. Figure 44 Following Omicron BA.5 attack, the mean viral titer in the load control group in NT was 10. 2.7 TCID 50 / ml, and the average viral titer in the lungs was 10. 4.4 TCID 50 / organ( Figure 45 In contrast, no virus was detected in the NT and lungs of mice that received all three vaccines (detection limit was 10). 0.5 TCID 50 However, approximately 10 TCID were detected in the NT in two mice from the parental trimeric group. 50 This indicates the presence of breakthrough infection. Data show that all three S6P.BA45-1192 trimeric vaccines provide protection against BA.5 challenge, with versions D17 and I1.VI offering complete protection.

[0534] Example 16 - Antibody response to covalently stable S6P.BA45-1192 trimer after 2 and 3 immunizations

[0535] The R-20 microneutralization assay, developed by Aggarwal et al. (2022) using HAT-24 cells and real omicron BA.5 SARS-CoV-2, was used to determine whether the third dose of immunogen increased neutralizing antibody titers. Comparison of sera after two and three doses (week 6 and week 8, respectively) showed that the mean neutralizing ID after the third dose was [missing information].50S Statistically, this represents a significant increase of approximately 3.3 to 4.6 times. Figure 46 A). The binding titers of omicron BA.5 RBD and S6P.BA45.AA-1192 trimers in vaccine serum were determined by chemiluminescent immunosorbent assay (CLIA). Figure 46 B shows that RBD binding activity decreased significantly by approximately 2.6-fold after the third dose of S2P.BA45.D17.AA-1192, but not for the other two immunogens. Regarding trimer-specific antibodies, only the I1.VI variant caused a slight increase in average titer of approximately 2-fold after the third dose. Figure 46 C). Data indicate that the third immunogen boosted neutralizing antibody titers, but this was not reflected in RBD and trimer-binding antibodies.

[0536] Example 17 - The covalently stable S6P.BA45-1192 trimer is an excellent immunogen in terms of inducing neutralizing antibodies.

[0537] The R-20 microneutralization assay was used to determine neutralizing antibody titers against homologous omicron BA.5 virus, as well as ancestral, XBB.1.5, and JN.1 variants, at week 8. Figure 47 The mutations present in the spike proteins of omicron BA.5, XBB.1.5, and JN.1 relative to the ancestral isolate Hu-1 are listed. Figure 48 The data shown in the figure indicate that, relative to the parent (432), the neutralizing titers of BA.5 induced by D17 and I1.VI immunogens were significantly higher (geometric mean ID). 50 The reciprocals are 6,503 and 5,128 respectively. Although the overall titer is approximately 1.5 log lower compared to the neutralizing titer of the homologous virus. 10 This may be due to mutations in the NTD and RBD of these variants. Figure 47 However, this trend of neutralizing potency was also observed in ancestral, omicron XBB.1.5, and JN.1 variants. For XBB.1.5 and JN.1 variants that appeared after BA.5, D17 and I1.VI immune sera retained geometrically average ID values. 50s All showed neutralizing activity greater than 1 / 70, while the serum neutralizing activity induced by the parent trimer was not significantly different from that of the carrier control group.

[0538] Example 18 – Three S6P.BA45-1192 immunogens elicit cross-variant reactive antibodies against RBD and NTD.

[0539] CLIA was used to study the ability of vaccine antibodies to recognize the RBD and NTD immunodominant antigenic domains from different SARS CoV-2 variants. Figure 49 The range of reciprocal titers of RBD-binding antibodies induced by the three immunogens is shown as follows: BA.5, 6141 to 8453; ancestor, 2559 to 4246; XBB.1.5, 1727 to 7808; and JN.1, 1250 to 2645. The data indicate a change in 14 amino acids in the JN.1 RBD compared to BA.5 (see [link to data]). Figure 47 It affects the binding ability of vaccine serum.

[0540] Figure 50 The NTD binding capacity of vaccine serum was demonstrated. In this experiment, NTDs from three variants (BA.5, ancestor, and JN.1) were investigated. The parental S6P.BA45.AA-1192 immunogen elicited binding titers against the NTDs of all three variants significantly higher than those of the covalently stable D17 and I1.VI immunogens (at least 3 times higher than those of BA.5 and JN.1 NTDs; and approximately 6 times higher than those of the ancestor). It should be noted here that the parental vaccine (S6P.BA45.AA-1192) elicited neutralizing antibody titers against NTD variant-matched viruses significantly lower than those of the covalently stable D17 and I1.VI vaccines (see [link to study]). Figure 48 These data indicate that high NTD titers are negatively correlated with neutralizing activity. Serum from the three immunogen groups showed binding titers very similar to those for BA.5 and JN.1 NTDs, but with approximately 1.5 log lower neutralizing activity against JN.1 virus compared to BA.5 virus. 10 These data suggest that the antibodies against NTD detected here are unlikely to explain... Figure 48 Strong neutralization of BA.5 virus and weak neutralization of JN.1 virus were observed.

[0541] Example 19 – Detection of antibodies against the highly conserved stem region of S2

[0542] The stem region of S2 (amino acids 1138 to 1208) is presumed to form a flexible link between the ordered S1-S2 trimer head and the transmembrane sequence (Turonova et al., 2020). The S2 stem is highly conserved in sabevirus and contains neutralizing antibody epitopes across clades (Zhou et al., 2023). The use of a synthetic stem peptide (amino acids 1138 to 1165) and a chimeric protein (MBP-stem [1138 to 1208]) containing a maltose-binding protein linked to the stem via three alanine residues was used to assess whether three immunogens elicited antibodies against this antigenic region. Figure 51The data shown indicate that the three immunogens elicited roughly equal antibody titers against the stem, with the MBP-stem (positions 1138-1208) peptide exhibiting approximately five-fold higher titers compared to the synthetic stem (positions 1138-1165) peptide. This observation suggests that the immunogens elicited antibody specificity beyond the epitopes at positions 1138-1165 of the core stem.

[0543] Example 20 - No antibodies against the conserved fusion peptide were detected in vaccine serum.

[0544] The fusion peptide (amino acids 808 to 832 of S2) has also been speculated to be a conserved neutralizing antibody epitope (Dacon et al., 2022). ELISA using the synthetic fusion peptide (FP808-832) containing amino acids 808 to 832 of the spike did not detect specific antibodies against this epitope in vaccine serum. Figure 51 ).

[0545] Viral attack and immunogenicity data in Figure 52 Summary.

[0546] Example 21 - The ability of D17 and I1.VI mutations to covalently stabilize the soluble S trimer of the emerging SARS CoV2 variant: omicron BA.2.86.

[0547] The omicron BA.2.86 variant was first reported in August 2023, and phylogenetic analysis indicated that its most recent ancestor is BA.2. Compared to BA.2, BA.2.86 S has 38 amino acid changes and is considered to occur in immunocompromised individuals with chronic infection due to the lack of any intermediate sequences in the database. BA.2.86 is the ancestor of the currently dominant globally circulating isolates JN.1, KP.1, KP.2, and KP.3. When compared to BA.4 / 5, BA.2.86 S has 28 point mutations, 3 deletions, and 4 amino acid insertions (see [link to relevant documentation]). Figure 47 These two variants of the S glycoprotein share 97.3% amino acid identity. Using BA.2.86 S as an example, the above-described method for generating covalently stable soluble SARS-CoV-2 S trimers was applied to emerging SARS-CoV-2 variants. pcDNA3 expression vectors targeting the spike sequences of S6P.BA286.AA-1192, S6P.BA286.D17.AA-1192, and S6P.BA286.I1.VI-1192, containing the N-terminal tPA precursor and C-terminal His6 tag, were prepared. The amino acid and DNA sequences of the expected mature S protein (i.e., excluding the tPA precursor and His6 tag) were obtained from [the following data]. Figure 53 and 54 As shown in the image.

[0548] The omicron BA.2.86 S glycoprotein was expressed in Expi293F cells, and as follows: Figure 41 The BA.4 / 5 counterparts were purified by TALON divalent cation affinity chromatography and Superose 6 SEC. Figure 55 A shows the SEC spectrum of the BA.2.86 spike after the TALON step. S6P.BA286.AA-1192 produced the major putative trimer peak eluted only before 440 kDa. The S6P.BA45.AA-1192 trimer was stable after freeze (-80°C) - thaw cycles. Figure 55 B), and the DSF indicates Tm is 42℃ ( Figure 41 C). S6P.BA286.AA-1192 and its BA.4 / 5 counterparts are almost identical in biophysical characteristics, except that the former is expressed in higher yields: 86 mg / L and 42 mg / L, respectively.

[0549] The SEC spectra of S6P.BA286.D17.AA-1192 and S6P.BA286.I1.VI-1192 show the main putative trimer peak eluted approximately midway between the 669 and 440 kDa markers. Figure 55 A). Fractions containing the putative trimer were combined, concentrated, and subjected to SEC after freeze-thaw cycles. A single symmetrical peak indicated that the S6P protein had been purified to homogeneity. Figure 55 B). The Tm of the purified S6P.BA286.D17.AA-1192 trimer was 57℃, while the Tm of S6P.BA286.I1.VI-1192 was 53℃. Figure 55 C), which is almost identical to the counterpart of BA.4 / 5 (see C). Figure 41 SDS-PAGE under non-reducing conditions indicated bands for two disulfide bond mutants > 250 kDa, while under reducing conditions, these bands dissociated into monomer molecules of approximately 170 kDa as expected, consistent with quantitative disulfide bond formation. Figure 55 D). The yields of the trimers S6P.BA286.D17.AA-1192 and S2P.BA286.I1.VI-1192 were 26 and 40 mg / L, respectively (Table 6).

[0550] Example 22 - Epitope spectrum of S6P.BA286-1192 trimer

[0551] Figure 56The binding of the spike ligand to the S6P.BA286-1192 trimer in BLI is illustrated. As before, ACE2-Fc and human NAb were attached to the anti-human IgG Fc capture biosensor, while the S6P glycoprotein (30 nM) was in the analyte phase. Multiple effects of the D17 and I1.VI mutations on ligand (ACE2-Fc, Omi-42, and SA55 (RBM)) binding were observed: a significantly reduced binding response was observed for D17; a slightly reduced binding response was observed for I1.VI, but with a decreased dissociation rate.

[0552] Omi-18 (RBM): Rapid on- and off-rates of the parent trimer were observed; the binding response of D17 was significantly reduced; the binding response of I1.VI was strong and the off-rate was significantly reduced.

[0553] C1520 (NTD), S2H97, SP1-77 (RBD flank): The binding response levels of the three spikes were similar, and the closing rates of D17 and I1.VI were reduced.

[0554] CV3-25, CC95-108 and CC99-103 (stem): Decreased binding response to D17 and I1.VI was observed, but the shutdown rate was also reduced.

[0555] Overall, the data suggest that D17 tends to reduce binding to ligands targeting RBM, possibly by inducing conformations that restrict access to the region. In contrast, RBM-dependent epitopes appear to be accessible in the I1.VI spike, and ligands binding to these epitopes show signs of being stabilized by mutations. In the case of stem epitopes, both D17 and I1.VI mutations restrict binding to these epitopes, but ligands binding to these antigenic epitopes show signs of being stabilized by these mutations.

[0556] Example 23 - Ability of soluble S-trimer in bat sabervirus PRD-0038 using ACE2-based covalently stable differences between D17 and I1.VI mutations.

[0557] The subgenus *Sabevirus* of the genus *β-coronavirus* consists of four clades: clade 1b, which includes SARS-CoV-2 and related Asian bat and pangolin viruses; clade 1a, which includes SARS-CoV and related Asian bat and civet viruses; clade 2, which is mainly composed of Asian bat viruses; and clade 3, which includes viruses from European and African bats. Sabeviruses in clades 1a, 1b, and 3 use ACE2 to enter cells, while viruses in clade 2 use an alternative receptor due to a deletion in their RBD that hinders ACE2 binding. Figure 57The phylogenetic relationships among the S glycoproteins of four members of the sabevirus clade are shown, as well as the percentage of amino acid identity between omicron BA.4 / 5 and prominent instances from the clade using ACE2.

[0558] The inventors selected the PRD-0038 spike to investigate whether D17 and I1.VI mutations could covalently stabilize a highly variably ACE2-dependent soluble S-trimer of bat sabevirus with the potential for spillover into the human population. This spike shares 72.6% amino acid identity with the BA.4 / 5 spike. The PRD-0038 spike uses Rhinolophus sp. ACE2 for entry; however, only two mutations (K482Y / T487W) in the RBD are required to confer human ACE2 binding ability (Lee et al., 2023). Structural alignment of the PRD-0038 spike trimer (PDB ID 8U29) with BA.4 / 5 (PDB ID 7XNQ) identified equivalent D17 and I1 mutant targets. Figure 58 The spike protein of SARS-CoV-2 is shown. Figure 58 The D17 (D571 / S967) and I1 (A570 / S967) mutation targets in A) correspond to PRD-0038 ( Figure 58 B) D560 / S950 and S559 / S950. C of D560 / S950 and S559 / S950. α -C α The distances were 5.3 Å and 5.0 Å, respectively. For comparative purposes, the same analysis was performed on bat sabeviruses of clade 1b BANAL-20-235 (Lan et al., 2020) and clade 1a WIV1 (Ge et al., 2013), which are capable of using human ACE2 and are more closely related to the BA.4 / 5 spike sequence. Studies of the spike trimer structures of BANAL-20-235 and WIV1 (PDB ID 8I3W and 8TC0, respectively) identified D17 and I1 equivalent targets, which have similar inter-C-terminal relationships to their counterparts in the PRD-0038 spike. α distance( Figure 58 (C and D).

[0559] pcDNA3 expression vectors containing N-terminal tPA precursors and C-terminal His6 tags of PRD-0038 soluble spikes (S6P.PRD.AA-1192, S6P.PRD.D17.AA-1192, and S6P.PRD.I1.VI-1192) were prepared. The amino acid and DNA sequences of the expected mature S protein (i.e., excluding the tPA precursor and His6 tag) were obtained from [the following data]. Figure 59 and 60As shown in the figure. The PRD-0038 spike was expressed in Expi293F cells and purified by TALON divalent cation affinity chromatography and Superose 6 SEC as described for its omicron BA.4 / 5 and BA.2.86 counterparts. Figure 61 A shows the SEC spectrum of the PRD-0038 spike after the TALON step. The PRD-0038 spike mutant exhibits a major putative trimer peak eluted from a standard close to 440 kDa. DSF indicates a Tm of 50.5 °C for the parent S6P.PRD.AA-1192, while D17 and I1.VI increase the Tm to 55.8 °C and 52 °C, respectively. Figure 61 B). SDS-PAGE under non-reducing conditions indicated bands for two disulfide bond mutants > 250 kDa, while under reducing conditions, these bands dissociated into the expected monomer molecular weight of approximately 170 kDa, consistent with quantitative disulfide bond formation. Figure 61 C). These data suggest that stem truncation of 1192, in conjunction with D17 or I1.VI mutations, can be used to generate covalently stable soluble spike trimers from differentially expressed sabeviruses, accompanied by increased thermal stability.

[0560] Example 24 - Epitope spectrum of S6P.PRD-1192 trimer

[0561] Figure 62 Epitope spectra of the disulfide-linked S6P.PRD-1192 trimer are shown, as determined in BLI. As before, a human monoclonal NAb was attached to an anti-human IgG Fc capture biosensor, with the S6P glycoprotein in the analyte phase. CR3022 bound to three S6P.PRD-1192 variant glycoproteins, exhibiting approximately equal affinity (KD) for the parent and the I1.VI PRD-0038 trimer, but a decrease of approximately 3-fold for D17 (KD is shown in the upper right corner of each sensor plot). CR3022 was isolated from SARS-CoV-infected patients (Ter Meulen et al., 2006) and recognizes cryptic epitopes of the RBD (Huo et al., 2020 and Zhou et al., 2020). Conserved stem regions of the PRD-0038 glycoprotein variant were probed using NAbs obtained from SARS-CoV-2-infected patients. In all cases, an affinity of S6P.PRD.I1.VI-1192 was observed to be 9 to 312-fold higher than that of the parent and the D17 trimer (KD highlighted). These data suggest that the I1.VI mutation plays a stabilizing role in stem-NAb interactions in the context of the PRD-0038 spike.

[0562] Example 25 - Expression of S6P.BA286-1192 trimeric protein derived from mRNA

[0563] To determine whether the S6P.BA286-1192 trimer protein expressed by mRNA shares similar characteristics with those expressed by DNA (see [link to relevant documentation]). Figure 55 and 56 mRNAs encoding S6P...

Claims

1. A coronavirus (CoV) vaccine antigen comprising a CoV S protein trimer having at least one non-endogenous inter-disulfide bond.

2. The CoV vaccine antigen of claim 1, wherein the non-endogenous protomer disulfide bond is formed between cysteine ​​residues selected from the following: i) Cysteine ​​(D17) at positions corresponding to amino acid numbers 571 and 967 of SEQ ID NO: 1 or SEQ ID NO:

2. ii) Cysteine ​​(I1) at positions corresponding to amino acid numbers 570 and 967 of SEQ ID NO: 1 or SEQ ID NO: 2; and iii) Cysteine ​​(L23) at positions corresponding to amino acid numbers 914 and 1123 of SEQ ID NO: 1 or SEQ ID NO:

2.

3. The CoV vaccine antigen of claim 1 or claim 2, wherein the non-endogenous protomer disulfide bond is formed between cysteine ​​residues at positions corresponding to amino acid numbers 914 and 1123 of SEQ ID NO: 1 or SEQ ID NO: 2 (L23).

4. The CoV vaccine antigen of claim 1 or claim 2, wherein the non-endogenous protomer disulfide bond is formed between cysteine ​​residues at positions corresponding to amino acid numbers 571 and 967 of SEQ ID NO: 1 or SEQ ID NO: 2 (D17).

5. The CoV vaccine antigen of claim 1 or claim 2, wherein the non-endogenous protomer disulfide bond is formed between cysteine ​​residues at positions corresponding to amino acid numbers 570 and 967 of SEQ ID NO: 1 or SEQ ID NO: 2 (I1).

6. The CoV vaccine antigen of any one of claims 1 to 5, wherein the melting temperature of the S protein trimer is increased by at least about 5°C compared to the S protein trimer lacking the interdisulfide bonds between the non-endogenous protomers.

7. The CoV vaccine antigen of any one of claims 1 to 6, wherein the melting temperature of the S protein trimer is increased by about 5°C to about 20°C, or about 5°C to about 15°C, or about 5°C to about 12.5°C, or about 5°C to about 10°C, or about 5°C to about 8°C, compared to the S protein trimer lacking the non-endogenous inter-disulfide bonds.

8. The CoV vaccine antigen of any one of claims 1 to 7, wherein the two protomers of the S protein trimer are bonded by disulfide bonds between non-endogenous protomers.

9. The CoV vaccine antigen of any one of claims 1 to 8, wherein each protomer of the S protein trimer is bonded to another protomer of the S protein trimer via a non-endogenous interprotein disulfide bond.

10. The CoV vaccine antigen of any one of claims 1 to 9, wherein the S protein trimer comprises an endogenous or non-endogenous transmembrane sequence.

11. The CoV vaccine antigen of any one of claims 1 to 10, wherein the promeron of the S protein trimer comprises a sequence selected from the following: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 132 and SEQ ID NO:

133.

12. The CoV vaccine antigen of any one of claims 1 to 11, wherein the S protein trimer is stable in the pre-fusion conformation.

13. Coronavirus (CoV) vaccine antigen, which contains a CoV S protein trimer with a C-terminus truncated in the stem region.

14. The CoV vaccine antigen of any one of claims 1 to 12, wherein the S protein trimer has a C-terminal truncation in the stem region.

15. The CoV vaccine antigen of claim 13 or claim 14, wherein the C-terminus is truncated between residues 1147 to 1207 corresponding to SEQ ID NO: 1 or SEQ ID NO:

2.

16. The CoV vaccine antigen of any one of claims 13 to 15, wherein the C-terminus is truncated between residues 1147 to 1193 corresponding to SEQ ID NO: 1 or SEQ ID NO:

2.

17. The CoV vaccine antigen of any one of claims 13 to 15, wherein the C-terminus is truncated after residues 1147, 1157, 1165, 1192, 1195, 1196, 1199, 1201 or 1204 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 and before residue 1208 corresponding to SEQ ID NO: 1 or SEQ ID NO:

2.

18. The CoV vaccine antigen of any one of claims 13 to 15 or 17, wherein the C-terminus is truncated after residues 1147, 1157, 1165, 1192, 1195, 1196, 1199, 1201 or 1204 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 and before residue 1208 corresponding to SEQ ID NO: 1 or SEQ ID NO:

2.

19. The CoV vaccine antigen of any one of claims 13 to 18, wherein the C-terminus is truncated after residue 1192 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 and before residue 1208 corresponding to SEQ ID NO: 1 or SEQ ID NO:

2.

20. The CoV vaccine antigen of any one of claims 13 to 19, wherein the promerum of the S protein trimer comprises or is composed of the following: Residues 16 to 1147, or residues 16 to 1157, or residues 16 to 1165, or residues 16 to 1192, or residues 16 to 1204, corresponding to SEQ ID NO: 1 or SEQ ID NO:

2.

21. The CoV vaccine antigen of claim 20, wherein the promeron of the S protein trimer comprises or is composed of the following: Residues 16 to 1192 corresponding to SEQ ID NO: 1 or SEQ ID NO:

2.

22. The CoV vaccine antigen of any one of claims 1 to 21, wherein when administered to a subject, the S protein trimer triggers a neutralizing antibody response.

23. The CoV vaccine antigen of claim 22, wherein the neutralizing antibody response is a neutralizing antibody response against one or more epitopes comprising part or all of the receptor-binding domain (RBD).

24. The CoV vaccine antigen of claim 22, wherein the neutralizing antibody response is a non-RBD neutralizing antibody response.

25. The CoV vaccine antigen of claim 24, wherein the non-RBD neutralizing antibody response comprises a neutralizing antibody response against one or more epitopes containing part or all of the stem region.

26. The CoV vaccine antigen of claim 24 or claim 25, wherein the non-RBD neutralizing antibody response comprises a neutralizing antibody response against one or more epitopes containing part or all of the N-terminal structural domain (NTD).

27. The CoV vaccine antigen of any one of claims 24 to 26, wherein the neutralizing antibody response comprises a neutralizing antibody response against one or more epitopes containing part or all of the RBD flanking regions.

28. The CoV vaccine antigen of claim 23, wherein the neutralizing antibody response comprises initiating a neutralizing antibody against one or more epitopes containing a portion or all of a receptor-binding motif (RBM).

29. The CoV vaccine antigen of any one of claims 1 to 23, wherein the S protein trimer does not elicit a neutralizing antibody response against the receptor-binding motif (RBM).

30. The CoV vaccine antigen of any one of claims 1 to 29, wherein the S protein trimer is capable of binding to an antibody comprising one or more epitopes containing part or all of the N-terminal domain (NTD).

31. The CoV vaccine antigen of claim 30, wherein the antibody is selected from: C1520 and antibodies that bind to epitopes bound by C1520.

32. The CoV vaccine antigen of any one of claims 1 to 31, wherein the S protein trimer is capable of binding to any antibody that includes one or more epitopes comprising a partial or complete receptor-binding domain (RBD).

33. The CoV vaccine antigen of claim 32, wherein the antibody is selected from: S2H92, SP177, Omi-18, Omi-42, an antibody that binds to an epitope bound by S2H92, an antibody that binds to an epitope bound by SP1-77, an antibody that binds to an epitope bound by Omi-18, and an antibody that binds to an epitope bound by Omi-42.

34. The CoV vaccine antigen of any one of claims 1 to 31, wherein the S protein trimer is capable of binding to any antibody that includes one or more epitopes comprising part or all of the RBD flanking regions.

35. The CoV vaccine antigen of claim 34, wherein the antibody is selected from: S2H97, SP1-77, antibodies that bind to epitopes bound by S2H92, and antibodies that bind to epitopes bound by SP1-77.

36. The CoV vaccine antigen of any one of claims 1 to 35, wherein the S protein trimer is capable of binding to any antibody containing one or more epitopes of part or all of the RBM.

37. The CoV vaccine antigen of claim 36, wherein the antibody is selected from: Omi-18, Omi-42, antibodies that bind to epitopes that bind to Omi-18, and antibodies that bind to epitopes that bind to Omi-42.

38. The CoV vaccine antigen of any one of claims 1 to 37, wherein the S protein trimer is capable of binding to any antibody that includes one or more epitopes comprising part or all of the stem region.

39. The CoV vaccine antigen of claim 38, wherein the antibody is CV3-25 or an antibody that binds to an epitope that is bound by CV3-25.

40. The CoV vaccine antigen of any one of claims 1 to 39, wherein the melting temperature of the S protein trimer is about 38°C to about 71°C, or about 40°C to about 71°C, or about 42°C to about 71°C, or about 45°C to about 71°C, or about 50°C to about 71°C, or about 55°C to about 71°C, or about 60°C to about 71°C.

41. The CoV vaccine antigen of claim 40, wherein the melting temperature is from about 40°C to about 71°C.

42. The CoV vaccine antigen of any one of claims 13 to 41, wherein when expressed in a recombinant expression system, the trimer is produced at a higher level compared to the trimer lacking the truncated S protein expressed in the same recombinant expression system.

43. The CoV vaccine antigen of any one of claims 13 to 42, wherein when expressed in a recombinant expression system, the level of the S protein trimer is increased by about 17% to about 36% compared to the level lacking the truncated S protein trimer.

44. The CoV vaccine antigen of any one of claims 13 to 43, wherein when expressed in a recombinant expression system, the level of the S protein trimer is increased to about 2 to about 10 times compared to the level lacking the truncated S protein trimer.

45. The CoV vaccine antigen of any one of claims 13 to 44, wherein the S protein trimer is produced at a level greater than about 300 μg / 50 mL.

46. ​​The CoV vaccine antigen of any one of claims 13 to 45, wherein the S protein trimer is produced at a level of about 300 μg / 50 mL to about 2000 μg / 50 mL.

47. The CoV vaccine antigen of any one of claims 1 to 46, wherein the S protein trimer is soluble.

48. The CoV vaccine antigen of any one of claims 13 to 42, wherein the vaccine antigen further comprises a 2P modification.

49. The CoV vaccine antigen of any one of claims 1 to 47, wherein the vaccine antigen further comprises 6P modification.

50. The CoV vaccine antigen of any one of claims 1 to 49, wherein the vaccine antigen is a pancoronavirus vaccine antigen.

51. The CoV vaccine antigen of any one of claims 1 to 50, wherein the vaccine antigen is a SARS-CoV-2 vaccine antigen.

52. The CoV vaccine antigen of claim 51, wherein the vaccine antigen is a SARS-CoV-2 omicron vaccine antigen.

53. The CoV vaccine antigen of claim 51, wherein the vaccine antigen is a SARS-CoV-2 non-omicron vaccine antigen.

54. The CoV vaccine antigen of any one of claims 1 to 53, wherein the S protein trimer further comprises a structural modification that reduces the size of the alanine cavity in the coiled-coil region of the S protein trimer.

55. The CoV vaccine antigen of claim 54, wherein the structural modification is within the coiled-helix region.

56. The CoV vaccine antigen of claim 54 or claim 55, wherein the structural modification creates an artificial hydrophobic core in the alanine cavity.

57. The CoV vaccine antigen of any one of claims 54 to 56, wherein at least one amino acid in the coiled-coil region of the S protein monomer of the S protein trimer is replaced by a more hydrophobic amino acid, or wherein at least two amino acids in the coiled-coil region of the S protein monomer of the S protein trimer are replaced by more hydrophobic amino acids.

58. The CoV vaccine antigen of claim 57, wherein at least one or at least two amino acids are located at position a and / or d of the heptapeptide repeat motif in the coiled-coil region of the S protein monomer.

59. The CoV vaccine antigen of any one of claims 54 to 58, wherein the more hydrophobic amino acid comprises one or more of the following properties: i) Higher hydrophobicity than alanine; ii) Hydrophobic amino acids larger than alanine; iii) Hydrophobicity greater than 47 at pH 2; iv) Hydrophobicity greater than 41 at pH 7; and v) is selected from: isoleucine, leucine, methionine, valine, phenylalanine, tyrosine, and tryptophan.

60. The CoV vaccine antigen of any one of claims 54 to 59, wherein at least one amino acid in the coiled-coil region of the S protein monomer is A1016.

61. The CoV vaccine antigen of claim 60, wherein A1016 is replaced by valine (A1016V or 16V).

62. The CoV vaccine antigen of any one of claims 54 to 61, wherein at least one amino acid in the coiled-coil region of the S protein monomer is A1020.

63. The CoV vaccine antigen of claim 62, wherein A1020 is replaced by isoleucine (A1020I or 20I).

64. The CoV vaccine antigen of any one of claims 54 to 63, wherein at least one amino acid in the coiled-coil region of the S protein monomer is A1016 replaced by valine and A1020 replaced by isoleucine (A1016V / A1020I or VI).

65. The CoV vaccine antigen of any one of claims 54 to 64, wherein at least one amino acid in the coiled-coil region of the S protein monomer is A1016 (A1016L or 16L) replaced by leucine.

66. The CoV vaccine antigen of any one of claims 13 to 65, wherein the S protein trimer comprises or is composed of the following: The non-endogenous protomer disulfide bond corresponding to residues 16 to 1192 of SEQ ID NO: 1 or SEQ ID NO: 2 is formed between residues D571 and S967 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 (D17) or between residues A570 and S967 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 (I1), and the melting temperature of the S protein trimer is from about 50°C to about 55°C.

67. The CoV vaccine antigen of any one of claims 13 to 66, wherein the S protein trimer comprises or is composed of sequences selected from or composed of sequences selected from: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO:

13.

68. The CoV vaccine antigen of any one of claims 13 to 67, wherein the S protein trimer comprises or consists of sequences selected from or composed of sequences selected from: SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO:

49.

69. The CoV vaccine antigen of any one of claims 13 to 68, wherein the S protein trimer comprises or is composed of sequences selected from the following: SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO:

72. SEQ ID NO: 102, SEQ ID NO: 103 SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 122, SEQ ID NO: 123 and SEQ ID NO:

124.

70. The CoV vaccine antigen of any one of claims 13 to 69, wherein the S protein trimer lacks a signal sequence.

71. The CoV vaccine antigen of any one of claims 13 to 70, wherein the C-terminus is truncated between residues 1162 to 1200 corresponding to SEQ ID NO: 1 or SEQ ID NO:

2.

72. The CoV vaccine antigen of any one of claims 13 to 71, wherein the C-terminus is truncated after residues selected from the group consisting of residues 1162, 1165, 1192, or 1199 corresponding to SEQ ID NO: 1 or SEQ ID NO:

2.

73. The CoV vaccine antigen of any one of claims 13 to 71, wherein the C-terminus is truncated after residue 1192 of SEQ ID NO: 1 or SEQ ID NO:

2.

74. Protein nanoparticles comprising the coronavirus (CoV) vaccine antigen as described in any one of claims 1 to 73.

75. Virus-like particles comprising the coronavirus (CoV) vaccine antigen as claimed in any one of claims 1 to 73.

76. Deoxyribonucleic acid or ribonucleic acid encoding the coronavirus vaccine antigen of any one of claims 1 to 73.

77. Ribonucleic acid encoding an S protein monomer of a coronavirus (CoV) vaccine antigen, wherein the vaccine antigen comprises a CoV S protein trimer having at least one non-endogenous protomer disulfide bond.

78. Ribonucleic acid encoding an S protein monomer of a coronavirus (CoV) vaccine antigen, wherein the vaccine antigen is a CoVS protein trimer, and wherein the S protein monomer of the CoV S protein trimer has a C-terminal truncation in the stem region.

79. A vector comprising any one of claims 76 to 78 deoxyribonucleic acid or ribonucleic acid.

80. A host cell comprising any one of claims 76 to 78 deoxyribonucleic acid or ribonucleic acid or the vector of claim 79.

81. A method for producing a coronavirus (CoV) vaccine antigen according to any one of claims 1 to 70, comprising culturing the host cells of claim 80 in a culture medium to produce the vaccine antigen.

82. A method for producing a coronavirus (CoV) vaccine, comprising culturing the host cells of claim 80 in a culture medium to produce ribonucleic acid or deoxyribonucleic acid as described in any one of claims 76 to 78.

83. A vaccine comprising a coronavirus (CoV) vaccine antigen as described in any one of claims 1 to 73 or 81, or a protein nanoparticle as described in claim 74, or a virus-like particle as described in claim 75, or deoxyribonucleic acid as described in claim 76, or ribonucleic acid as described in any one of claims 76 to 78, or a vector as described in claim 73.

84. The vaccine of claim 83, wherein the vaccine is selected from: inactivated vaccines; live attenuated vaccines; protein subunit vaccines and RNA vaccines.

85. The vaccine of claim 83 or claim 84, wherein the vaccine further comprises at least one additional CoV vaccine antigen or at least one additional ribonucleic acid encoding an S protein monomer of a coronavirus (CoV) vaccine antigen.

86. A method for inducing an immune response against a coronavirus (CoV) in a subject, the method comprising delivering to the subject a vaccine antigen of any one of claims 1 to 73 or 81, or deoxyribonucleic acid of claim 76, or ribonucleic acid of any one of claims 76 to 78, or a vaccine of any one of claims 83 to 85.

87. A method for enhancing an immune response against a coronavirus (CoV) in a subject, the method comprising delivering to the subject a vaccine antigen of any one of claims 1 to 73 or 81, or deoxyribonucleic acid of claim 76, or ribonucleic acid of any one of claims 76 to 78, or a vaccine of any one of claims 83 to 85.

88. A method for preventing coronavirus (CoV) infection or reducing the likelihood of coronavirus (CoV) infection in a subject, the method comprising delivering to the subject a vaccine antigen of any one of claims 1 to 73 or 81, or deoxyribonucleic acid of claim 76, or ribonucleic acid of any one of claims 76 to 78, or a vaccine of any one of claims 83 to 85.

89. A method for preventing symptoms of coronavirus (CoV) infection or reducing the likelihood or severity of symptoms of coronavirus (CoV) infection in a subject, the method comprising delivering to the subject a vaccine antigen of any one of claims 1 to 73 or 81, or deoxyribonucleic acid of claim 76, or ribonucleic acid of any one of claims 76 to 78, or a vaccine of any one of claims 83 to 85.

90. A method for reducing the severity and / or duration of coronavirus (CoV) infection in a subject, the method comprising delivering to the subject a vaccine antigen of any one of claims 1 to 73 or 81, or deoxyribonucleic acid of claim 76, or ribonucleic acid of any one of claims 76 to 78, or a vaccine of any one of claims 83 to 85.

91. A method for preventing or reducing viral shedding in human individuals infected with coronavirus (CoV), the method comprising delivering to the subject a vaccine antigen of any one of claims 1 to 73 or 81, or deoxyribonucleic acid of claim 76, or ribonucleic acid of any one of claims 76 to 78, or a vaccine of any one of claims 83 to 85.

92. The method of any one of claims 86 to 91, wherein delivery is via intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intranasal, sublingual, tonsillar, oral, pulmonary, surface, or other extraintestinal mucosal route.

93. The CoV vaccine antigen of any one of claims 1 to 73 or claim 81, or the deoxyribonucleic acid of claim 76, or the ribonucleic acid of any one of claims 76 to 78, or the vaccine of any one of claims 83 to 85, wherein it is used for one or more of the following: i) Inducing an immune response against CoV in the target population; ii) Enhance the immune response against CoV in the target population; iii) To prevent or reduce the likelihood of CoV infection in individuals; iv) Preventing CoV symptoms or reducing the likelihood or severity of CoV symptoms in subjects; v) Reduce the severity and / or duration of CoV infection in subjects; vi) Preventing or reducing viral shedding in the target; and vii) Treating CoV infection in subjects.

94. A kit, device, surface, or test strip comprising a coronavirus (CoV) vaccine antigen as described in any one of claims 1 to 73 or 81, or ribonucleic acid as described in any one of claims 76 to 78.

95. Use of the coronavirus (CoV) vaccine antigen of any one of claims 1 to 73 or claim 81, or the deoxyribonucleic acid of claim 76, or the ribonucleic acid of any one of claims 76 to 78, in the preparation of a medicament for use in one or more of the following: i) Inducing an immune response against CoV in the target population; ii) Enhance the immune response against CoV in the target population; iii) To prevent or reduce the likelihood of CoV infection in individuals; iv) Preventing CoV symptoms or reducing the likelihood or severity of CoV symptoms in subjects; v) Reduce the severity and / or duration of CoV infection in subjects; vi) Preventing or reducing viral shedding in the target; and vii) Treating CoV infection in subjects.

96. A method for increasing the yield of S protein trimer, comprising modifying the CoV S protein trimer to include a stem region C-terminus truncated.

97. A method for stabilizing a CoV S protein trimer in a pre-fusion conformation, comprising modifying the CoV S protein trimer to include at least one non-endogenous protomer disulfide bond.

98. A method for increasing the melting temperature of the CoV S protein trimer, comprising modifying the CoV S protein trimer to include a stem region C-terminus truncated.

99. A method for increasing the melting temperature of a CoVS protein trimer stable in its pre-fusion conformation, comprising modifying the CoVS protein trimer to include a C-terminal truncation of the stem region.

100. A method for enhancing a neutralizing antibody response, comprising modifying a CoV S protein trimer to include at least one interprotomeric disulfide bond and / or modifying said CoV S protein trimer to include a C-terminal truncation of the stem region.

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