Vaccine antigens

Abstract

The field of the present specification generally relates to SARS-CoV-2 vaccine spike protein antigens and methods of using and producing these antigens. The present invention also relates to vectors and polynucleotides encoding SARS-CoV-2 vaccine antigens, as well as vaccines, kits, devices, and strips comprising coronavirus vaccine antigens. The spike protein from SARS-CoV-2 has substituted prolines (2P or S-2P) at positions 986, 987, and additional alanine cavity-filling mutations at positions A1016 and A1020.

Description

[Technical field]

[0001] The field of the present specification relates generally to coronavirus vaccine antigens and methods of using and producing coronavirus vaccine antigens. The present invention also relates to vectors and polynucleotides encoding coronavirus vaccine antigens, as well as vaccines, kits, devices, and strips comprising coronavirus vaccine antigens. [Background technology]

[0002] Bibliographic details of the references in the subject specification are also listed at the end of the specification.

[0003] The reference to any prior art in this specification is not to be construed as an acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0004] SARS-CoV-2 has killed over 4 million people worldwide, with a strong age-dependent infection mortality rate. First-generation vaccines delivering viral spike glycoprotein (S) sequences from ancestral SARS-CoV-2 for in vivo expression and neutralizing antibody (NAb) induction have proven highly effective in preventing symptomatic and severe COVID-19 and have been deployed worldwide. Highly effective S vaccine platforms include (Pfizer-BioNTech, BNT162b2, Moderna mRNA-1273) (Baden et al., 2021; Polack et al., 2020), adenovirus 26 and adenovirus 5 (Ad26, Ad5) (Sputnik V, Janssen COVID-19) (Logunov et al., 2021; Sadoff et al., 2021), and chimpanzee adenovirus (Astrazeneca / Oxford, ChAdOx1 nCoV-19) (Emary et al., 2021; Madhi et al., 2021; Voysey et al., 2021). Sites of vulnerability to neutralizing antibodies (NAbs) within S have been revealed by the isolation of monoclonal NAbs from COVID-19 patients. The ACE2 receptor binding domain (RBD) of S is the immunodominant antibody target in natural infection, and highly potent NAbs directed against the RBD can block infection through ACE2 receptor mimicry (e.g., B38), through steric blocking of ACE2 binding (e.g., H4), or by binding to a quaternary epitope formed by two RBD monomers (e.g., 2-43). The N-terminal domain (NTD) of S1 has been identified as a vulnerable supersite and contains multiple antigenic sites (Andreano et al., 2020; Cerutti et al., 2021; Liu, et al., 2020; McCallum et al., 2021). This region shows a high degree of adaptability, acquiring deletions, insertions, and glycan additions to evade antibodies.Undefined neutralizing epitopes have also been observed within S1 and S2 ( Brouwer et al., 2020 , Jennewein et al., 2021 ).

[0005] Naturally acquired immunity to SARS-CoV-2 is thought to drive the emergence of variants of concern (VOCs) with mutations in the RBD and NTD that reduce the neutralizing capacity of convalescent and vaccine-induced immune sera and human monoclonal NAbs (Plante et al., 2021). Major mutations observed in the RBD of VOCs include K417T / N, N439K, L452R, Y453F, S477N, T478K, E484K / Q, and N501Y, while deletions of amino acids 69–70, 156–157, and 242–245 have been observed in the NTD. Vaccine efficacy may also vary in VOCs depending on the viral S sequence and vaccine modality. Thus, ChAdOx1-nCOV-19 vaccine efficacy was reduced from 81.5% to 70.4% in the case of alpha / B.1.1.7 isolates (Emary et al., 2021), while efficacy was reduced to 10.4% in the case of beta / B.1.351 isolates (Madhi et al., 2021). The reduced efficacy of ChAdOx1 nCOV-19 against these VOCs seems to correlate with the reduced in vitro neutralizing capacity of vaccine sera against VOCs (Dejnirattisai et al., 2021; Supasa et al., 2021). The neutralizing capacity of mRNA vaccine recipient sera is also reduced for VOCs (Alter et al., 2021; Dejnirattisai et al., 2021; Garcia-Beltran et al., 2021; Liu et al., 2021; Supasa et al., 2021; Tada et al., 2021). However, full vaccination with BNT162b2 provides a high level of protection against infection and disease caused by alpha and beta variants (Abu-Raddad et al., 2021), but when breakthrough infections are occurring, these have been found to be associated with VOCs (Kustin et al., 2021).Current vaccines have so far retained substantial efficacy against most VOCs, but as the pandemic progresses in the setting of partially immune human populations, boosting with VOC-adapted vaccines may be required to maintain immunity against emerging viral variants.

[0006] Thus, there is a need for improved antigens for eliciting immune responses against coronaviruses, and in particular, there is a need for improved antigens for eliciting immune responses against coronavirus VOCs. Summary of the Invention

[0007] The term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be understood to provide explicit support for both or either meaning. As used herein, the term "about," unless stated to the contrary, refers to + / - 10%, more preferably + / - 5%, and even more preferably + / - 1% of the specified value.

[0008] Throughout this specification, the use of "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0009] As used herein, the singular forms "a," "an," and "the" include singular and plural referents unless the context clearly dictates otherwise. Each embodiment herein applies mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0010] Nucleotide and amino acid sequences are referred to by sequence identifier numbers (SEQ ID NO:). <400> 1 (SEQ ID NO:1), <400> 2 (SEQ ID NO:2), etc. A sequence listing is provided after the claims.

[0011] In an aspect, the invention provides a coronavirus vaccine antigen comprising a coronavirus S protein trimer, the S protein trimer modified to contain a structural modification that reduces the size of an alanine cavity within the coiled-coil region of the S protein trimer, and the S protein trimer elicits a neutralizing antibody response.

[0012] In aspects, the invention provides a coronavirus vaccine antigen comprising a coronavirus S protein trimer, wherein at least one amino acid within the region of the S protein monomer that forms the coiled-coil of the S protein trimer is replaced with a more hydrophobic amino acid.

[0013] In an aspect, the invention provides a vector or polynucleotide encoding an S protein monomer of a coronavirus (CoV) vaccine antigen described herein. In one embodiment, the polynucleotide is codon optimized for expression in a host cell or vaccine recipient cell.

[0014] In one embodiment, the invention provides a polynucleotide complement of a polynucleotide encoding an S protein monomer of a coronavirus (CoV) vaccine antigen described herein.

[0015] In an aspect, the invention provides a host cell comprising a vector or polynucleotide as described herein. In one embodiment, the host cell is a host cell for in vitro expression that is not a vaccine recipient cell.

[0016] In an aspect, the invention provides a method for producing a coronavirus (CoV) vaccine antigen described herein, comprising culturing a host cell described herein in a medium.

[0017] In an aspect, the present invention provides protein nanoparticles comprising a coronavirus (CoV) vaccine antigen as described herein.

[0018] In an aspect, the invention provides a virus-like particle comprising a coronavirus (CoV) vaccine antigen as described herein.

[0019] In an aspect, the invention provides a vial or a solid or semi-solid surface containing a vector or polynucleotide, an antigen, a protein nanoparticle, or a VLP as described herein.

[0020] In an aspect, the invention provides a vaccine comprising a coronavirus (CoV) vaccine antigen as described herein, or a vector or polynucleotide as described herein, or a proteinaceous nanoparticle as described herein, or a virus-like particle as described herein.

[0021] In an aspect, the invention provides a method of inducing an immune response against a coronavirus (CoV) in a subject, the method comprising administering a vaccine described herein.

[0022] In an aspect, the invention provides a method of enhancing an immune response to a coronavirus (CoV) in a subject, the method comprising administering a vaccine described herein.

[0023] In an aspect, the invention provides a method of preventing or reducing the likelihood of a coronavirus (CoV) infection in a subject, the method comprising administering a vaccine as described herein.

[0024] In an aspect, the invention provides a method of preventing or reducing the likelihood of a coronavirus (CoV) infection or the severity of a symptom thereof in a subject, the method comprising administering a vaccine as described herein.

[0025] In an aspect, the invention provides a method of reducing the severity and / or duration of a coronavirus (CoV) infection in a subject, the method comprising administering a vaccine as described herein.

[0026] In an aspect, the invention provides a method of preventing or reducing viral shedding in a human individual infected with a coronavirus (CoV), the method comprising administering a vaccine as described herein.

[0027] In aspects, the invention provides a CoV vaccine antigen as described herein or a vaccine as described herein for use in one or more of: i) preventing or reducing the likelihood of CoV infection in a subject; ii) preventing or reducing the likelihood of severity of CoV symptoms in a subject; iii) reducing the severity and / or duration of CoV infection in a subject; iv) preventing or reducing viral shedding in a subject; and v) treating a CoV infection in a subject.

[0028] In aspects, the invention provides kits, devices, surfaces, or strips comprising the coronavirus (CoV) vaccine antigens described herein.

[0029] In aspects, the invention provides the use of a coronavirus (CoV) vaccine antigen in the manufacture of a medicament for one or more of: i) preventing or reducing the likelihood of CoV infection in a subject; ii) preventing or reducing the likelihood of severity of CoV symptoms in a subject; iii) reducing the severity and / or duration of CoV infection in a subject; iv) preventing or reducing viral shedding in a subject; and v) treating a CoV infection in a subject.

[0030] In an aspect, the invention provides the use of an antigen or coding sequence in the manufacture of a preparation for treatment, prevention or testing in relation to coronavirus (CoV) infection in a population. In one embodiment, the stable trimeric S protein described herein is used to prepare neutralizing antibodies.

[0031] In an aspect, the present invention provides a soluble S protein trimer that lacks a heterologous trimerization sequence, the S protein trimer being modified to include a structural modification that reduces the size of an alanine cavity within the coiled-coil region of the S protein trimer, and the S protein trimer elicits a neutralizing antibody response.

[0032] In an aspect, the present description provides a method for manufacturing a semiconductor device comprising the steps of: (i) form as non-disulfide-linked trimers; (ii) form as stable non-disulfide-linked trimers; (iii) forms a trimer that is thermally stable at about 58° C. (iv) form trimers that are more resistant to destruction by treatment with 0.8% w / v SDS, with or without B2ME, at 25°C than a control (e.g., S2P-FHA); (v) form trimers that are more resistant to destruction by boiling with 0.8% w / v SDS without B2ME than an appropriate control (e.g., S2P-FHA); and (vi) forming a stable, soluble trimer without a heterologous trimerization module.

[0033] In one embodiment, it is contemplated that the modified coronavirus S antigens described herein exhibit reduced off-target reactivity because the structural changes induced by the modified coiled-coil region enhance trimer stability, obviating the need for the use of heterologous trimerization domains to stabilize the expressed trimers.

[0034] In aspects, the description provides a polynucleotide comprising a polynucleotide sequence encoding at least a modified coronavirus S protein antigen as described herein and capable of being expressed in a recipient cell. In one embodiment, the modified antigen exhibits enhanced exposure of broadly neutralizing epitopes in an isolate-dependent manner compared to a suitable control. [Brief description of the drawings]

[0035] [Figure 1-1] Figure 1A) Represents the 3D structure of the SARS-CoV-2 S ectodomain drawn with coordinating PDB ID 6VSB (Walls et al., 2020). The RBM is the ACE2 receptor binding motif with receptor interacting amino acids shown as spheres in grey at the top of the structure, the RBD is the receptor binding domain in black at the top of the structure with beta strands shown as thick arrows, and the NTD is the N-terminal domain in grey with beta-sheets represented as thick arrows. The central coiled coil of S2 is shown as three perpendicular helices in dark grey with an Ala cavity highlighted in light grey. [Figure 1-2] Figure 1B) Heptad repeat motifs within the coiled-coil sequences of three betacoronaviruses. [Figure 1-3] Figure 1C) Comparison of the central coiled-coil in the pre-fusion (PDB ID 6VSB (Walls et al., 2020) and post-fusion (PDB 6XRA (Cai et al., 2020) conformations. The HR1 helix, shown in white in the right-hand structure, forms the base of the coiled-coil extension that protrudes upward toward the plasma membrane in the post-fusion conformation (Cai et al., 2020). [Figure 1-4] FIG. 1D) Schematic of how the Ala cavity in pre-fusion S can be filled after substitution with a hydrophobic amino acid. [Diagram 2] Figure 2 shows the expression, binding properties and stability of S2P-FHA. A) Superose6 size exclusion chromatography (SEC) of purified S2P-FHA trimer. B) SDS-PAGE of purified S2P-FHA under reducing conditions. C) Binding of avidin-captured biotinylated S2P-FHA trimer with ACE2-Fc and various human monoclonal antibodies in ELISA. D) Differential scanning fluorimetry of purified S2-FHA protein performed using SYPRO Orange. The rate of change of fluorescence over time [-d(RFU) / dt] as a function of temperature is shown. [Figure 3-1] Figure 3 shows expression and stability screening of Ala cavity mutants. A) SDS-PAGE under reducing conditions of S2P-FHA mutants purified from culture supernatants using TALON divalent cation affinity resin. 10% polyacrylamide gels were stained with Coomassie blue. [Figure 3-2] (Continued from Figure 3) B) Differential scanning fluorescence measurements of purified S protein performed using SYPRO Orange, showing the rate of change of fluorescence over time [-d(RFU) / dt] as a function of temperature. [Figure 3-3] (Continued from Figure 3) B) Differential scanning fluorescence measurements of purified S protein performed using SYPRO Orange, showing the rate of change of fluorescence over time [-d(RFU) / dt] as a function of temperature. [Figure 4-1] FIG. 4 shows purification and characterization of selected Ala cavity mutants. A) Superose6 SEC of S2P-FHA and selected Ala cavity mutants after elution from TALON resin. Calibration standards are thyroglobulin (669 kDa), ferritin (440 kDa), and IgG (150 kDa). B) Superose6 SEC of purified trimers after freeze (-80°C)-thaw cycles. C) Differential scanning fluorimetry of purified S proteins performed using SYPRO Orange. The rate of change of fluorescence over time [-d(RFU) / dt] as a function of temperature is shown. Representative of at least two independent experiments. [Figure 4-2] (Continued from Figure 4) D) SDS-PAGE of purified S2P-FHA variant trimers under non-reducing (top) and reducing (bottom) conditions. 10% polyacrylamide gels were stained with Coomassie blue. [Figure 5-1] Figure 5 shows biolayer interferometry of human monoclonal anti-S2 IgG binding to ACE2-Fc and Ala cavity mutants. A) Binding of S2P-FHA, 1016L, and 1016 / 20VI analytes with S ligand immobilized on anti-human IgG capture biosensor. Association was 300 seconds followed by dissociation for 300 seconds. S2P = S2P-FHA. [Figure 5-2](Continued from FIG. 5) A) Binding of S2P-FHA, 1016L, and 1016 / 20VI analytes to the S ligand immobilized on an anti-human IgG capture biosensor. Association was for 300 seconds, followed by dissociation for 300 seconds. S2P=S2P-FHA. [Figure 5-3] (Continued from Figure 5) B) Relative binding of 300 nM S2P-FHA analyte with various ligands after 240 seconds of association shown as a heat map. [Figure 6-1] Figure 6 shows the immunogenicity of Ala cavity mutants. A) Immunization protocol. B) ELISA titers of final bleed vaccine sera against plates bound with RBD, S1 monomer, and S2P-FHA trimer. Immunogen groups are indicated below the graph. Endpoints were determined as 10x the background OD obtained in the absence of primary antibody. Bars are geometric means. S2P=S2P-FHA [Figure 6-2] (Continued from Figure 6) C) Pseudovirus neutralization ID50 of vaccine sera at week 16. S genotypes used in the S-HIV pseudotype assay are shown below the graph. Wilcoxon matched rank test was used to determine whether the differences in ID50 observed between groups were significant: ns not significant, * P<0.05, ** P<0.01, ** P<0.01. B. Geometric mean neutralization ID50 and fold reduction in mean neutralization for 1.351 vs. Hu-1 pseudotypes are shown below the graph. S2P=S2P-FHA. D) Binding titers of sera against Hu-1 RBD and RBD-NKY (N417N / E484K / N501Y) mutants. Endpoints were determined as 10x the background OD obtained in the absence of primary antibody. Bars are geometric means. ns not significant. Kruskal-Wallis test. S2P=S2P-FHA [Figure 7] Figure 7 shows the ancestral Hu-1 S-pseudovirus neutralization ID50 of vaccine sera. ns: not significant. **: P<0.01, Kruskal-Wallis test. [Figure 8]Figure 8 shows pseudovirus neutralization ID50 of vaccine sera at week 16 for mutant S pseudotypes. The S genotypes used in the S-HIV pseudotype assay are indicated below the graph. Friedman test was used to determine whether differences in ID50 observed between groups were significant: ns not significant, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Hu-1=ancestral Hu-1. [Figure 9-1] Figure 9 shows the specificity of induced antibodies assessed by competitive ELISA. A) Competitive ID50 of vaccine sera. 0.5 log10 serially diluted terminal bleed vaccine sera were mixed with a constant amount of ACE2-Fc and human monoclonal anti-S IgG before incubation with avidin-captured biotinylated S2P-FHA and Ala cavity mutant. Immunogen groups are indicated below the graph. A) Kruskal-Wallis test was used to determine the significance of the ID50 differences observed between groups: ns is not significant, ** is P<0.01. [Figure 9-2] (Continued from Figure 9) B) Location of antibody epitopes in the S2P-FHA trimer. S2P = S2P-FHA. B) Location of ACE2-Fc and human monoclonal anti-S IgG binding sites (epitopes) in the S trimer. [Figure 10] FIG. 10 shows the amino acid sequence of SEQ ID NO:1. [Figure 11] FIG. 11 shows the amino acid sequences of S2P (SEQ ID NO: 2) and S2P-FHA (SEQ ID NO: 3). [Figure 12] FIG. 12 shows the nucleotide sequence of S2P (SEQ ID NO:4). [Figure 13] FIG. 13 shows the nucleotide sequence of S2P-FHA (SEQ ID NO:5). [Figure 14]Figure 14 shows that the subject mutant S antigens induce stronger immune responses against wild-type (Hu-1) virus and against highly resistant variants (beta) than the current conventional vaccine encoding the wild-type virus. Pseudovirus neutralizing ID50 of vaccine sera at week 16 for mutant S pseudotypes compared to vaccine responses generated in humans using conventional vaccines. The S genotypes used in the S-HIV pseudotype assay are shown below the graph. Data show neutralizing antibody responses against matched WT (Hu-1) viruses and highly resistant variants of concern (beta variants) from guinea pigs vaccinated with S2P-FHA protein trimers containing 1016 / 20VI (Burnet VI spike on the left) compared to sera obtained 3-5 weeks after two doses of conventional vaccine in humans (exemplary conventional vaccines (two doses of mRNA or adenovirus vaccine given to humans) on the right). Wilcoxon matched rank test was used to determine whether differences in ID50 observed between groups were significant: ns not significant, P≧0.05, ****P<0.0001. [Figure 15] FIG. 15 shows ELISA binding titers of vaccine sera. A) ELISA binding titers of vaccine sera against biotinylated RBD monomer corresponding to Hu-1, delta, and omicron BA.1 variants (indicated on the x-axis) captured on avidin-coated ELISA plates. Immunogen group is indicated below the variant name. Endpoint was determined as 5-fold background OD obtained in the absence of primary antibody. Horizontal bars are geometric mean binding titers in each immunogen group. B) ELISA binding titers of vaccine sera against S2P-FHA corresponding to Hu-1, delta, and omicron BA.1 variants (indicated on the x-axis) bound to ELISA plates. Immunogen group is indicated below the variant name. Endpoint was determined as 5-fold background OD obtained in the absence of primary antibody. Horizontal bars are geometric mean binding titers in each immunogen group. *, P<0.05 compared to Hu-1 glycoprotein as determined by Kruskal-Wallis test. If no P value is shown, the difference in titers is not statistically significant. [Figure 16-1]FIG. 16 shows the results of the neutralization assay. A) Pseudovirus neutralization ID50 of vaccine sera (ID50=reciprocal dilution of serum causing 50% neutralization). The S variant genotype used in the S-HIV pseudotype assay is indicated below the x-axis. The immunogen group from which the sera were obtained is indicated above the graph. The ID50 neutralization titers obtained with sera from individual animals are indicated by the various symbols. Bars are the geometric mean ID50 in each immunogen group. A Kruskal-Wallis test was used to determine whether the differences in ID50 observed between groups were significant: ns is not significant. [Figure 16-2] (Continued from Fig. 16) B) Neutralization assays performed in a high-throughput format on HAT-24 cells using Hu-1, Delta, Omicron BA.1, and Beta authentic infectious SARS-CoV-2 viruses in a microneutralization assay. The SARS-CoV-2 variants used are indicated below the x-axis. The immunogen groups from which sera were obtained are indicated above the graph. The ID50 neutralization titers obtained with sera from individual animals are indicated by the various symbols. The horizontal bars are the geometric mean ID50 for each immunogen group. A Kruskal-Wallis test was used to determine whether the differences in ID50 observed between variants were significant: ns is not significant, * is P<0.05, ** is P<0.01, *** is P<0.001, **** is P<0.0001. [Figure 17] FIG. 17 shows the mean fold reduction in the geometric mean of biotin-RBD and S2P-FHA ELISA binding titers, neutralization ID50, and mean titers across glycoproteins and viral variants compared to Hu-1 from the data presented in FIGS. 15 and 16. [Figure 18-1]FIG. 18 shows that introduction of the 1016 / 20VI (VI) mutation into S2P-FHA derived from the SARS-CoV-2 OmicronBA.1 variant of concern (herein referred to as S2P.omicron-FHA) confers hyperstability to the glycoprotein trimer. A) Superose6 SEC profiles of S2P.omicron-FHA, S2P.omicron.VI-FHA expressed in Expi-293F cells, and S2P-FHA (from Hu-1) expressed in 293FS cells after purification by TALON affinity chromatography. The elution positions of the molecular weight markers thyroglobulin (669 kDa), ferritin (440 kDa), and aldolase (158 kDa) are indicated by arrows above the graph. The dashed box indicates the fractions pooled to obtain the pure trimer shown in B. B) Superose6 SEC profiles of putative S2P.omicron-FHA, S2P.omicron.VI-FHA, and S2P-FHA trimer obtained from the experiment shown in A. The dashed box in A indicates fractions that were concentrated and subjected to freeze (-80°C)-thaw cycles before reelution in Superose6 to obtain B. The elution positions of the molecular weight markers thyroglobulin (669 kDa), ferritin (440 kDa), and aldolase (158 kDa) are indicated by arrows above the graph. C) Differential scanning fluorimetry of purified S2P-FHA protein performed using SYPRO Orange to determine the temperature of thermal unfolding. Representative of at least two independent experiments. [Figure 18-2](Continued from FIG. 18) D) SDS-PAGE of S2P.Omicron-FHA and S2P.Omicron.VI-FHA trimer under non-reducing and reducing (1% v / v beta-mercaptoethanol). In this experiment, samples were not boiled prior to electrophoresis. m is a marker. E) SDS-PAGE of S2P.Omicron-FHA and S2P.Omicron.VI-FHA trimer under non-reducing and reducing (1% v / v beta-mercaptoethanol) conditions. Samples were boiled for 5 min prior to electrophoresis. The position of the main S2P.Omicron.VI-FHA band under various conditions is indicated by an arrow. m is a marker. F) SDS-PAGE of S2P.Omicron-FHA, S2P.Omicron.VI-FHA, and S2P-FHA trimer under non-reducing (left gel) and reducing (1% v / v beta-mercaptoethanol) (right gel) conditions. SDS-containing sample buffer was added to the samples with and without the addition of 1% beta-mercaptoethanol, and the samples were left at room temperature (25°C) or boiled (100°C) for 3 min prior to electrophoresis. Thyroglobulin that had been chemically cross-linked with 1 mM bis(sulfosuccinimidyl) suberate was included to show the expected position of S2P.omicron.VI-FHA trimer (669 kDa). The putative S2P.omicron.VI-FHA trimer band is indicated by an arrow. Note that S2P.omicron.VI-FHA is destroyed to its monomeric molecular weight only after boiling in the presence of beta-mercaptoethanol and SDS. [Figure 19] Figure 19 shows binding of human neutralizing monoclonal antibodies to streptavidin-captured biotinylated S2P.omicron-FHA (panel left) and biotinylated S2P.omicron.VI-FHA (panel right) trimers in an ELISA. The specificity of the monoclonal is indicated next to the legend. [Figure 20-1]Figure 20 shows that the 1016 / 20VI (VI) mutation allows trimerization of the prefusion stabilized S ectodomain (residues 16-1208) in the absence of the T4 foldon trimerization tag. A) Cryo-EM structure of the prefusion S trimer ectodomain obtained from solubilized full-length S (Cai et al. 2020). The receptor binding domain is shown in black, and the central coiled coil of S2 (in which the 1016 / 20VI mutation was introduced) with its alanine cavity is highlighted in dark grey. The three helices at the bottom of the trimer are part of the stem that is visualized in the cryo-EM structure. The dashed rectangle indicates the part of the stem that is not visualized in the cryo-EM structure. The crystal structure of the foldon domain solved in isolation (Guthe et al. 2004) has been added for illustrative purposes. The structure of residues 1217-1237 of the S2 transmembrane domain (TMD) in lipid bicelles as resolved by nuclear magnetic resonance (Fu and Chou, 2021) is also shown for illustrative purposes. The vertical lines indicate the range of constructs analyzed by SEC, thermofluor assay, and SDS-PAGE in panels B-D, respectively. [Figure 20-2](Continued from FIG. 20) B) Size exclusion chromatography of S2P-FHA, S2P-1208.H6, and S2P.VI-1208.H6. First panel: Superose6 elution profile of S2P-FHA purified from 293FS cells by TALON affinity chromatography. Arrows indicate the elution positions of molecular weight standards: thyroglobulin (669 kDa), ferritin (440 kDa), and aldolase (158 kDa). Second panel: Superose6 elution profile of S2P-1208.H6 (lacking the Foldon domain) purified from 293FS cells by TALON affinity chromatography. Third panel: Superose6 elution profile of S2P.VI-1208.H6 (containing the 1016 / 20VI mutation and lacking the Foldon domain) purified from 293FS cells by TALON affinity chromatography. Fourth panel: Superose6 elution profile of S2P.VI-1208.H6 (containing the 1016 / 20VI mutation and lacking the foldon domain) purified from Expi293F cells by hiTRAP affinity chromatography. C) Differential scanning fluorescence measurements of purified S trimers after freeze (-80 °C)-thaw cycles performed using SYPRO Orange to determine the temperature of thermal unfolding. Representative of at least two independent experiments. D) SDS-PAGE of purified proteins under non-reducing and reducing conditions. m is the marker. [Figure 21-1] Figure 21A) Schematic of the S2P-1273, S2P-FHA, and S2P-1208.H6 constructs. L is the native leader peptide, NTD is the N-terminal domain of S1, RBD is the receptor binding domain, (P)GSAS is the mutated cleavage furin site, tPAL is tissue plasminogen activator leader peptide, TMD is the transmembrane domain, FHA is the Foldon-His8-avitag sequence. [Figure 21-2](Continued from FIG. 21) B) SDS-PAGE-Western blot of Hu-1, delta, and omicron BA.1 S2P-1273, and S2P.VI-1273 glycoproteins expressed in 293T cells. S proteins were detected with rabbit anti-S1 polyclonal antibody. The left gel is a 10% polyacrylamide gel. The right gel is a 4-12% polyacrylamide gradient gel. [Figure 22] Figure 22 shows the binding of ACE2-Fc and human monoclonal NAbs to the S2P-1273 glycoprotein expressed on the surface of transfected 293T cells, as determined by FACS. Transfected cells were gently detached from the culture plate, and intact cells were stained with ACE2-Fc and various human monoclonal NAbs, as well as AlexaFluor-conjugated anti-human immunoglobulin. Cells were counterstained with propidium iodide to allow the exclusion of dead cells from the analysis. HC33.1 is an HCV-specific NAb and is used as an isotype (IgG1) control. [Figure 23-1] FIG. 23A) Gating of unstained (nil), moderately (lo) fluorescent and highly (hi) fluorescent S2P-1273 expressing cells. [Figure 23-2] (Continued from Figure 23) B) Representative data showing the distribution of S2P-1273 expressing cells stained with five NAbs directed against various epitopes in ACE2-Fc and S within the nil, lo, and hi gates in graphical format. [Figure 24] FIG. 24 shows the amino acid sequence of S2P. omicron-FHA (SEQ ID NO:6). [Diagram 25] FIG. 25 shows the DNA sequence of S2P. omicron-FHA (SEQ ID NO:7). [Figure 26] FIG. 26 shows the amino acid sequence of S2P-1208.H6 (SEQ ID NO:8). [Figure 27] FIG. 27 shows the DNA sequence of S2P-1208.H6 (SEQ ID NO:9). [Figure 28] FIG. 28 shows the protein sequence of S2P-1273 (SEQ ID NO:10). [Figure 29] FIG. 29 shows the DNA sequence of S2P-1273 (SEQ ID NO:11). [Diagram 30] FIG. 30 shows the protein sequence of S2P.delta-1273 (SEQ ID NO:12). [Diagram 31] FIG. 31 shows the DNA sequence of S2P.delta-1273 (SEQ ID NO:13). [Diagram 32] FIG. 32 shows the protein sequence of S2P. omicron-1273 (SEQ ID NO: 14). [Diagram 33] FIG. 33 shows the DNA sequence of S2P. omicron-1273 (SEQ ID NO:15). [Diagram 34] Figure 34 shows an example of an mRNA vaccine sequence encoding the S2P-1208 open reading frame (SEQ ID NO: 16). In this sequence, "U" represents pseudouridine or 1-methylpseudouridine. [Diagram 35] Figure 35 shows an example of an mRNA vaccine sequence encoding the S2P.VI-1273 open reading frame (SEQ ID NO: 17). In this sequence, "U" represents pseudouridine or 1-methylpseudouridine. [Diagram 36] Figure 36 shows an example of an mRNA vaccine sequence encoding the S2P.delta.VI-1273 open reading frame (SEQ ID NO: 18). In this sequence, "U" represents pseudouridine or 1-methylpseudouridine. [Figure 37] Figure 37 shows an example of an mRNA vaccine sequence (SEQ ID NO: 19) encoding the S2P.omicron.VI-1273 open reading frame. "U" in this sequence represents pseudouridine or 1-methylpseudouridine. [Figure 38]Figure 38 shows SDS-PAGE / Western blot analysis of S2P-1273 and S2P.omicron-1273 from transfected 293T cell lysates after various heat treatments in the presence of 1.2% (w / v) SDS and 0.25% (v / v) beta-mercaptoethanol. AA: constructs containing Ala at amino acid positions 1016 and 1020, VI: constructs containing Val and Ile at amino acid positions 1016 and 1020, respectively. The positions of S monomers and trimers, respectively, are shown, including AA and VI forms of purified S2P.omicron-FHA trimers treated with 0.67% SDS for 5 min at room temperature. Samples were electrophoresed on 5% SDS-PAGE gels, transferred to nitrocellulose, and blotted with rabbit anti-S1 and anti-rabbit IRDye800. Blots were scanned on a LiCOR Odyssey instrument. [Figure 39] Figure 39 shows that the 1016 / 20VI (VI) mutation stabilizes trimerization of the prefusion stabilized OmicronBA.1S ectodomain (residues 16-1208) in the absence of the T4 Foldon trimerization tag. A) Superose 6 size exclusion chromatography of S2P.Omicron.BA.1-1208.H6 (top) and S2P.Omicron.BA1.VI-1208.H6 (bottom) purified from Expi293F cells by hiTRAP affinity chromatography. B) Superose 6 size exclusion chromatography of S2P.Omicron.BA.1-1208.H6 (top) and S2P.Omicron.BA1.VI-1208.H6 (top) trimers purified in A after freeze (-80°C)-thaw cycles. C) Differential scanning fluorescence measurements of purified S trimers after freeze (-80°C)-thaw cycles using SYPRO Orange to determine the temperature of thermal unfolding. D) SDS-PAGE under non-reducing conditions and Coomassie blue staining of purified proteins. WT: Ala at amino positions 1016 and 1020, VI: Val and Ile at amino positions 1016 and 1020, respectively. [Diagram 40]Figure 40 shows biolayer interferometry of human monoclonal anti-S IgG binding to ACE2-Fc and Ala cavity mutants. A) Binding of S2P-FHA and S2P.VI-FHA analytes derived from Hu-1 sequence with S ligand immobilized on anti-human IgG capture biosensor. B) Binding of S2P-FHA and S2P.VI-FHA analytes derived from Omicron.BA.1 sequence with S ligand immobilized on anti-human IgG capture biosensor. Association was for 300 seconds followed by dissociation for 300 seconds. [Diagram 41] Figure 41 is the protein sequence of S2P. omicron-1208 (SEQ ID NO:25). [Diagram 42] Figure 42 shows the protein sequence of the DNA sequence of S2P. omicron-1208 (SEQ ID NO:26).

[0036] [Table 1-1] [Table 1-2]

[0037] Considerations of the embodiment Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Any materials and methods similar or equivalent to those described herein can be used to practice or test the present disclosure. Practitioners are particularly directed 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, eds., Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications, 1986; Kontermann and Dubel (Ed), Antibody Engineering, Vol 1-2, Ed., Springer Press, 2010) for definitions and terms of the art and other methods known to those skilled in the art.

[0038] The sequence of the coronavirus spike (S) protein from the ancestral Hu-1 strain is described by Wu, F., et al Nature 579(7798), 265-269(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.

[0039] As used herein, "antigen" refers to a substance capable of stimulating an immune response.

[0040] As used herein, "protection" refers to immunity or partial immunity against coronavirus infection.

[0041] "Alanine cavity" or "cavity" herein refers to the observed region and reduced interactions between monomers of the trimer structure within the coiled coil of SARS-CoV S protein due to the absence of amino acids with non-polar side chains bulkier than alanine or aromatic residues. In one embodiment, the alanine cavity includes A1016 and A1020 as set forth in any one of SEQ ID NO:1-3.

[0042] As used herein, with reference to exemplary embodiments of the invention, reference to S2P includes embodiments with or without the FHA sequence, unless specifically specified.

[0043] As used herein, "trimerization sequence" refers to a sequence found in the C-terminal region of an S protein monomer that promotes trimerization of the S protein trimer. In some embodiments, the trimerization domain is a heterologous sequence not found in coronaviruses. In one embodiment, the trimerization sequence is a heterologous sequence not found in SARS-COV2. In some embodiments, the trimerization sequence is the trimer Foldon domain of bacteriophage T4 fibritin or a modified version thereof (Figure 20A). In some embodiments, the trimerization sequence is a coiled coil, an artificial coiled coil, or a modified coiled coil. In some embodiments, the trimerization sequence is designed de novo, and in some embodiments, the trimerization sequence is the trimer Foldon domain of bacteriophage T4 fibritin or a modified version thereof. In some embodiments, the trimerization sequence is a native CoV trimerization sequence (in some embodiments, it is a native transmembrane domain). In some embodiments, the trimerization sequence comprises, or consists of, residues 1209 to 1256 of the S protein monomer. In some embodiments, the trimerization sequence comprises, or consists of, residues 1217 to 1237 of the S protein monomer.

[0044] coronavirus The term "coronavirus family" refers to viruses commonly known as "coronavirus" or "CoV", which are enveloped, positive single-stranded RNA viruses. There are two subfamilies of coronavirus family: the Retrovirinae and the Orthocoronavirus. In one embodiment, the CoV is selected from the genus Alphacoronavirus (alphaCoV), the genus Betacoronavirus (betaCoV), the genus Gammacoronavirus (gammaCoV), and the genus Deltacoronavirus (deltaCoV). In one embodiment, the alphaCoV 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, the beta-CoV 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), mouse 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 subtypes or variants thereof.

[0045] In one embodiment, the CoV is SARS-CoV-2 or a subtype or variant thereof. In one embodiment, the SARS-CoV-2 is SARS-CoV-2hCoV-19 / Australia / VIC01 / 2020. In one embodiment, the SARS-CoV-2 comprises a sequence set forth in NCBI Reference Sequence: NC_045512.2. In one embodiment, the SARS-CoV-2 comprises a sequence set forth in Gen Bank: MN908947.3, or a variant thereof. Examples of SARS-CoV-2 variants are described, for example, 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, and Sanyaolu et al., 2021.

[0046] In one embodiment, the CoV variant is at least 90% identical to the parent sequence. In one embodiment, the variant is at least 92% identical to the parent sequence. In one embodiment, the variant is at least 93% identical to the parent sequence. In one embodiment, the variant is at least 94% identical to the parent sequence. In one embodiment, the variant is at least 95% identical to the parent sequence. In one embodiment, the variant is at least 96% identical to the parent sequence. In one embodiment, the variant is at least 97% identical to the parent sequence. In one embodiment, the variant is at least 98% identical to the parent sequence. In one embodiment, the variant is at least 99% identical to the parent sequence. In one embodiment, the parent strain (also called the ancestral strain) is the Hu-1 strain, as reported by Wu, F., et al Nature 579(7798), 265-269(2020). In some embodiments, the parent strain is SARS-CoV-2 hCoV-19 / Australia / VIC01 / 2020. In some embodiments, the parent strain is BetaCoV / Ancestral / WIV04 / 2019.

[0047] In one embodiment, the CoV is a "variant of interest," also referred to as a "VOI." As used herein, a VOI is a coronavirus variant that is predicted or associated with known genetic changes that affect viral characteristics such as transmissibility, disease severity, immune escape, diagnostic escape, or therapeutic escape, and that has been identified to cause significant community transmission or multiple disease clusters (in the case of the SARS-CoV-2 COVID19 cluster) in multiple countries with increasing relative prevalence with increasing case numbers over time, or other clear epidemiological impacts that suggest an emerging risk to global public health.

[0048] In one embodiment, the CoV is a "variant of concern," also referred to as a "VOC." As used herein, a VOC is a coronavirus variant that is associated with one or more of the following changes of global public health importance: an increase in transmissibility or a detrimental change in epidemiology (in the case of SARS-CoV-2, the detrimental change is in COVID-19 epidemiology), an increase in virulence or a change in clinical disease symptoms, or a reduction in the effectiveness of public health and social measures or available diagnostics, vaccines, and therapies.

[0049] In an embodiment, the CoV is a VOC or VOI as reported in Vasireddy et al. (2021), Winger et al. (2021), or Sanyaolu et al. (2021). In an embodiment, the CoV is classified as a VOC, VOI, or VHC by a health regulatory body, such as the World Health Organisation (WHO), the United States Center for Disease Control (CDC), the European Centre for Disease Prevention and Control (ECDC), or an equivalent local government health regulatory body in a particular jurisdiction. In an embodiment, the coronavirus is classified as a VOC or VOI by the WHO. In an embodiment, the coronavirus is classified as a VOC, VOI, or VHC by the CDC. In an embodiment, the coronavirus is classified as a VOC or VOI by the ECDC.

[0050] In an embodiment, the CoV is a "VHC", also referred to as a "VHC". In an embodiment, the VHC as clear evidence that preventive or medical countermeasures have significantly reduced effectiveness compared to previously circulating variants. In addition to the characteristics of a VOC, a VHC may have one or more of the following effects on medical countermeasures: demonstrated failure of diagnostic test targets, evidence suggesting a significant reduction in vaccine efficacy, a disproportionately high number of vaccine breakthrough cases, or very low vaccine-induced protection against disease, significantly reduced susceptibility to multiple emergency use authorizations or approved therapeutics, and more severe clinical disease and increased hospitalizations.

[0051] In embodiments, when the CoV is a SARS-CoV-2 VOC, the VOC is selected from the group consisting of the following mutations: 69del, 70del, 144del, E484K / Q, S494P, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, K1191N, D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y, D614G, A701V, T1 9R, V70F, T95I, G142D, E156-, F157-, R158G, A222V*, W258L*, K417N / T*, L452R, T478K, D614G, P681H / R, D950N, L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, E484K, H655Y including one or more of A67V, del69-70, T95I, del142-144, Y145D, del211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F.

[0052] In embodiments, where the CoV is a SARS-CoV-2 VOC, the VOC comprises one or more of the following RBD mutations: K417T, N439K, L452R, Y453F, S477N, T478K / Q, and N501Y. In embodiments, the VOC comprises one or more of the following NTD mutations: 70del, 156-157del, and 242-245del.

[0053] In an embodiment, the VOC is B.1.1.7 or a variant thereof. In an embodiment, the VOC is B.1.351 or a variant thereof. In an embodiment, the VOC is B.1.351.2 or a variant thereof. In an embodiment, the VOC is B.1.351.2 or a variant thereof. In an embodiment, the VOC is B.1.351.3 or a variant thereof. In an embodiment, the VOC is P1 or a variant thereof. In an embodiment, the VOC is P1.1 or a variant thereof. In an embodiment, the VOC is P1.2 or a variant thereof. In an embodiment, the VOC is B.1.617.2 or a variant thereof. In an embodiment, the VOC is AY.1 or a variant thereof. In an embodiment, the VOC is AY.2 or a variant thereof. In an embodiment, the VOC is AY.3 or a variant thereof. In an embodiment, the VOC is B.1.1.529 or a variant thereof. In an embodiment, the VOC is BA.1 or a variant thereof. In an embodiment, the VOC is BA.2 or a variant thereof. In an embodiment, the VOC is BA.3 or a variant thereof. In an embodiment, the VOC is BA.4 or a variant thereof. In an embodiment, the VOC is BA.5 or a variant thereof.

[0054] In embodiments, when the CoV is a SARS-CoV-2 VOI, the VOI comprises 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 and Q957R, N501Y, P681R, P681H, E484Q, P681R, S477N, L452Q, and F490S. In embodiments, the VOI is B.1.525 or a variant thereof. In an embodiment, the VOI is B.1.526 or a variant thereof. In an embodiment, the VOI is B.1.617.1 or a variant thereof. In an embodiment, the VOI is C37 or a variant thereof. In an embodiment, the VOI is B.1.427 or a variant thereof. In an embodiment, the VOI is B.1.429 or a variant thereof. In an embodiment, the VOI is P2 or a variant thereof. In an embodiment, the VOI is B.1.525 or a variant thereof. In an embodiment, the VOI is P3 or a variant thereof. In an embodiment, the VOI is B.1.620 or a variant thereof. In an embodiment, the VOI is B.1.621 or a variant thereof. In an embodiment, the VOI is C.37 or a variant thereof.

[0055] CoV infection can cause respiratory, enteric, hepatic, and neurological diseases in different animal species, including camels, cattle, cats, and bats. CoVs can be transmitted from one individual to another through contact of viral droplets with mucous membranes. Typically, viral droplets become airborne and are inhaled through the respiratory tract, including the nasal airways. Typically, the individual is a human individual. In some embodiments, the individual is a livestock or pet animal. Typically, during the infectious period, CoVs can be found in the upper respiratory tract, e.g., the nasal cavity. In some instances, CoVs can be found in the lower respiratory tract, e.g., the bronchi and / or alveoli.

[0056] In embodiments, CoV infection causes one or more symptoms selected from one or more of fever, cough, sore throat, shortness of breath, virus-excretion respiratory failure, runny nose, nasal congestion, fatigue, bronchitis, headache, muscle pain, dyspnea, moderate pneumonia, severe pneumonia, and acute respiratory distress syndrome (ARDS). In embodiments, 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 embodiments, SARS-CoV-2 infection can cause one or more symptoms selected from one or more of fever, cough, sore throat, shortness of breath, virus-excretion respiratory failure, runny nose, nasal congestion, fatigue, bronchitis, headache, muscle pain, dyspnea, moderate pneumonia, severe pneumonia, and acute respiratory distress syndrome (ARDS). In embodiments, CoV infection is asymptomatic.

[0057] SARS-CoV-2 In embodiments, the coronavirus vaccine antigen described herein induces an immune response against SARS-CoV-2. In embodiments, the coronavirus vaccine antigen described herein comprises the SARS-CoV-2 S protein trimer.

[0058] SARS-CoV-2 has four major 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 within the ribonucleoprotein. The S protein recognizes host cell receptors to initiate virus entry.

[0059] The viral S glycoprotein mediates receptor attachment and virus-cell membrane fusion and is the 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, which are derived from a polyprotein precursor, S, by furin cleavage of oligobasic motifs as it transits the Golgi. ACE2 receptor attachment is mediated by the RBD within the large subunit, S1, whereas membrane fusion is mediated by the small subunit, S2, which contains the fusion peptide. S1 and S2 form a heterodimer through non-covalent interactions, and the coiled-coil-forming α-helix of S2 (amino acids 986-1033, designated CH) forms the core of the trimer (Wrapp et al., 2020) (Figure 1A). The transmembrane sequence at the C-terminus of S2 stabilizes the trimer and anchors it to the viral or cellular membrane (Fu et al., 2021). The ACE2 RBD is located on top of the S1 glycoprotein trimer and presents an ACE2-binding ready orientation in the "up" and an inactive orientation in the "down" (Ke et al., 2020). After receptor attachment, S2 is cleaved by TMPRSS2 protease at the cell surface, releasing the fusion peptide and full fusion activation. The S glycoprotein mediates membrane fusion via a class I mechanism, whereby the activation trigger (ACE2 binding by S1, TMPRSS2 cleavage of S2) refolds the S2 subunit of the metastable prefusion trimer into a stable trimer of hairpins, bringing the N-terminal fusion peptide and the C-terminal transmembrane sequence together so that their associated membranes fuse (Cai et al., 2020).

[0060] Class I viral fusion glycoproteins, such as S of betacoronaviruses, Env of retroviruses, and HA of orthomyxoviruses, contain a central coiled coil, which serves as a scaffold for the conformational changes required for the membrane fusion process (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) (Figure 1A). In the case of SARS-CoV-2, prior to fusion, the coiled coil comprises amino acids 988-1031 as shown in SEQ ID NO:1 (NCBI reference sequence YP_009724390.1). This region of an exemplary SARS-CoV-2 strain is shown in Figure 1B. After fusion, the coiled-coil sequence is extended to include amino acids 913 to 1031 as shown in SEQ ID NO: 1 (NCBI Reference Sequence YP_009724390.1). In some embodiments, residues 986 and 987 are modified to proline (K986P, V987P).

[0061] The inner surface positions of the coiled coil are usually occupied by hydrophobic residues in 3-4 repeats. In the case of SARS-CoV and SARS-CoV-2 S, these positions are mostly occupied by polar residues that mediate few interhelical contacts in the prefusion trimer (Figure 1B). In the postfusion trimer, the N-terminal 2 / 3 of the coiled coil is held together by the packing of the HR1 helix, which extends the coiled coil towards the N-terminus by 110 Å. In this conformation, the inner surface residues are close enough to form hydrogen bonds (Figure 1C). Ile1013 forms a small hydrophobic core by interhelical contacts with I1013 and L1012. These interactions form a hydrophobic ceiling above the cavity formed by A1016 and A1020, which occupy central positions of the coiled coil (Figure 1A-D).

[0062] Vaccine antigens In an aspect, the invention provides a coronavirus vaccine antigen comprising a CoV S protein trimer, the S protein trimer modified to contain a structural modification that reduces the size of an alanine cavity within the coiled-coil of the S protein trimer, and the S protein trimer elicits a neutralizing antibody response.

[0063] In aspects, the structural modifications stabilize the S protein trimer. As used herein, "stabilized" refers to increasing one or more of thermal stability, longevity, immunogenicity and production stability, yield or homogeneity of the S protein trimer, and denaturation stability. In embodiments, the stability is increased in vitro and / or in vivo stability. In embodiments, the in vivo stability is increased (when administered to a subject or upon assembly in a subject, e.g., after translation from a nucleic acid, such as an mRNA vaccine). In embodiments, the stability is increased in vitro (e.g., during the production process).

[0064] In embodiments, the structural modification stabilizes the S protein trimer by reducing the size of the alanine cavity in the coiled coil. In embodiments, the alanine cavity is partially filled or fully filled. In embodiments, the alanine cavity is conformationally altered. In embodiments, the size of the alanine cavity 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 embodiments, the size of the alanine cavity is reduced by at least 5%. In embodiments, the size of the alanine cavity is reduced by at least 10%. In embodiments, the size of the alanine cavity is reduced by at least 20%. In embodiments, the size of the alanine cavity is reduced by at least 30%. In embodiments, the size of the alanine cavity is reduced by at least 40%. In embodiments, the size of the alanine cavity is reduced by at least 50%. In embodiments, the size of the alanine cavities is reduced by at least 60%. In embodiments, the size of the alanine cavities is reduced by at least 70%. In embodiments, the size of the alanine cavities is reduced by at least 80%. In embodiments, the size of the alanine cavities is reduced by at least 90%. In embodiments, the size of the alanine cavities is reduced by 100%.

[0065] In an embodiment, the size of the alanine cavities 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 an embodiment, the size of the alanine cavities is reduced by about 10% to 100%. In an embodiment, the size of the alanine cavities is reduced by about 10% to 90%. In an embodiment, the size of the alanine cavities is reduced by about 20% to 90%. In an embodiment, the size of the alanine cavities is reduced by about 20% to 80%. In an embodiment, the size of the alanine cavities is reduced by about 30% to 80%. In an embodiment, the size of the alanine cavities is reduced by about 40% to 80%. In an embodiment, the size of the alanine cavities is reduced by about 50% to 80%.

[0066] In embodiments, the structural modification increases the stability of the S protein trimer compared to an S protein trimer lacking the structural modification.

[0067] In embodiments, the structural modification increases the temperature at which the S protein trimer degrades compared to an S protein trimer lacking the structural modification, in embodiments, degrade or degradation refers to the exposure of hydrophobic residues in the core of the S protein trimer.

[0068] In embodiments, the structural modification increases the melting temperature of the S protein trimer compared to an S protein trimer lacking the structural modification. In embodiments, the structural modification increases the thermal stability of the S protein trimer. In embodiments, the structural modification increases the melting temperature of the S protein trimer by about 5°C to about 25°C. In embodiments, the structural modification increases the melting temperature of the S protein trimer by about 5°C to about 23°C. In embodiments, the structural modification increases the melting temperature of the S protein trimer by about 5°C to about 23°C. In embodiments, the structural modification increases the melting temperature of the S protein trimer by about 10°C to about 23°C. In embodiments, the structural modification increases the melting temperature of the S protein trimer by about 10°C to about 20°C. In embodiments, the structural modification increases the melting temperature of the S protein trimer by about 5°C to about 15°C. In embodiments, the structural modification increases the melting temperature of the S protein trimer by about 5°C to about 10°C.

[0069] In embodiments, the structural modifications increase the stability of the S protein trimer to denaturing conditions, such as boiling in the presence of a detergent (e.g., sodium dodecyl sulfate) or treatment with a detergent (e.g., sodium dodecyl sulfate) with and without the addition of 2 beta-mercaptoethanol at room temperature.

[0070] In one embodiment, the present disclosure provides a method for improving the stability and / or expression of a coronavirus S antigen.

[0071] In an embodiment, the CoV vaccine antigen is soluble. In an embodiment, the CoV vaccine antigen described herein does not include an FHA sequence. In an embodiment, the CoV vaccine antigen is stabilized in a pre-fusion S protein trimer confirmation. In an embodiment, the ACE2 receptor binding domain (RBD) of the S protein trimer is in a downward (non-ACE2 binding ready) orientation. In an embodiment, the RBD-down orientation generates neutralizing antibodies that recognize the S-trimer in the RBD-down conformation in addition to RBD-up directed neutralizing antibodies.

[0072] In embodiments, the CoV vaccine antigen lacks a trimerization sequence. In embodiments, the CoV vaccine antigen lacks a transmembrane domain. In embodiments, the CoV vaccine antigen lacks a foldon sequence / domain.

[0073] In embodiments, when the RBD is in a downward orientation, other non-RBD epitopes are in a favorable position for generating additional non-RBD neutralizing antibodies.

[0074] In embodiments the structural modification is within the coiled-coil region. In embodiments the structural modification stabilizes the coiled-coil region.

[0075] In embodiments, structural modifications in the coiled-coil in S2 have an allosteric effect on the immunogenicity of S1 that enhances the immune response to the CoV variants described herein.

[0076] In embodiments, the CoV vaccine antigen is suitable for intradermal administration. In embodiments, the CoV vaccine antigen is suitable for oral administration. In embodiments, the CoV vaccine antigen is suitable for pulmonary administration. In embodiments, the CoV vaccine antigen is suitable for nasal administration.

[0077] Coronavirus vaccine antigen S protein monomer In embodiments, the S protein monomer in the S-protein trimer can be an ancestral SARS-CoV-2 sequence described herein (e.g., NCBI Reference Sequence: YP_009724390.1) or a more recent variant such as a VOC, VOI, or VHC described herein (e.g., delta, beta, omicron). In embodiments, the S protein monomer is an ancestral SARS-CoV-2 sequence that has been modified to include one or more mutations present in a VOC, VO1, or VHC described herein. In embodiments, the modifications are one or more of S13I, L18F, T19R, T20N, P26S, A67V, delH69-V70, D80A, T95I, D138Y, G142D, delY144, W152C, E154K, E156del, F157del, R158G, R190S, D215G, del242-245, D253G, R246I, K 417N / 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.

[0078] In embodiments, the S protein monomer comprises residues 1 to 1208 of the amino acid sequence SEQ ID NO: 1, or a sequence at least 90% identical thereto. In embodiments, the S protein monomer comprises residues 1 to 1208 of the amino acid sequence SEQ ID NO: 2, or a sequence at least 90% identical thereto. In embodiments, the S protein monomer comprises residues 1 to 1208 of the amino acid sequence SEQ ID NO: 3, or a sequence at least 90% identical thereto.

[0079] In embodiments, the S protein monomer comprises the amino acid sequence of residues 1 to 1237 of SEQ ID NO:1, or a sequence at least 90% identical thereto.

[0080] In embodiments, the S protein monomer comprises residues 1 to 1237 of the amino acid sequence of SEQ ID NO:3, or a sequence at least 90% identical thereto.

[0081] In one embodiment, the S protein monomer comprises the amino acid sequence of residues 1 to 1256 of SEQ ID NO:1, or a sequence at least 90% identical thereto.

[0082] In embodiments, the S protein monomer comprises residues 1 to 1256 of the amino acid sequence of SEQ ID NO:3, or a sequence at least 90% identical thereto.

[0083] In embodiments, the S protein monomer comprises the amino acid sequence of SEQ ID NO:6, or a sequence at least 90% identical thereto.

[0084] In embodiments, the S protein monomer comprises the amino acid sequence of SEQ ID NO:8, or a sequence at least 90% identical thereto.

[0085] In embodiments, the S protein monomer comprises the amino acid sequence of SEQ ID NO:25, or a sequence at least 90% identical thereto.

[0086] In embodiments, the S protein monomer comprises the amino acid sequence of SEQ ID NO:26, or a sequence at least 90% identical thereto.

[0087] In an embodiment, the S protein monomer comprises a sequence encoding a coronavirus transmembrane domain. In an embodiment, the S protein monomer comprises a sequence encoding a SARS-COV2 transmembrane domain. In an embodiment, the transmembrane domain comprises residues 1217-1237 of the amino acid sequence of SEQ ID NO:1, or a sequence at least 90% identical thereto. In an embodiment, the transmembrane domain comprises residues 1217-1237 of the amino acid sequence of SEQ ID NO:3, or a sequence at least 90% identical thereto. In an embodiment, the transmembrane domain comprises residues 1209-1256 of the amino acid sequence of SEQ ID NO:1, or a sequence at least 90% identical thereto. In an embodiment, the transmembrane domain comprises residues 1209-1256 of the amino acid sequence of SEQ ID NO:3, or a sequence at least 90% identical thereto.

[0088] In embodiments, the S protein monomer does not include a sequence encoding a coronavirus transmembrane domain.

[0089] In embodiments, the S protein monomer comprises a 2P mutation as described herein.In embodiments, the S protein monomer does not comprise a 2P mutation as described herein. In embodiments, the S protein monomer comprises the amino acid sequence of one or more of the VOC and / or VOI mutations described herein.

[0090] In embodiments, the S protein monomer comprises S protein residues 1-1208 of the SARS-CoV-2 VOC. In embodiments, the S protein monomer comprises S protein residues 1-1208 of the SARS-CoV-2 VOI. In embodiments, the S protein monomer comprises S protein residues 1-1208 of the SARS-CoV-2 VHC.

[0091] In embodiments, the S protein monomer does not include a trimerization sequence. In embodiments, the S protein monomer does not include a transmembrane domain sequence. In embodiments, the S protein monomer does not include a Foldon sequence. In embodiments, the S protein monomer does not include an FHA.

[0092] Structural modifications The structural modification to the alanine cavity is effected by using one or more of the following: amino acid substitutions in the S protein, disulfide bonds, hydrogen bonds, pi-stacking (π-π stacking), salt bridges, van der Waals interactions, the use of hydrophobic residue substitutions or additions, or proline stabilization. In one embodiment, the structural modification to the alanine cavity is effected by amino acid substitution of one or more amino acids that form the alanine cavity. In an embodiment, the structural modification is a substitution of one or more amino acids with more hydrophobic amino acids. In an embodiment, the structural modification is a substitution of one or more amino acids with more hydrophobic amino acids in the coiled-coil region.

[0093] In an embodiment, one or two or three of the S protein monomers in the S protein trimer contain substitutions of one or more amino acids in the coiled-coil region with more hydrophobic amino acids. In an embodiment, one of the S protein monomers in the S protein trimer contains substitutions of one or more amino acids in the coiled-coil region with more hydrophobic amino acids. In an embodiment, two of the S protein monomers in the S protein trimer contain substitutions of one or more amino acids in the coiled-coil region with more hydrophobic amino acids. In an embodiment, three of the S protein monomers in the S protein trimer contain substitutions of one or more amino acids in the coiled-coil region with more hydrophobic amino acids.

[0094] Amino acid substitutions in the alanine cavity In embodiments, the structural modifications create an artificial hydrophobic core in the coiled-coil region. In embodiments, the structural modifications create an artificial hydrophobic core that includes residues of an alanine cavity. In embodiments, the structural modifications create an artificial hydrophobic core in an alanine cavity. In embodiments, the amino acids at positions 1016 and 1020 contribute to the formation of the artificial hydrophobic core.

[0095] In embodiments, the artificial hydrophobic core is created by replacing amino acids in the coiled-coil region with more hydrophobic amino acids, hi one embodiment, polar residues are replaced with bulkier hydrophobic residues.

[0096] As used herein, a "more hydrophobic amino acid" refers to an amino acid that is more hydrophobic than the amino acid present in the coronavirus strain position that is being substituted. For example, if the modified / substituted amino acid is alanine, it can be replaced with a more hydrophobic amino acid, such as isoleucine, leucine, methionine, valine, phenylalanine, tyrosine, and tryptophan.

[0097] The hydrophobicity index is a measure of the relative hydrophobicity, or how soluble an amino acid is in water, and is described, for example, in Sereda et al. (1994) and Monera et al., (1995). The normalized hydrophobicity of different amino acids at pH 2 and pH 7, such that the most hydrophobic residue is given a value of 100 relative to glycine (value 0), is provided in the table below. [Table 2]

[0098] In embodiments, the more hydrophobic amino acid is a hydrophobic amino acid. In embodiments, the hydrophobic amino acid is an aliphatic hydrophobic amino acid. In embodiments, the hydrophobic amino acid is an aromatic hydrophobic amino acid.

[0099] In an embodiment, at least one amino acid in the coiled-coil region of an S protein monomer in an S protein trimer is substituted with a more hydrophobic amino acid. In an embodiment, at least one S protein monomer in an S protein trimer comprises a substitution. In an embodiment, at least two S protein monomers in an S protein trimer comprise a substitution. In one embodiment, three S protein monomers in an S protein trimer comprise a substitution.

[0100] In embodiments, at least two amino acids in the coiled-coil region of the S protein monomers in the S protein trimer are substituted with more hydrophobic amino acids. In embodiments, at least one S protein monomer in the S protein trimer comprises the substitution(s). In embodiments, at least two S protein monomers in the S protein trimer comprise the substitution(s). In embodiments, three S protein monomers in the S protein trimer comprise the substitution(s).

[0101] In an embodiment, at least one amino acid or at least two amino acids are located at positions a and / or d of the heptad repeat motif of the coiled-coil region of the S protein monomer. The locations of positions a and d in the heptad repeat motif are shown in FIG. 1B. For SARS-COV-2, positions a and d correspond to amino acids 988, 991, 995, 998 1002, 1005, 1009, 1013, 1016, 1020, 1023, 1027, 1031 of SEQ ID NO: 1. In an embodiment, the substitution with a more hydrophobic amino acid occurs at position 1016. In an embodiment, the substitution with a more hydrophobic amino acid occurs at position 1020. In an embodiment, the substitution with a more hydrophobic amino acid occurs at positions 1016 and 1020. In an embodiment, A1016 or A1020 is substituted with leucine, valine, isoleucine, or phenylalanine. In an embodiment, A1016 is substituted with leucine (A1016L), valine (A1016V), or isoleucine (A1016I). In an embodiment, A1020 is substituted with isoleucine (A1020I). In an embodiment, A1016 is substituted with leucine (A1016L) or valine (A1016V) and A1020 is substituted with isoleucine (A1020I). In an embodiment, A1016 is substituted with leucine (A1016L). In an embodiment, A1016 is substituted with valine (A1016V). In an embodiment, A1020 is substituted with isoleucine (A1020I). In an embodiment, A1020 is not substituted with tryptophan (W). In embodiments, A1016 is substituted with valine and A1020 is substituted with isoleucine (referred to herein as "A1016V / A1020I" or "1016 / 20VI" or "VI").

[0102] In embodiments, the more hydrophobic amino acid comprises one or more of the following characteristics: i) more hydrophobic than alanine, ii) a more hydrophobic amino acid than alanine, ii) a hydrophobicity greater than 47 at a pH of 2, iii) a hydrophobicity greater than 41 at a pH of 7, and iv) selected from isoleucine, leucine, methionine, valine, phenylalanine, tyrosine, and tryptophan. In embodiments, the amino acid is selected from isoleucine, leucine, valine. In embodiments, the amino acid is isoleucine. In embodiments, the amino acid is leucine. In embodiments, the amino acid is valine. In embodiments, the amino acid is methionine. In embodiments, the amino acid is phenylalanine. In embodiments, the amino acid is tyrosine. In embodiments, the amino acid is tryptophan.

[0103] In embodiments, the more hydrophobic amino acid comprises a higher hydrophobicity than alanine. In embodiments, the more hydrophobic amino acid is greater than alanine. In embodiments, the more hydrophobic amino acid comprises a hydrophobicity of greater than 47 at a pH of 2. In embodiments, the more hydrophobic amino acid comprises a hydrophobicity of greater than 41 at a pH of 7. In embodiments, the more hydrophobic amino acid is selected from isoleucine, leucine, methionine, valine, phenylalanine, tyrosine, and tryptophan. In embodiments, the more hydrophobic amino acid is isoleucine. In embodiments, the more hydrophobic amino acid is leucine. In embodiments, the more hydrophobic amino acid is methionine. In embodiments, the more hydrophobic amino acid is valine. In embodiments, the more hydrophobic amino acid is phenylalanine. In embodiments, the more hydrophobic amino acid is tyrosine. In embodiments, the more hydrophobic amino acid is tryptophan.

[0104] For the avoidance of doubt, where optimal hydrophobicity has been achieved by amino acid substitutions in the alanine cavities (including A1016 and A1020), further conservative amino acid mutations can be made in those regions without affecting the desired performance of the spike proteins described herein. Conservative amino acid substitutions are known in the art.

[0105] Further modifications / further stabilization modifications In embodiments, the CoV vaccines described herein include one or more additional modifications to enhance one or more of the stability, immunogenicity, expression, and purification of the S protein trimer, or, if the vaccine is a polynucleotide-based vaccine, the additional modifications enhance the in vivo stability and expression of the polynucleotide comprising the S protein coding sequence or a soluble form thereof.

[0106] In embodiments, further modifications to the antigen or its coding molecule are selected from proline stabilization, a furin cleavage site, a trimerization sequence, repeats or spacers, or the nucleotide sequence encoding same.

[0107] In embodiments, the proline stabilizing 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.

[0108] In an embodiment, the further modification is the insertion of a furin cleavage site. In an embodiment, the mutation PG682SAS is introduced to insert a furin cleavage site (e.g., PG682SAS replaces RR682RAR with delta, PG682SAS replaces HR682RAR with omicron). In an embodiment, the further modification is the addition of FHA. In an embodiment, the further modification is the addition of a purification tag.

[0109] Antigen combination In an embodiment, the subject modified S antigen induces a broadly neutralizing immune response against the strain from which it is derived and one or more other strains circulating in the community. In another aspect, the antigen or vaccine comprising the antigen or coding sequence delivers one or more antigens of interest to a subject and induces an effective functional and polyfunctional immune response against homologous or heterologous strains, including, for example, T cell and antibody responses. In one embodiment, the coronavirus antigens from one or more strains are selected from one or two or three or four of spike, nucleocapsid, membrane, and envelope proteins. In one embodiment, amino acid and / or nucleotide sequences encoding two, three, or four of the SARS-CoV proteins N, M, E, and S are used. In an exemplary embodiment, N, M, E, and S are used. In an embodiment, one or two or more different SARS-CoV variants are combined. In an embodiment, multiple variants and multiple antigens are used. In one embodiment, the antigen, or a vaccine comprising the antigen or coding sequence, is administered in conjunction with one or more B cell and / or T cell epitopes.

[0110] Contemplated herein are cell lines capable of expressing the modified S antigen disclosed herein together with one or more of the N, M, E antigens, or their coding sequences.

[0111] The combined administration of a subject antigen together with one or more of the N, M, E antigens, or their coding sequences for one or more variants of interest / concern, can be simultaneous or spaced apart, in the same or different compositions, optionally involving the combined use of simultaneous or sequential protein and nucleic acid administration protocols.

[0112] Spike RBD-only vaccine antigens (e.g., 319-545) are also contemplated in protein or nucleic acid vaccine formats. In one embodiment, administration of the present modified S antigen is combined with an antigen representing only the RBD portion of the coronavirus in protein or nucleic acid form (e.g., mRNA). The RBD domain can be administered in monomeric, dimeric, or multimeric form and may include immune enhancing elements such as the Fc fragment of human IgG.

[0113] In another embodiment, the modified RBD of the modified S antigen trimers of the invention are produced or administered as an RBD-only antigen in a suitable vaccine format. In particular, the structural modification of S2 to a coiled coil beneficially alters the structure and immunogenicity of the S1 RBD region, and thus the production or administration of RBD forms based on the RBD forms produced by the subject modified S antigens is contemplated.

[0114] In another embodiment, the modified S1 of the modified S antigen trimer of the invention is produced or administered as an S1-only antigen in a suitable vaccine format. In particular, the structural modification of S2 to a coiled coil alters the structure and immunogenicity of the S1 region, and therefore, production or administration of an S1 form based on the form produced by the subject modified S antigen is contemplated. In one embodiment, a portion of the S1 region is produced or administered without the RBD. The S1 domain, or S1 minus the RBD domain, can be administered in a monomeric, dimeric, or multimeric form and may include immune enhancing elements such as the Fc fragment of human IgG.

[0115] Antibody response The S protein is the main protein used as a target antigen in COVID-19 vaccines. In theory, antibodies could target the S protein to inhibit viral infection at multiple stages during the viral entry process. The RBD is the main target of neutralizing antibodies (NAbs) that interfere with viral receptor binding. To date, most of the NAbs against SARS-CoV-2 target the RBD. In addition, NAbs targeting the N-terminal domain have been reported in SARS-CoV-2 and MERS-CoV infections, making it 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 the fusion protein required for virus-host membrane fusion.

[0116] As used herein, a "broadly neutralizing antibody" refers to an antibody that provides cross-protection against at least one, and preferably multiple, coronavirus variants (e.g., multiple SARS-COV-2 variants). In an embodiment, at least one of the variants is a VOC or a VOI or a VHC.

[0117] NAbs are called functional antibodies because they have a functional antiviral effect.

[0118] The ability of a vaccine to induce NAb or effective immune responses against homologous strains or emerging variants of concern is a major factor influencing the successful deployment of a vaccine program against SARS-CoV-2.

[0119] The ability of a vaccine to elicit NAb or effective immune responses against homologous and heterologous strains or emerging variants of concern is a major factor influencing the successful deployment of a vaccine program against SARS-CoV-2. The present application enables the production and use of coronavirus S antigen variants described herein that elicit enhanced immunogenicity against a broader range of variants, including ancestral and naturally occurring and emerging variants of concern.

[0120] Decreased vaccine efficacy against variants of concern has been observed and can be assessed, for example, by screening NAbs of the virus in vaccinated subjects against one or more strains or variants of the virus. As determined herein, in one embodiment, the structurally modified antigens enabled herein can generate functional antibody responses or neutralizing antibody titers in subjects against heterologous strains, including variants of concern, that are at least as good as against homologous strains.

[0121] In one embodiment, an antigen as defined herein induces neutralizing antibody titers against a VOC that are at the same or similar levels as those generated against one or more non-variant strains.

[0122] At least 100% of the homologous antibody titer. In one embodiment, the titer is greater than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the corresponding homologous titer.

[0123] Methods for Producing Coronavirus Vaccine Antigens The antigens described herein may be produced by recombinant or synthetic routes known in the art.

[0124] In one embodiment, a nanoparticle is provided that comprises an antigen as described herein fused to a polyhedrin targeting peptide from CPV or other suitable virus. Other nanoparticles are known in the art, including SOR particles, luminazine synthase particles, and pyruvate dehydrogenase particles.

[0125] Antigens can be linked to carriers or nanoparticles for increased immunogenicity. Suitable carriers are known in the art.

[0126] Virus-like particles 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 particle" refers to a vaccine that contains viral surface proteins but lacks the viral genome and one or more structural proteins. Human and hepadnavirus HBV are good examples. Antigen-containing VLPs can be formed spontaneously, for example, upon recombinant expression of proteins, and can be characterized using conventional techniques.

[0127] Vaccines in the form of liposomes are included. As used herein, the term "liposome" refers to a single or multilamellar lipid structure that encloses an aqueous interior. Lipids that can form liposomes include fats or any substance that has fat-like properties. Dynamic laser light scattering is a method used to measure the size of liposomes that is well known to those skilled in the art. A detailed description of adjuvants can be found in Cox and Coulter, "Advances in Adjuvant Technology and Application", in Animal Parasite Control Utilizing Biotechnology, Chapter 4, Ed. Young, WK, CRC Press 1992, and in Cox and Coulter, Vaccine 15(3):248- 256, 1997.

[0128] Antigens can be delivered in the form of viral or non-viral vectors. The term "vector" as used herein includes any delivery moiety into which at least an antigen coding sequence is inserted, including viral or baculoviral vectors such as plasmid vectors, cosmid vectors, phage vectors such as lambda phage, virus-like particles, adenoviruses, adeno-associated viruses (AAV), alphaviruses, flaviviruses, herpes simplex viruses (HSV), measles viruses, CMV, rhabdoviruses, retroviruses, lentiviruses, Newcastle disease virus (NDV), poxviruses, and picornaviruses, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI artificial chromosomes (PAC). Vectors include expression vectors and cloning vectors. In an embodiment, the primary coronavirus vaccine regimen is a viral vector vaccine. As used herein, a "viral vector vaccine" uses a viral backbone to insert SARS-CoV-2 genes or parts thereof into a host organism. These vaccines deliver genes to target cells where they are expressed and can tamper with the immune response. In one embodiment, the vector is a replicating vector. In an embodiment, the vector is a non-replicating vector (a vector that does not integrate into a host cell). In an embodiment, the vector is selected from adenovirus, poxvirus, measles virus, and vesicular stomatitis virus.

[0129] Expression vectors include plasmids and viral vectors, and generally contain the desired coding sequence and the appropriate NA sequence required for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a particular desired NA fragment, and may lack functional sequences required for expression of the desired DNA fragment.

[0130] In one embodiment, the vector is a viral vector or a non-viral vector. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses, and attenuated forms thereof, each of which has its own advantages and disadvantages known in the art. Viral vectors include, but are not limited to, adenovirus vectors and poxvirus vectors. Typically, for viral vectors, the number of cells is about 5×10 7 ~5×10 12 Virus particles, typically about 5 x 10 9 ~5×10 10 100 viral particles are administered.

[0131] The antigen coding sequence may be inserted into any suitable vector, the purpose of which, at least in one embodiment, is to deliver the coding nucleic acid to the host environment and facilitate protein expression and presentation to the host immune response. Vectors may be replicating or non-replicating.

[0132] An expression vector generally comprises transcriptional and translational regulatory nucleic acid operably linked to a nucleic acid molecule encoding an antigen. "Operably linked" in this context means that the transcriptional and translational regulatory DNA is positioned relative to the coding sequence of the antigen in such a manner that transcription is initiated. Generally, this means that the promoter and transcriptional initiation or initiation sequence are located 5' to the protein coding region. The transcriptional and translational regulatory nucleic acid is generally appropriate for the cell used to express the foreign protein, for example, transcriptional and translational regulatory nucleic acid sequences from mammalian cells, particularly humans, are preferably used to express proteins in mammals and humans. Numerous types of suitable expression vectors and suitable regulatory sequences are known in the art.

[0133] The viral vector may comprise a vaccinia vector, such as a synthetic modified vector based on the Copenhagen vaccinia vector or modified Vaccinia Ankara (MVA). The viral vector may comprise MVA or Copenhagen derivatives known in the art when used as a vaccine boost in a prime-boost regime. The viral vector may comprise an adeno-associated virus (AAV) or a lentivirus. The viral vector may be an attenuated viral vector. For example, essential genes for replication may be deleted and immunomodulatory molecules may be inserted.

[0134] The viral vector may be maintained in a BAC for ease of manipulation, and the DNA encoding the antigen may be linear or circular.

[0135] Non-viral vectors or attachments / conjugates include lipids, carbohydrates, proteins, peptides, nanoparticles, liposomes, virus-like particles, virosomes, emulsions. Amphiphilic agents such as lipids can exist in aggregates as micelles, insoluble monolayers, liquid crystals or lamellar layers in aqueous solution.

[0136] The antigen may be administered in the form of its encoding nucleic acid. The nucleic acid molecules described herein may be in any form, such as DNA, cDNA, genomic DNA, or RNA, including in vitro transcribed or synthetic RNA, mRNA or PNA, or mixtures thereof. Nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules, and modified forms thereof. The nucleic acid molecules may be single-stranded or double-stranded, linear or covalently closed to form a circle. In an embodiment, the nucleic acid is RNA. The RNA may be modified by stabilizing sequences, capping, and polyadenylation. It may be RNA or DNA and delivered as a plasmid to express the antigen and induce an immune response. The RNA may be modified to enhance delivery via lipid nanoparticles. The RNA may be modified to increase the stability of the RNA molecule. Examples of RNA of the invention are provided in Figures 35-37. RNA-based approaches are generally preferred, and these may include amplifying or non-self-amplifying constructs. The nucleic acid molecules are usually at least 10 bases in length and may be single-stranded or double-stranded. "cDNA" refers to a DNA that is complementary or identical to an mRNA, in either single- or double-stranded form. "Encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of another polynucleotide or polypeptide.

[0137] In some embodiments, RNA encoding an antigen is administered. In some embodiments, the RNA encodes an antigen that includes a coronavirus transmembrane domain as described herein. In some embodiments, the RNA encodes an antigen that lacks a coronavirus transmembrane domain as described herein. In some embodiments, the RNA encodes an antigen that includes a trimerization domain as described herein. In some embodiments, the RNA encodes an antigen that lacks a trimerization domain as described herein.

[0138] In some embodiments, the polynucleotide administered by transient in vivo transfection is a chemically modified RNA in which at least one type of nucleotide, e.g., a percentage of cytosine (e.g., 10%, 30%, 50%, or 100%), is chemically modified to increase its stability in vivo. For example, in some cases, the modified cytosine is 5-methylcytosine. Such polynucleotides are particularly useful for delivery / transfection to cells in vivo, especially when combined with a transfection / delivery agent. In some cases, the chemically modified RNA is a chemically modified RNA in which the majority (e.g., all) of the cytosines are 5-methylcytosines and the majority (e.g., all) of the uracils are pseudouracils. In some embodiments, the non-natural cysteines are engineered (e.g., via recombinant gene technology) to create disulfide bonds. The synthesis and use of such modified RNAs are described, for example, in WO2011 / 130624. Methods for in vivo transfection of DNA and RNA polynucleotides are known in the art, for example, as summarized in Liu et al. (2015) and Youn et al. (2015).

[0139] The term "RNA" refers to a molecule that comprises ribonucleotide residues, and preferably consists entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material at the end of the RNA, or internally, for example, at one or more nucleotides of the RNA. Nucleotides in an RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0140] Thus, in one embodiment, the G / C content of the coding region of a nucleic acid coding region is modified, in particular increased, compared to the G / C content of the coding region of that particular wild-type coding sequence, i.e., of an unmodified mRNA. Preferably, the encoded amino acid sequence of the mRNA is unmodified compared to the encoded amino acid sequence of the particular wild-type mRNA.

[0141] The optimized mRNA-based composition may include 5' and 3' untranslated regions (5'-UTR, 3'-UTR) that optimize translation efficiency and intracellular stability as known in the art, as well as an open reading frame encoding an S protein. In one embodiment, removal of uncapped 5'-triphosphates may be achieved by treating the RNA with a phosphatase. The RNA may have modified ribonucleotides to increase its stability and / or reduce cytotoxicity. For example, in one embodiment, 5-methylcytidine is partially or completely substituted for cytidine in the RNA. Alternatively or additionally, pseudouridine is partially or completely substituted for uridine, preferably completely. These modifications may also reduce indiscriminate immune inactivation that may prevent translation of the RNA. In one embodiment, the term "modification" refers to providing an RNA with a 5' cap or a 5' cap analog. The term "5' cap" refers to the cap structure found at the 5' end of an mRNA molecule and generally consists of a guanosine nucleotide attached to the mRNA via an unconventional 5' to 5' triphosphate linkage. 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" includes 5' cap analogs that are modified to resemble an RNA cap structure and have the ability to stabilize RNA and / or enhance translation of RNA. Providing an RNA with a 5' cap or 5' cap analog can be achieved by in vitro transcription of a DNA template in the presence of the 5' cap or 5' cap analog, and the 5' cap is co-transcriptionally incorporated into the generated RNA strand, or the RNA can be generated, for example, by in vitro transcription, and the 5' cap can be attached to the RNA post-transcriptionally, for example, using vaccinia virus capping enzyme.

[0142] Further modifications of the RNA can be alterations of the 5' or 3' untranslated region (UTR), such as extension or truncation of a naturally occurring UTR, such as an X-region tail, or introduction of a UTR not associated with the coding region of the RNA, e.g., replacement or insertion of an existing 3'-UTR with one or more, preferably two copies of a 3'-UTR from a globin gene, such as alpha2-globin, alpha-globin, beta-globin, etc. RNA with an unmasked polyA sequence is translated more efficiently than RNA with a masked polyA sequence.

[0143] The term "poly(A) tail" or "polyA sequence" refers to a sequence of adenyl (A) residues that may be located at the 3' end of an RNA molecule, and an "unmasked polyA sequence" means that the polyA sequence at the 3' end of an RNA molecule ends with an A of the polyA sequence and is not followed by any nucleotides other than A located at the 3' end, i.e. downstream, of the polyA sequence. Furthermore, a long polyA sequence of about 120 base pairs provides optimal transcriptional stability and translational efficiency of the RNA.

[0144] Thus, in order to increase the stability and / or expression of the RNA, it may be modified to be present in conjunction with a heterologous polyA sequence, preferably having a length of 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 polyA sequence has a length of about 120 adenosine residues. In order to further increase the stability and / or expression of the RNA used according to the invention, the polyA sequence may not be masked.

[0145] In addition, incorporating a 3' untranslated region (UTR) into the 3' untranslated region of the RNA molecule can result in improved translation efficiency. Synergistic effects can be achieved by incorporating two or more of such 3' untranslated regions. The 3' untranslated regions can be autologous or heterologous to the RNA into which they are introduced. In one particular embodiment, the 3' untranslated region is derived from the human β-globin gene.

[0146] The combination of the above mentioned modifications, i.e., optionally, incorporation of a polyA sequence, unmasking of a polyA sequence, and incorporation of one or more 3' untranslated regions, synergistically affects RNA stability and increased translation efficiency.

[0147] To increase the expression of RNA, the coding region may be modified to increase GC content, increase mRNA stability, and perform codon optimization, thus improving translation in cells. The modified mRNA may be enzymatically synthesized and packaged into nanoparticles, such as lipid nanoparticles, and administered, for example, intramuscularly. Self-replicating RNA or protamine-complexed RNA approaches have also been shown to generate immune responses against viral infections.

[0148] Nucleic acid molecules can be encapsulated in microcapsules, colloidal drug delivery systems (e.g., liposomes, microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions prepared, for example, by coacervation techniques or by interfacial polymerization. Such techniques are known in the art and are disclosed in Remington, the Science and Practice of Pharmacy, 20th Edition, Remington, J., ed. (2000).

[0149] Various approaches are known for the systemic administration of nucleic acids as nanoparticles or colloidal systems. In non-viral approaches, cationic liposomes are used to induce DNA / RNA condensation and promote cellular uptake. Cationic liposomes usually consist of a cationic lipid, such as DOTAP, and one or more helper lipids, such as DOPE. So-called "lipoplexes" can be formed from cationic (positively charged) liposomes and anionic (negatively charged) nucleic acids. In the simplest case, lipoplexes form spontaneously by mixing nucleic acid and liposomes in a specific mixing protocol, but various other protocols may be applied. In one embodiment, nanoparticle RNA formulations, such as RNA lipoplexes, are produced with a defined particle size, and the net charge of the particles is zero or close to negative. For example, electrically neutral or negatively charged lipoplexes from RNA and liposomes result in substantial RNA expression in the spleen or immune cells after systemic administration, as disclosed in WO2013 / 143683. In one embodiment, the nanoparticles comprise at least one lipid. In one embodiment, the nanoparticle comprises at least one cationic lipid. The cationic lipid may be monocationic or polycationic. Any cationic amphiphilic molecule, for example a molecule that comprises at least one hydrophilic and lipophilic portion, is a cationic lipid within the meaning of the present 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 helper lipid. The helper lipid may be a neutral or anionic lipid. The helper lipid may be a natural lipid, such as a phospholipid or an analogue of a natural lipid, or a completely synthetic lipid or lipid-like molecule that has no similarity to a natural lipid. In one embodiment, the cationic lipid and / or the helper lipid are bilayer-forming lipids.

[0150] In one embodiment, the at least one cationic lipid comprises 1,2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA) or an analog or derivative thereof, and / or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or an analog or derivative thereof.

[0151] In one embodiment, the at least one helper lipid comprises 1,2-di(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or an analogue or derivative thereof, cholesterol (Choi) or an analogue or derivative thereof, and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or an analogue or derivative thereof.

[0152] In one embodiment, the molar ratio of the at least one cationic lipid to the at least one helper 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 cationic lipid is obtained by multiplying the molar amount of cationic lipid by the number of positive charges in the cationic lipid. In the nanoparticles described herein, the lipids may form a complex with RNA and / or encapsulate RNA. In one embodiment, the nanoparticles comprise lipoplexes or liposomes. In one embodiment, the lipids are included in vesicles that encapsulate the RNA. The vesicles may be multilamellar vesicles, unilamellar vesicles, or a mixture thereof. The vesicles may be liposomes.

[0153] Lipid nanoparticles (LNPs) are generally known as nano-sized particles composed of a combination of different lipids (an aqueous volume is encapsulated by an amphiphilic lipid bilayer, e.g., single, monolayer or multilayer, multilayer). Many different types of lipids can be included in the LNP. In some embodiments, the lipid can be one or more of an ionizable lipid, a phospholipid, a structural lipid, a neutral lipid, and a PEG lipid. For example, mRNA is encapsulated in the LNP. In another example, mRNA is bound to the LNP. For example, mRNA is absorbed into the LNP.

[0154] 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). As used herein, the term "ionizable lipid" or "ionizable lipids" refers to a lipid having at least one protonated or deprotonated group. For example, the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH (e.g., above physiological pH). In embodiments, the lipid nanoparticle comprises an ionizable lipid as described in Table 1 of Schoenmaker et al. (2021).

[0155] Suitable ionizable lipids can have anionic, cationic, or zwitterionic hydrophilic head groups. Exemplary phospholipids (anionic or zwitterionic) for use in the present disclosure include, for example, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. In one example, the lipid is a cationic lipid. Exemplary cationic lipids include, but are not limited to, dioleoyltrimethylammoniumpropane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750). In one example, the phospholipid is 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxyl)benzyl-4-(dimethylamino)butanoate (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 can be saturated or unsaturated. In one embodiment, the lipid nanoparticles do not include cationic lipids.

[0156] Those skilled in the art will understand that PEGylated lipid is lipid modified with polyethylene glycol.Exemplary PEGylated lipid includes, but is 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 lipid includes PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipid, and combinations thereof.

[0157] Suitable neutral or zwitterionic lipids for use in the present disclosure will be apparent to those of skill in the art and include, for example, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3 ... DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsP C), 1-hexadecyl-sn-glycero-3-phosphocholine (C16LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, Examples of such lipids include 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocohexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. The lipids can be saturated or unsaturated.

[0158] Exemplary structural lipids include, but are not limited to, cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and alpha-tocopherol. In an embodiment, the structural lipid is a sterol. In an embodiment, the structural lipid is cholesterol. In one embodiment, the structural lipid is campesterol.

[0159] Compositions, routes of administration, and dosages One of skill in the art will appreciate that the coronavirus vaccine antigens described herein, or vectors or polynucleotides encoding the coronavirus vaccine antigens described herein, can be formulated into a pharmaceutical composition. In embodiments, the pharmaceutical composition is a vaccine composition.

[0160] In an aspect, the present specification provides a pharmaceutical composition comprising a polynucleotide comprising a sequence of nucleotides encoding a coronavirus S protein trimer antigen, wherein the S protein trimer has been modified to include a structural modification that reduces the size of an alanine cavity within the coiled-coil region of the S protein trimer described herein.

[0161] In aspects, the present specification provides a pharmaceutical composition comprising a polynucleotide comprising a sequence of nucleotides encoding a coronavirus S protein trimer antigen, wherein at least one amino acid within a region of an S protein monomer that forms the coiled coil of the S protein trimer is replaced with a more hydrophobic amino acid, as described herein.

[0162] Such compositions may include a coronavirus vaccine antigen, vector, or polynucleotide described herein and one or more pharma- ceutically acceptable carriers. Remington's Pharmaceutical Sciences by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the immunogens of the present disclosure. In general, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations usually contain injectable fluids that contain pharma- ceutically and physiologically acceptable carriers, such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, or the like, as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions (such as immunogenic compositions) to be administered may contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate. In certain embodiments, carriers suitable for administration to a subject may be sterile and / or suspended or otherwise contained in a unit dosage form containing one or more measured doses of a composition suitable for inducing a desired immune response. They may also be accompanied by a drug for its use for therapeutic purposes. The unit dosage form may be, for example, a sealed vial containing sterile contents, or in a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration, or a solid or controlled release dosage.

[0163] In an embodiment, the composition comprises a vaccine antigen as described herein. In an embodiment, the composition comprises a vector as described herein. In an embodiment, the composition comprises a polynucleotide as described herein. In an embodiment, the polynucleotide is DNA. In an embodiment, the polynucleotide is RNA. In an embodiment, the polynucleotide is mRNA.

[0164] In embodiments, the composition comprises a lipid nanoparticle, hi embodiments, the lipid nanoparticle encapsulates a polynucleotide described herein.

[0165] In embodiments, where the composition is a vaccine composition, it may contain one or more other epitopes for eliciting an immune response, such as B cell and / or T cell epitopes.

[0166] In embodiments, a composition is formulated to be compatible with its intended route of administration, e.g., local or systemic, including intradermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intranasal, sublingual, tonsillar, oral, pulmonary, topical, or other parenteral and mucosal routes.

[0167] In an embodiment, the composition is formulated to be stable at refrigerator temperature. In an embodiment, the composition is formulated to be suitable for transport and / or storage at refrigerator temperature. In an 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 an embodiment, the composition is formulated to be stable at room temperature. In an 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 an embodiment, the composition is formulated to be suitable for non-cold chain transport and / or storage. In an embodiment, the composition is formulated to be suitable for room temperature storage and / or evaporation. In an embodiment, the composition is formulated to be suitable for transport and / or storage at temperatures higher than room temperature, for example, about 25°C to 40°C (due to countries where cold chain and low temperature storage and transport trains are not available).

[0168] Oral, nasal, and pulmonary administration includes administration via inhalation and spray delivered to the above sites. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent, such as water for injection, saline solution, fixed 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 acetic acid, citric acid, or phosphate, and agents for adjusting tonicity, such as sodium chloride or dextrose. pH can be adjusted using acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0169] Compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, nonaqueous solutions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride can also be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, such as aluminum monostearate or gelatin.

[0170] Sterile injectable solutions can be prepared by incorporating the coronavirus vaccine antigen, vector, or polynucleotide described herein in the required amount in a suitable solvent or buffer with one or a combination of the ingredients listed above, followed by sterilization by filtration as required. In general, dispersions are prepared by incorporating the polynucleotide into a sterile vehicle, containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for preparing sterile injectable solutions, suitable methods of preparation include vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredients from the solution previously sterile-filtered.

[0171] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the coronavirus vaccine antigen, vector, or polynucleotide described herein can be incorporated with an excipient and used in the form of a spray, tablet, troche, or capsule, such as a gelatin capsule. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The sprays, tablets, pills, capsules, troches and the like can contain any of the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrant such as alginic acid, PRIMOGEL, corn starch; a lubricant such as magnesium stearate or sterols; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate, or orange flavor, or compounds of a similar nature.

[0172] Suitable formulations for administration by nasal inhalation include, when the carrier is a solid, a coarse powder having a particle size ranging, for example, from about 1 to about 500 microns, which is administered by means of a spray, nebulizer, inhaler, or via inhalation. Suitable formulations where the carrier is a liquid for administration by a nebulizer include aqueous or oily solutions of the drug. For administration by inhalation, the drug can also be delivered in the form of droplets or an aerosol spray from a pressurized container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in US 6,468,798.

[0173] Formulations suitable for administration by oral inhalation include coarse powders having a fine particle size, for example, in the range from about 20 to about 500 microns, where the carrier is a solid, which may be administered by oral inhalation from a container holding the powder close to the mouth, or where the carrier is a liquid for administration by a nebulizer, which may include aqueous or oily solutions of the agent.

[0174] Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration may be achieved through the use of nasal sprays, drops, or suppositories. For transdermal administration, the coronavirus vaccine antigens, vectors, or polynucleotides described herein are formulated into ointments, salves, gels, or creams, as generally known in the art.

[0175] Intradermal delivery of vaccines by needle or needle-free approach offers advantages in terms of ease of administration, and intradermal administration approaches are contemplated to effectively target immune-competent cells. Liquid formulations can be provided in prefilled or non-prefilled syringes, or require such a disposable syringe jet injector, a hollow microneedle attached to the syringe, and a needle adapted for intradermal delivery. Prefilled syringes with single ID needles are commercially available. Alternatively, solid or biodegradable microneedles coated or impregnated with vaccine, or composed of vaccine, such as patches or other mini-needle / spike devices, can be used. These are inserted into the dermal layer of the skin, either where the vaccine coating dissolves or where the microneedle itself dissolves in place. Vaccine antigens can be provided as liquid or semi-liquid formulations, or as solid or powder formulations. Jet injectors work by generating a high-pressure stream that flushes the liquid vaccine formulation into deeper skin layers. However, approaches to deliver vaccines in solid form may also prove promising. One such method is the ballistic approach, in which solid vaccine particles or vaccine-coated gold particles are accelerated towards the skin by a needle-free device, such that the particles are deposited in the epidermal and dermal layers of the skin.

[0176] Intramuscular administration can be via any intramuscular method known to those of skill in the art, including, for example, intramuscular injection.

[0177] The compositions can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0178] The composition may include an adjuvant. When administered in a mixture with one or more adjuvants, the immune response to the antigen can be enhanced. Immune adjuvants typically function in one or more of the following ways: (1) immunomodulation, (2) enhanced presentation, (3) CTL generation, (4) targeting, and / or (5) depot generation.

[0179] Exemplary adjuvants that 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, ISCOMS, LPS derivatives such as MPL and their derivatives such as 3D-MPL, GLA, and AGP, mycobacteria-derived proteins such as muramyl dipeptide or muramyl tripeptide, certain saponins from Quillaja saponaria such as QS21, QS7, and ISCOPREP™ saponins, ISCOMATRIX™ adjuvants, and peptides such as thymosin alpha 1. In addition to the saponin component, the adjuvants may include sterols such as beta-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol. In some embodiments, the adjuvant is presented in the form of an oil-in-water emulsion, for example, containing squalene, alpha-tocopherol, and a surfactant, or in the form of a liposome. AddaVax is a squalene-based oil-in-water nanoemulsion based on a formulation of MF-59, which has been found to be useful in influenza vaccines. Adjuvants AS03, MF59, and CpG1018 are already used in licensed vaccines. Other suitable adjuvants include lecithin and calomer homopolymer, Matrix M, ASO1, ALFQ. Co-stimulatory molecules, including CpG Moffitt and TLR agonists, B7, OX-40L, G-CSF, are contemplated. Adjuvants are discussed in Liang et al Front. Immunol. 6 November 2020.

[0180] In embodiments, the composition comprises an adjuvant selected from one or more of an aluminum salt-based adjuvant, an emulsion adjuvant, or a TLR agonist. Examples of such adjuvants are described, for example, in Liang et al. (2020).

[0181] Suitable dosage ranges for intravenous administration of viral vectors, for example, are generally about 0.001 to 10 micrograms of nucleic acid. Suitable dosage ranges for intranasal administration are generally about 0.01 pg / kg body weight to 10 mg / kg body weight. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight, and oral compositions preferably contain 10% to 95% active ingredient.

[0182] A subject may receive one dose of the composition or two or three doses of the composition at scheduled intervals.

[0183] Antibodies generated against an antigen of interest can be used in therapy or for screening. Antibodies include immunoglobulins, antigen-binding fragments thereof, or derivatives that specifically bind and recognize an antigen or an antigenic fragment thereof, or a dimer or multimer of an antigen. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multi- (and bi)specific antibodies, and antibody fragments. 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 that are either produced by modification of whole antibodies or synthesized de novo using recombinant DNA methodology (see Kontermann and Dubel (Ed), Antibody Engineering, Vol 1-2, Ed., Springer Press, 2010).

[0184] The term epitope refers to a specific peptide sequence on a molecule that is antigenic, thereby eliciting a specific immune response. An epitope is a region of an antigen to which B cells and / or T cells respond. Antibodies can bind to specific antigen epitopes that can be formed from both contiguous or non-contiguous amino acids.

[0185] Methods of Prevention and / or Treatment In an aspect, the present invention provides a method of preventing and / or treating a coronavirus infection in a subject.

[0186] As used herein, the term "prevention" or "prophylaxis" refers to reducing the likelihood of contracting or developing an infection or its symptoms. Prevention need not be complete and does not mean that a subject will not eventually contract or develop an infection or its symptoms.

[0187] As used herein, the term "treating" or "treatment" refers to at least partially obtaining a desired therapeutic outcome. In embodiments, treatment includes preventing or delaying the appearance of one or more symptoms of CoV infection. In embodiments, treatment includes inhibiting or reducing the onset of one or more symptoms of CoV infection.

[0188] Reference to a "subject" or "subjects" includes subjects susceptible to or at risk of exposure to a coronavirus infection. A subject may be infected or non-infected and may be asymptomatic or not in need of treatment. In an embodiment, a subject is susceptible to or at risk of exposure to SARS-CoV-2 infection. For example, a subject can be a mammal, a bird, an arthropod, a chordate, an amphibian, or a reptile. Exemplary subjects include, but are not limited to, humans, primates, livestock (e.g., sheep, cows, chickens, horses, donkeys, pigs), pet animals (e.g., dogs, cats), laboratory 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 subject is a mammal. In embodiments, the subject is a human. In embodiments, the human is a fetus, an infant, a child, an early adult, and an adult. In embodiments, the adult is an elderly adult. In embodiments, the adult is one or more of: over 60 years of age, over 65 years of age, over 70 years of age, over 75 years of age, over 80 years of age, over 85 years of age, and over 90 years of age. In embodiments, the subject has had a past coronavirus infection. In embodiments, the subject has had a past SARS-CoV-2 infection. In embodiments, the subject is receiving a first-line coronavirus treatment regimen as described herein. In embodiments, the subject is receiving a first-line and a second-line coronavirus treatment regimen. In embodiments, the subject is receiving a first-line coronavirus treatment regimen, a second-line coronavirus treatment regimen, and a third-line coronavirus treatment regimen. In embodiments, the subject is immunocompromised. In embodiments, the subject has a respiratory condition.

[0189] In an aspect, the invention provides a method of inducing an immune response against coronavirus in a subject, the method comprising administering a vaccine described herein.In an aspect, the invention provides a method of enhancing an immune response against coronavirus in a subject, the method comprising administering a vaccine described herein.

[0190] In an aspect, the invention provides a method of preventing or reducing the likelihood of coronavirus infection in a subject, the method comprising administering a vaccine described herein.

[0191] In an aspect, the invention provides a method of preventing or reducing the likelihood of coronavirus infection or the severity of a symptom thereof in a subject, the method comprising administering to the subject a vaccine as described herein.

[0192] In aspects, the invention provides methods of reducing the severity and / or duration of a coronavirus infection in a subject, the method comprising administering to the subject a vaccine as described herein. As used herein, the phrase "reducing the severity of infection" or similar phrases includes 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 a subject. As used herein, the phrase "duration of coronavirus infection" refers to the period of time an individual has CoV infection or symptoms caused by CoV infection.

[0193] In an aspect, the invention provides a method of preventing or reducing viral shedding in a human individual infected with a coronavirus, the method comprising administering to the subject a vaccine as described herein.

[0194] In an embodiment, the present invention provides a vaccine that is a primary vaccine regimen. As used herein, a "primary vaccine regimen" is the first vaccine regimen administered to a subject to generate a response against a particular pathogen. In the context of SARS-COV-2, a primary vaccine is the first vaccine regimen administered to a subject to generate an immune response against ancestral strains and / or their variants.

[0195] In an embodiment, the present invention provides a booster vaccine for a primary coronavirus vaccine regimen. In an embodiment, the present invention provides a booster vaccine for cases where a subject has received two or more previous coronavirus vaccine regimens. In an embodiment, the booster acts by enhancing the immune response elicited by the primary vaccine regimen. In an embodiment, the booster acts by enhancing the immune response against VOCs or VOIs or VHCs to which the primary vaccine regimen generates less, little or no protective immune response. In an 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 an embodiment, the booster is administered sequentially or in combination with one or more other booster vaccines.

[0196] Primary Vaccine Regimens In some embodiments, the vaccine described herein is administered after the subject has received a primary coronavirus vaccine regimen. Those skilled in the art will understand that the primary coronavirus vaccine regimen can be any coronavirus vaccine regimen that provides protection against coronavirus infection. In embodiments, the coronavirus vaccine is a SARS-CoV-2 coronavirus vaccine.

[0197] In embodiments, the primary coronavirus vaccine regimen is selected from a) a single dose vaccine regimen, b) a two dose vaccine regimen, c) a single dose of a two dose vaccine regimen, or d) a combination thereof.

[0198] In embodiments, the primary coronavirus vaccine regimen is selected from a) an RNA-based vaccine, b) a DNA-based vaccine, c) a viral vector vaccine, d) an inactivated vaccine, e) a live attenuated vaccine, and f) a protein subunit vaccine.

[0199] In embodiments, the primary coronavirus vaccine regimen is an RNA-based vaccine. As used herein, an "RNA-based vaccine" delivers instructions for the expression of a coronavirus antigen, e.g., the S protein or a portion thereof, in human cells via an RNA molecule, such as mRNA.

[0200] In embodiments, the primary coronavirus vaccine regimen is a DNA-based vaccine. As used herein, a "DNA-based vaccine" delivers instructions for the expression of a coronavirus antigen, such as the S protein or a portion thereof, in human cells via a DNA molecule.

[0201] In embodiments, the primary coronavirus vaccine regimen is a viral vector vaccine, in embodiments, the vector is selected from adenovirus, poxvirus, measles virus, and vesicular stomatitis virus.

[0202] In an embodiment, the primary coronavirus vaccine regimen is an inactivated virus vaccine. Inactivated vaccines are made by inactivating the virus with, for example, chemical UV light and / or heat, so that the virus is no longer transmissible. Such vaccines are often desirable because they present several epitopes for immune recognition and generation of an immune response.

[0203] In embodiments, the primary coronavirus vaccine regimen is a live-attenuated coronavirus. As used herein, "live-attenuated" refers to.

[0204] In an embodiment, the vaccine is a protein-based vaccine, such as a protein subunit vaccine or a virus-like article. As used herein, a "protein subunit vaccine" comprises an immunogenic antigen that can stimulate a host immune response. When the coronavirus is SARS-CoV-2, the protein subunit vaccine may comprise an S1 protein or a portion thereof, an RBD domain, an S2 protein or a portion thereof. In an embodiment, the protein-based vaccine is a virus-like particle or nanoparticle.

[0205] Combination treatment The coronavirus vaccine antigens or vaccines described herein may be administered to a subject in combination with one or more additional vaccine antigens or vaccines that may generate an immune response against an infectious pathogenic organism, such as influenza, SARS-CoV-2, or specific VOCs, VOIs, or VHCs thereof. Administration may be concomitant (simultaneous) or sequential in any order.

[0206] Kits, Devices, Surfaces, or Strips The subject coronavirus antigens are captured on solid or semi-solid surfaces for assay purposes, including epidemiological, diagnostic, purification, drug screening, vaccine screening applications, etc. Many such applications and methods of immobilizing antigens on surfaces are known in the art and are encompassed.

[0207] As used herein, the term "complement" or "complementary" is used according to its plain and ordinary meaning and refers to a nucleotide (e.g., an RNA nucleotide or a DNA nucleotide) or a sequence of nucleotides that can base-pair with a complementary nucleotide or sequence of nucleotides. As described herein and generally known in the art, the complementary (matching) nucleotide of adenosine is thymidine in DNA, or alternatively, in RNA, the complementary (matching) nucleotide of adenosine is uracil, and the complementary (matching) nucleotide of guanosine is cytosine. Nucleotides can also be non-naturally occurring or modified bases. Thus, a complement can include a sequence of nucleotides that base-pair with the corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of the complement can partially or completely match the nucleotides of the second nucleic acid sequence. If the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, then the complement will base-pair with each nucleotide of the second nucleic acid sequence. When the nucleotides of the complement partially match those of the second nucleic acid sequence, only some of the nucleotides of the complement will base pair with the nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding sequences and non-coding sequences, where the non-coding sequence contains nucleotides that are complementary to the coding sequence, thus forming the complement of the coding sequence. Further examples of complementary sequences are sense and antisense sequences, where the sense sequence contains nucleotides that are complementary to the antisense sequence, thus forming the complement of the antisense sequence. Pairing of purine-containing nucleotides (e.g., A or G) with pyrimidine-containing nucleotides (e.g., T or C) is considered complementary. AT and CG pairing functions to form double or triple hydrogen bonds between the amine and carbonyl groups at the complementary bases. Complementarity of sequences can be partial, where only some of the nucleic acids match according to base pairing, or complete, where all of the nucleic acids match according to base pairing.Thus, two sequences that are complementary to each other may have a certain percentage of nucleotides that are complementary to each other (e.g., about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher complementarity over a particular region). In one embodiment, two sequences are complementary when they are fully complementary, having 100% complementarity. In one embodiment, two sequences are complementary when they are functionally complementary, i.e., successfully anneal under appropriate conditions, and have at least 80%-99%, or at least 70%-95% base pairing. EXAMPLES

[0208] Example 1 - Materials Material Examples 1-9 Recombinant protein. A synthetic gene encoding the SARS-CoV-2 (Hu-1 isolate) S ectodomain, corresponding to the S2P protein reported by Wrapp et al., 2020, was obtained from GeneART-ThermoFisher Scientific. The gene contains S residues 16–1208, a furin cleavage site mutation, R 682 RAR→G 682SAS, and diPro substitutions at positions 986 and 987. The C-terminus of S2P was appended with a foldon (YIPEAPRDGQAYVRKDGEWVLLSTFL) (SEQ ID NO: 20), an octa-His, and an avitag (GLNDIFEAQKIEWHE) sequence (collectively referred to as FHA or FHA tag SEQ ID NO: 21), each separated by a GSGS linker. The synthetic S2P gene was ligated downstream of a DNA sequence encoding a tissue plasminogen activator leader via NheI in pcDNA3 (Invitrogen). Mutations were introduced into the S2P expression vector using synthetic genes encoding mutant S2P subfragments produced by GeneART-ThermoFisher Scientific. Synthetic genes encoding the S1 subunit (amino acids 16-682) and the receptor binding domain (RBD, amino acids 332-532) were obtained by GeneART-ThermoFisher Scientific and ligated to the tissue plasminogen activator leader via NheI in pcDNA3. Both proteins encode a C-terminal 6His tag and an Avitag sequence. hACE2-Fc is a recombinant fusion protein containing amino acids 19-615 of the human ACE2 ectodomain linked to the Fc domain of human IgG1 via a GS linker. Synthetic genes encoding hACE2-Fc were obtained from GeneART-ThermoFisher Scientific and ligated downstream of the tissue plasminogen activator leader via NheI in pcDNA3. The DNA sequences of the S and hACE2 clones were verified by fluorescent Sanger sequencing (BigDye, ABI).

[0209] Recombinant protein expression and purification. S2P expression vectors were transfected into 293Freestyle cells using 293fectin as recommended by the manufacturer (ThermoFisher Scientific). Cells were cultured at 34°C for 5 days, after which the transfection supernatant was cleared of cells by centrifugation and filtration through a 0.45 μm nitrocellulose filter. S2P proteins were then purified by divalent cation affinity chromatography using TALON resin (Merck), followed by size-exclusion chromatography (SEC) using a Superose6 Increase10 / 300 column coupled to an AKTApure device (Cytiva). S1 and RBD proteins were produced by transfection of Expi293 cells with the appropriate expression vectors using Expifectamine according to the manufacturer's instructions (ThermoFisher Scientific). After 4 days of culture at 34°C, the transfection supernatant was cleared of cells by centrifugation and filtration through a 0.45 μm nitrocellulose filter. S1 and RBS proteins were purified by divalent cation affinity chromatography using TALON resin (Merck) followed by SEC using a Superdex200 16 / 600 column coupled to an AKTApure device (Cytiva). hACE2-Fc was produced in Expi293 cells like S1 and RBD and purified from clarified culture supernatant using protein G-agarose (Genscript). hACE2-Fc was further purified by SEC on a Superdex200 16 / 600 column coupled to an AKTApure device (Cytiva). All proteins were concentrated using Amicon centrifugal filter units. Protein solutions were filter-sterilized using 0.45 μm nitrocellulose filters and protein aliquots were stored at −80° C. Protein purity was assessed by SDS-PAGE and SEC.

[0210] Recombinant monoclonal antibodies (mAbs). pCDNA3-based IgG1 heavy and light chain expression vectors (Center et al. 2020) containing the variable regions of SARS-CoV-2-directed mAbs CR3022 (Muelen, PloS Med 2006), CB6 (Shi, Nature 2020), H4 and B38 (Wu et al., 2020), 2-51 (Liu, et al., 2020), COVA2-15, COVA2-33, COVA1-25, COVA1-22, COVA2-14 (Brouwer et al., 2020) were generated in-house using synthetic gene fragments encoding the mAb heavy and light chain variable regions produced by GeneART-ThermoFisher Scientific. mAbs were produced by transfection of Expi293 cells with equal amounts of matched heavy and light chain vectors using Expifectamine according to the manufacturer's instructions (ThermoFisher Scientific). After 5 days of incubation at 37°C, transfection supernatants were cleared of cells by centrifugation and filtration through 0.45 μm nitrocellulose filters. IgG was purified by affinity chromatography using protein G-agarose (Genscript) and exchanged into PBS. Antibodies were concentrated using Amicon centrifugal filter units. IgG solutions were filter sterilized using 0.45 μm nitrocellulose filters and aliquots were stored at -80°C.

[0211] Differential scanning fluorimetry. Protein thermal stability was assessed using differential scanning fluorimetry (Niesen Nature Protocols 2007). 10 μg of protein was diluted in duplicate to 25 μL with 5× concentration of SYPRO Orange protein gel stain (Sigma Aldrich). Samples were then heated in an Mx3005P qPCR system from 25° C. to 95° C. in 0.5° C. increments, 1 min per increment. Triplicate measurements of fluorescence were taken at the end of each increment. Excitation was at 492 nm and emission was at 610 nm. Melting temperatures were determined to be the minimum of the negative first derivative of the melting curve.

[0212] Biolayer Interferometry. BLI-based measurements (BLI) were determined using the OctetRED System (ForteBio, Fremont CA). Antibodies were diluted to 10 μg / ml in kinetic buffer and immobilized on anti-human IgG Fc capture biosensors (AHC, ForteBio). Kinetic assays were performed at 30°C using standard kinetic acquisition speed settings (5.0 Hz, averaging at 20) with a sample plate shaking speed of 1,000 rpm. The kinetic experiment included 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). Fitting curves were constructed using ForteBio Data Analysis 10.0 software using a 1:1 binding model and double reference subtraction was used for correction.

[0213] Immunization. Guinea pigs (inbred tricolor) matched for sex, weight, and age were immunized subcutaneously at weeks 0, 4, and 14 with a 1:1 mixture of 30 μg S2P protein in PBS and (v / v) AddaVax adjuvant (InvivoGen, San Diego, CA) at the same time. A negative control group was immunized as above with a 1:1 (v / v) mixture of PBS and adjuvant. Blood was collected via the saphenous vein 2 weeks after the second dose and by terminal cardiac puncture 2 weeks after the third dose and allowed to clot for serum preparation. Serum was stored at -80°C and heat inactivated at 56°C for 30 min before being used for immunological assays. Animals were housed and all procedures were performed at the Preclinical, Imaging, and Research Laboratories at the South Australian Health and Medical Research Institute (Gilles Plains, Australia). All animal experiments were performed in accordance with the 8th edition of the Australian Code for the Care and Use of Animals for Scientific Purposes and were approved by the SAHMRI Animal Ethics Committee, project number SAM-20-030.

[0214] ELISA. Nunc maxisorp 96-well plates were coated with S2P, S1, and RBD protein solutions (2 μg / ml, PBS) overnight at 4 °C. Plates were washed with PBS and blocked with BSA (10 μg / ml, PBS) for 1 h at room temperature. Plates were washed again and then incubated with serially diluted serum samples for 2-4 h at room temperature. Antibody binding was detected using horseradish peroxidase-labeled rabbit anti-guinea pig antibodies (Dako, Glostrup, Denmark) and 3,3',5,5',5,5'-dithiobis-(2-nitrobenzoic acid) tetramethylbenzidine dihydrochloride (TMB). Antibody binding with different antigens was compared by fitting curves with nonlinear regression using Prism version 9 software, and titers were obtained by interpolation of optical density (OD) values ​​above 10-fold background defined by binding with BSA.

[0215] Pseudotyped virus production. S-pseudotyped HIV luciferase reporter virus was prepared according to the method of Jackson et al., 2020. Plasmids for the production of S-HIV pseudoparticles were kindly donated by Professor Doria-Rose of the NIH Vaccine Research Center, and included the WH-Human1_EPI_402119 expression plasmid (Genbank No. MN908947.3) with codon-optimized full-length S, the packaging plasmid pCMVΔR8.2 and the luciferase reporter plasmid pHR' CMV Luc (Naldini PNAS 1996;93:11382), as well as the TMPRSS2 plasmid (Bottcher JVI 2006,80:9869). The four plasmids were co-transfected into HEK293T cells, and after 18 hours of incubation, the medium was replaced with fresh Dulbecco's modified minimal essential medium containing 10% fetal bovine serum (DMF10) and cultured for an additional 3 days. The clarified supernatant containing the retroviral pseudotyped virus was filtered through a 0.45 μm membrane filter. Mutations observed in variants of concern were introduced into the S open reading frame in WH-Human1_EPI_402119 by overlap extension polymerase chain reaction.

[0216] Neutralization assay. Neutralization was performed according to the method of Jackson et al., 2020. Heat-inactivated serum (56°C for 30 min) was serially diluted in DMF10, and each dilution was mixed with an equal volume of S-pseudotyped HIV luciferase reporter virus and incubated in triplicate at 37°C for 1 h. The virus-serum mixture was added to 293T-ACE2 cell monolayers attached the previous day to poly-L-lysine-coated 96-well plates at 5,000 cells / well and incubated at 37°C for 2 h before adding an equal volume of DMF10. After 3 days, tissue culture medium was removed, monolayers were washed once with PBS, lysed with cell culture lysis reagent (Promega), and luciferase was measured using luciferase substrate (Promega) on a Clariostar plate reader (BMG LabTechnologies). The average percentage of entry was calculated as (RLU plasma + virus) / (RLU medium + virus) x 100. The percentage of entry was plotted against the reciprocal dilution of plasma in Prism v8.3.1 and the curves were fitted to a one-site specific binding Hill plot. The reciprocal dilution of plasma required to prevent 50% virus entry was calculated from the non-linear regression line (ID50). The lowest amount of detectable neutralizing antibody is a titer of 200. All samples that did not reach 50% neutralization were assigned an arbitrary value of 100.

[0217] Serum-mAb cross-competitive ELISA. Biotinylated S2P protein was produced in 293Expi-BirA cells (293Expi cells stably expressing BirA) and purified as described above for S2P. For competitive ELISA, Nunc maxisorp 96-well plates were coated with streptavidin (Sigma) (5 μg / ml in 50 mM carbonate buffer) overnight at 4°C, after which they were blocked with BSA (10 mg / ml in PBS) for 1 h at room temperature. After two washes, plates were incubated with biotinylated S2P trimer (2 μg / ml in 5 mg / ml BSA / PBS containing 0.05% Tween 20) for 1 h at room temperature. Serially diluted vaccine sera were mixed with subsaturating amounts of hACE2-Fc and anti-SARS-CoV-2 S mAb and incubated with streptavidin-biotinylated S2P coated plates for an additional 2 h at room temperature. mAb binding was detected using horseradish peroxidase-conjugated goat anti-human IgG F(ab')2 (Thermofisher-Scientific) or horseradish peroxidase-conjugated anti-human IgA, IgG, IgM (Dako, Glostrup, Denmark) for hACE2-Fc. The substrate was TMB. Color reactions were measured using a Multiskan Ascent plate reader (Thermo Electron, Waltham, MA). Antibodies binding to different antigens were compared by fitting curves with nonlinear regression using Prism version 9 software, and ID50s were obtained by interpolation.

[0218] Statistical methods. Data were compared statistically using the nonparametric Kruskal-Wallis test with Dunn's multiple comparisons in Prism 9. For neutralization assays, paired samples were compared using the Friedman test.

[0219] Material Examples 10-11 Recombinant proteins. Synthetic genes encoding the SARS-CoV-2 Hu-1, Delta (B.1.617.2) and Omicron BA.1 (B.1.1.529) receptor binding domains (S residues 332-532 according to the Hu-1 numbering system) were obtained from GeneART-ThermoFisher Scientific. The RBD was C-terminally appended with a GGSGS-OctaHis-GSGS-avitag (GLNDIFEAQKIEWHE) sequence. GSGSGS = SEQ ID NO:22, GSGS = SEQ ID NO:23. The synthetic RBD-His8-avitag gene was ligated downstream of the DNA sequence encoding the tissue plasminogen activator leader via NheI in pcDNA3 (Invitrogen). Synthetic genes encoding the SARS-CoV-2 Delta and Omicron BA.1 isolate S ectodomains, corresponding to the S2P protein reported by Wrapp et al., 2020, were obtained from GeneART-ThermoFisher Scientific. The gene encodes S residues 16 to 1208 (according to the Hu-1 numbering system), furin cleavage site mutations, R681RRAR→P681GSAS in delta and H681RRAR→P681GSAS in omicronBA.1, and diPro'2P' substitutions at positions 986 and 987. The C-terminus of S2P is appended with foldon (YIPEAPRDGQAYVRKDGEWVLLSTFL; SEQ ID NO: 20), octa-His, and avitag (GLNDIFEAQKIEWHE; SEQ ID NO: 21) sequences (collectively referred to as FHA or FHA tag), each separated by a GSGS linker. A synthetic DNA sequence encoding a GSGS linker and a 6His tag were added to the 3' end of a synthetic gene encoding S residues 16 to 1208 of the Hu-1 isolate, the furin cleavage site mutation, R682RAR→G682SAS, and diPro substitutions at positions 986 and 987, to yield S2P-1208.H6. The synthetic S2P gene was ligated downstream of a DNA sequence encoding the tissue plasminogen activator leader via NheI in pcDNA3 (Invitrogen).Mutations were introduced into the S2P expression vector using synthetic genes encoding mutated S2P subfragments produced by GeneART-ThermoFisher Scientific.

[0220] Recombinant protein expression and purification. Biotinylated RBD and S2P-FHA proteins were produced by transfection of Expi293-BirA cells with the appropriate expression vectors using Expifectamine according to the manufacturer's instructions (ThermoFisher Scientific). After 4 days of incubation at 34°C, transfection supernatants were cleared of cells by centrifugation and filtration through 0.45 m nitrocellulose filters. RBD and S2P-FHA proteins were purified by divalent cation affinity chromatography using TALON resin (Merck) followed by size exclusion chromatography using Superdex200 16 / 600 or Superose6 columns, respectively, coupled to an AKTApure device (Cytiva). Non-biotinylated S2P-FHA and S2P-1208.H6 proteins were produced by transfection of either FreeStyle™ 293 cells (293Freestyle) or Expi293F™ (Expi293F) cells with the appropriate expression vector using 293Fectin or Expifectamine, respectively, according to the manufacturer's instructions (ThermoFisher Scientific). Cells were cultured at 34° C. for 5 days, after which transfection supernatants were cleared of cells by centrifugation and filtration through 0.45 μm nitrocellulose filters. S2P proteins were then purified by divalent cation affinity chromatography using TALON resin (Merck), followed by size exclusion chromatography using a Superose6 Increase 10 / 300 column coupled to an AKTApure device (Cytiva). Where necessary, the TALON affinity chromatography step was replaced by a chromatography step using a HiTrap Chelating HP Immobilized Metal Affinity Column (Cytiva) coupled to an AKTApure instrument (Cytiva), in which the S2P protein was eluted using an imidazole gradient.

[0221] Recombinant monoclonal antibodies (mAbs). pCDNA3-based IgG1 heavy and light chain expression vectors containing the variable regions of SARS-CoV-2-directed mAbs COVOX222 (Dejnirattisai et al. 2021), S2H97 and S2E12 (Starr et al. 2021), CV3-25 (Jennewein et al. 2021), and the control HCV-directed mAb, HC33.1 (Center et al. 2020), were generated in-house using synthetic gene fragments encoding the mAb heavy and light chain variable regions produced by GeneART-ThermoFisher Scientific. mAbs were produced by transfection of Expi293F cells with equal amounts of matched heavy and light chain vectors using Expifectamine according to the manufacturer's instructions (ThermoFisher Scientific). After 5 days of incubation at 37°C, transfection supernatants were cleared of cells by centrifugation and filtration through 0.45 μm nitrocellulose filters. IgG was purified by affinity chromatography using protein G-agarose (Genscript) and exchanged into PBS. Antibodies were concentrated using Amicon centrifugal filter units. IgG solutions were filter sterilized using 0.45 μm nitrocellulose filters and aliquots were stored at -80°C.

[0222] Avidin capture ELISA. Nunc maxisorp 96-well plates were coated with avidin (Rockland) (5 μg / ml in 50 mM carbonate buffer) overnight at 4° C., after which they were blocked with BSA (10 mg / ml in PBS) for 1 h at room temperature. After four washes, plates were incubated with biotinylated RBD protein (2 μg / ml in 5 mg / ml BSA / PBS containing 0.05% Tween 20) for 1 h at room temperature. Serially diluted vaccine sera were incubated with the avidin-biotinylated RBD coated plates for an additional 2 h at room temperature. Antibody binding was detected using horseradish peroxidase-labeled rabbit anti-guinea pig IgG (DAKO). The substrate was TMB (3,3′,5,5′-tetramethylbenzidine). The color reaction was measured using a Multiskan Ascent plate reader (Thermo Electron, Waltham, MA). Antibody binding with different antigens was compared by fitting curves with nonlinear regression using Prism version 9 software, and endpoint titers were determined as five times the background OD obtained in the absence of primary antibody.

[0223] Streptavidin capture ELISA. Nunc maxisorp 96-well plates were loaded with streptavidin

[0224] (Sigma) (5 μg / ml in 50 mM carbonate buffer) overnight at 4°C, after which they were blocked with BSA (10 mg / ml in PBS) for 1 h at room temperature. After two washes, the plates were incubated with biotinylated S2P-FHA trimer (2 μg / ml in 5 mg / ml BSA / PBS containing 0.05% Tween 20) for 1 h at room temperature. Serially diluted human mAbs were incubated with streptavidin-biotinylated S2P-FHA coated plates for an additional 2 h at room temperature. mAb binding was detected using horseradish peroxidase-labeled goat anti-human IgG F(ab')2 (Thermofisher-Scientific). The substrate was TMB (3,3',5,5'-tetramethylbenzidine). The color reaction was measured using a Multiskan Ascent plate reader (Thermo Electron, Waltham, MA).

[0225] Live virus neutralization assay. The rapid high content SARS-CoV-2 microneutralization assay (R-20 assay) platform using HAT-24 cells developed by Aggarwal et al. 2021 was used to determine the live virus neutralization ID of vaccine sera. 50 HAT-24 cells were trypsinized and resuspended in DMEM-5% fetal bovine serum medium containing 5% v / v Hoechst-33342 live nuclear dye (Invitrogen, R37605), and plated in 384-well plates (Corning, CLS3985) at 1.6 × 10 cells. 4guinea pig serum was serially diluted (2-fold) in DMEM-5% FBS and mixed in duplicate with an equal volume of SARS-CoV-2 virus solution at twice the median lethal dose (2xLD50). After 1 h of virus-serum incubation at 37°C, 40 μL was added to an equal volume of pre-seeded cells. The cell plates were then incubated for 20 h before being imaged directly with an InCell Analyzer HS2500 high content fluorescence microscope system (Cytiva). Cell nuclei counts were obtained with IN Carta automated image analysis software (Cytiva) and the percentage of virus neutralization was calculated with the formula: N% = (D-(1-Q)) x 100 / D, where "Q" is the number of nuclei in the well divided by the uninfected control (defined as having 100% neutralization) and D = 1-Q for the average number of positive infected controls (defined as having 0% neutralization). The cutoff for determining the neutralization endpoint titer of diluted serum samples was set at the last serial dilution that reached ≧50% neutralization for the average of technical duplicates.

[0226] Chemical cross-linking. Thyroglobulin (0.5 mg / ml in PBS, Cytiva) was chemically cross-linked with 1 mM bis(sulfosuccinimidyl) suberate (Thermo Fisher) in PBS for 1 h on ice. The reaction was quenched with 30 mM glycine in PBS (pH 7.2) for 30 min on ice.

[0227] Construction of S2P-1273 expression vectors. Synthetic genes encoding S residues 1-1273 of Hu-1, Delta, and Omicron BA.1 variants were produced by GeneART-Thermo Fisher. The genes contained a KpnI restriction site at the 5' end followed by the TATCGCCACC (SEQ ID NO: 24) sequence (before the ATG start codon) and an XbaI site at the 3' end (after the TAA stop codon). The synthetic genes encoded the furin cleavage site mutations, R682RAR→G682SAS in Hu-1, R681RRAR→P681GSAS in Delta, and H681RRAR→P681GSAS in Omicron BA.1, as well as di-Pro'2P' substitutions at positions 986 and 987. The synthetic genes were cloned into the KpnI-EcoRV sites of the CMV promoter-driven expression vector pSHUTTLE (Agilent).

[0228] Western blotting of S2P-1273 glycoprotein expressed in 293T cells. 293T cells were transfected with S2P-1273 expression vector using FUGENE HD (Promega) according to the manufacturer's instructions. 48 hours after transfection, cells were washed with ice-cold PBS, centrifuged at 10,000 rpm for 90 seconds, and pellets were lysed in lysis buffer (1% TritonX100 in PBS containing 1 mM ethylenediaminetetraacetic acid) for 30 minutes on ice. Lysates were cleared by centrifugation at 10,000 rpm for 10 minutes at 4°C and subjected to SDS-PAGE in the presence of 3% beta-mercaptoethanol. Proteins were transferred to nitrocellulose using the iBLOT2 system (Thermo Fisher), and membranes were blocked with 5% nonfat dry milk in PBS. Filters were probed with rabbit anti-S1 polyclonal antibody (Sino Biological) and anti-rabbit IRDye800CW (Odyssey). The filters were then scanned with a LI-CORE imager.

[0229] Flow cytometry. 293T cells were transfected with S2P-1273 expression vector using FUGENE6 (Promega) according to the manufacturer's instructions. 48 hours after transfection, attached cells were washed with PBS and then detached using Versen solution. Cells were resuspended in 800 μl of FACS buffer (5% v / v fetal bovine serum in PBS containing 2 mM ethylenediaminetetraacetic acid). Cells were added to u-bottom 96-well culture plates and incubated with 5 μg / ml human mAb in FACS buffer for 1 hour at room temperature. Cells were washed twice in ice-cold FACS buffer by centrifugation at 400×g for 5 minutes. Cells were then incubated with AlexaFluor647 goat anti-human (H+L) (Invitrogen) for 30 minutes at room temperature in the dark. Cells were washed twice in ice-cold FACS buffer by centrifugation at 400×g for 5 minutes. Cells were resuspended in 100 μl of FACS buffer. Before each flow cytometry run, propidium iodide was added to a final concentration of 2.5 μg / ml to allow for the exclusion of dead cells during analysis. Cells were applied to the Canto II flow cytometer immediately after the addition of propidium iodide. 10,000 events were captured for each antibody-S2P-1273 protein combination. FlowJo software was used for data analysis.

[0230] Material Example 12 Western blotting of S2P-1273 and S2P.omicron.BA.1-1273 glycoproteins expressed in 293T cells after treatment at various temperatures. 293T cells were transfected with S2P-1273 and S2P.omicron-1273 expression vectors using FUGENE HD (Promega) according to the manufacturer's instructions. 48 hours after transfection, cells were washed with ice-cold PBS, centrifuged at 10,000 rpm for 90 seconds, and pellets were lysed in lysis buffer (1% TritonX100 in PBS containing 1 mM ethylenediaminetetraacetic acid) for 30 minutes on ice. Lysates were clarified by centrifugation at 10,000 rpm for 10 minutes at 4°C. The clarified lysates were divided into equal volumes and adjusted to contain final concentrations of 1.2% (w / v) SDS and 0.25% (v / v) beta-mercaptoethanol. Samples were treated at various temperatures for 5 min and subjected to SDS-PAGE on 5% polyacrylamide gels. Proteins were transferred to nitrocellulose using the iBLOT2 system (Thermo Fisher) and membranes were blocked with 5% nonfat dry milk in PBS. Filters were probed with rabbit anti-S1 polyclonal antibody (Sino Biological) and anti-rabbit IRDye800CW (Odyssey). Filters were then scanned with a LI-CORE imager.

[0231] Recombinant S2P.omicron-1208.H6 protein. A synthetic gene encoding S residues 16-1208 (Hu-1 numbering system) of the OmicronBA.1 isolate, the furin cleavage site mutation, H681RRAR→P681GSAS, and di-Pro substitutions at positions 986 and 987 were added to the 3' end with a synthetic DNA sequence encoding a GSGS linker and a 6His tag to yield S2P.omicron-1208.H6. The synthetic gene was obtained from GeneART ThermoFisher Scientific. The S2P gene was ligated downstream of the DNA sequence encoding the tissue plasminogen activator leader via NheI in pcDNA3 (Invitrogen). The synthetic genes encoding the mutated S2P subfragments produced by GeneART ThermoFisher Scientific were used to introduce the mutations into the S2P expression vector.

[0232] Expression and purification of S2P.omicron-1208.H6 protein. S2P.omicron-1208.H6 protein was produced by transfection of Expi293F (Expi293F) cells with the appropriate expression vector using Expifectamine according to the manufacturer's instructions (ThermoFisher Scientific). Cells were cultured at 34°C for 7 days, after which the transfection supernatant was cleared of cells by centrifugation and filtration through a 0.45 μm nitrocellulose filter. S2P.omicron-1208 protein was then purified by divalent cation affinity chromatography using a HiTrap Chelating HP Immobilized Metal Affinity Column (Cytiva) coupled to an AKTApure device (Cytiva). S2P protein was eluted using an imidazole gradient. S2P.omicron-1208 trimer was purified by size-exclusion chromatography using a Superdex200 16 / 600 or Superose6 column coupled to an AKTApure instrument (Cytiva).

[0233] Example 2 - Characterization of S2P A CMV promoter-driven expression vector was used to express S residues 16 to 1208, a furin cleavage site mutation, and R 682 RAR -> G 682 We produced a soluble form of the S glycoprotein, known as S2P (Wrapp et al., 2020), containing SAS, di-Pro substitutions at positions 986 and 987. Foldon, octa-His, and avitag sequences were added to the C-terminus to obtain S2P-FHA. After partial purification by divalent cation affinity chromatography, SEC of the S2P-FHA protein showed a major peak co-elution with thyroglobulin (669 kDa), which was collected as a homogenous protein, as shown by SEC and SDS-PAGE (Figure 2A and B). The biotinylated form of purified S2P-FHA showed binding activity with hACE2-Fc (human ACE2 residues 19-615 bound to the Fc domain of human IgG1) and various human mAbs in ELISA (Figure 2C). Thermofluorescence assays showed that the S2P protein had a relatively low thermal stability, exhibiting a melting temperature of 43.6 °C (Figure 2D).

[0234] Example 3 - Effect of Ala cavity substitution on thermal stability The effect of replacing Ala1016 / 1020 with more bulky hydrophobic residues (Val, Leu, Ile, Phe) was evaluated on the stability and antigenic structure of the SARS-CoV-2 S trimer (two examples are shown in Figure 1D). In S2P-FHA and 293F-expressed glycoproteins partially purified by divalent cation affinity chromatography, alanine 1016 and A 1020were substituted singly and doubly by Val, Ile, Leu, and Phe. SDS-PAGE showed a range of yields: S2P-FHA, 1016V, 1020V, 1020I>1016 / 20VV, 1016I, 1020I, 1016L, 1020L, 1016 / 20VI>1016 / 20II, 1016 / 20LL, 1016 / 20VL, 1016 / 20VF, 1016 / 20IF>1016 / 20FF (Figure 3A). A thermofluorescence assay was used to examine the thermal stability of S2P-FHA and mutants. S2P-FHA contained a major species with a melting temperature of 43.6°C and a minor species with a more stable melting temperature of 58°C (Figure 3B). Substitution of a hydrophobic residue at position 1016 increases the proportion of the 58°C species with A1016L being the most stabilizing mutation. 1020 The predominant form at this substitution remained the 43.6 °C species. All double substitutions were associated with stable forms, except for 1016 / 20VV.

[0235] Representative mutants were purified to homogeneity and reanalyzed in a thermofluorescence assay (Figure 4A-C). Thermofluorescence data obtained with partially pure proteins were largely reproduced with the purified trimers. For mutants, an increasing proportion of the 58 °C form compared to the 43.6 °C form was observed for the following mutants: 1016 / 20VI = 1016 / 20II > 1016L > 1016V > S2P-FHA (Figure 4C). Interestingly, this hierarchy of stability was inversely correlated with trimer yield (Figure 4B). Figure 4D confirms the purity of the purified trimers as assessed by SDS-PAGE under non-reducing and reducing conditions.

[0236] Example 4 - Antigenic properties of Ala cavity mutants The 1016L and 1016 / 20VI variants exhibit favorable thermal properties and can be purified with reasonable yields. Their antigenic properties were therefore compared to those of S2P by examining their binding with hACE2-Fc and recombinant human anti-S monoclonal antibodies (mAbs) by biolayer interferometry (BLI). hACE2-Fc and mAbs were bound to an anti-human IgG Fc capture (AHC) biosensor while the S2P protein was in the analyte phase. No measurable off-rates were evident for the majority of the S ligands, likely due to affinity effects, preventing KD values ​​from being obtained (Figure 5A). Comparison of sensograms showed a series of reductions in the overall binding capacity for 1016L and 1016 / 20VI compared to S2P-FHA in hACE2-Fc and mAbs that interfere with the S-ACE2 interaction, such as H4, B38, and CB6. This binding pattern was also observed for CR3022, a mAb directed against an epitope at the base of the RBD that is exposed when the RBD is in the "up" orientation. In contrast, a series of increases in binding were observed for 1016L and 1016 / 20VI, respectively, for COVA1-25, directed against an epitope in S that is external to the RBD (Figure 5A). The data are summarized in a heat map (Figure 5B) showing the change in wavelength observed after 240 seconds of association with 30 nM analyte. These data suggest that 1016L and 1016 / 20VI promote an RBD-down conformation within the S2P-FHA trimer, exposing an epitope within S but outside the RBD.

[0237] Example 5 - Immunogenicity of Ala Cavity Mutants Guinea pigs were used to investigate whether the Ala cavity mutation could affect the magnitude and specificity of the antibody response to the S2P-FHA trimer. Inbred guinea pigs were immunized with 30 μg of S2P-FHA, 1016L, and 1016 / 20VI in Addavax adjuvant at weeks 0, 4, and 14 and bled at weeks 6 and 16 (Figure 6A). Antibody binding titers against the RBD, S1, and S2P-FHA (Hu-1 sequence) proteins were determined in week 16 sera by ELISA. No significant differences were observed between immunogen groups, with a geometric mean binding titer of 1.3 × 10 5 ~9.5×10 5 The range was 0.01 (Figure 6B).

[0238] Neutralizing activity in vaccine sera was determined using S-pseudotyped HIV luciferase reporter virus and 293-ACE2 target cells as reported by Jackson et al., 2020. Comparison of week 6 and week 16 sera (drawn 2 weeks after the first and second boosts, respectively) pseudotyped to contain the S2P-FHA immunogen-matched S glycoprotein (Hu-1) isolate demonstrated potent neutralizing activity in S2P-FHA-, 1016L-, and 1016 / 20VI-immune sera, with mean IDs ranging from 1,700 to 1,900 for week 6 sera and 6,000 to 9,100 for week 16 sera. 50 These data show that the mean neutralizing ID 50This corresponds to an approximately 3- to 5.4-fold increase in neutralization activity against beta (B.1.351) VOC S protein-containing pseudotypes (Jackson, 2020 No. 20) was next evaluated. The mean neutralization capacity of S2P-FHA- and 1016L-immune sera was reduced 3.7- and 2.8-fold, respectively, against beta S pseudotypes compared to the vaccine strain (Hu-1), whereas the only 1.8-fold reduction observed with 1016 / 20VI sera was not statistically significant. These data suggest that the 1016 / 20VI immunogen induces NAb specificity that is not affected by beta variant mutations. ELISA was used to compare binding of sera to Hu-1 and the beta variant RBD, the latter of which contained the K417N, E484K, and N501Y ("NKY") mutations (Figure 6C). No significant differences in binding titers were observed to the two antigens, indicating that the reduced beta variant neutralization by S2P-FHA and 1016L sera cannot be explained simply by sensitivity to the NKY mutation in the RBD.

[0239] We next evaluated the neutralizing activity of vaccine sera against S pseudotypes bearing individual mutations observed in the primary variants of concern (VOC and VOI), including N439K, S477N, E484K, and N501Y. These mutations were combined with D614G, present in the majority of pandemic isolates, and sera showed an overall increase in neutralizing capacity against D614G, which was largely retained in 1016 / 20VI with the addition of N439K, S477N, and N501Y (Figure 8). Interestingly, the reduction in neutralizing capacity against the variants was not evident compared to Hu-1 S pseudotypes. Thus, the reduction in neutralizing capacity against the beta variant pseudotype observed with S2P-FHA and 1016L sera could not be attributed to the individual component mutations present in this variant.

[0240] Example 6 - Specificity of antibody responses To gain an understanding of the specificity of the antibody response in vaccinated animals, a serum monoclonal antibody cross-competition assay was used. Biotinylated S2P-FHA captured on a streptavidin-coated ELISA plate was incubated with a mixture containing subsaturating amounts of human monoclonal antibodies and a dilution series of serum. The assay was developed using anti-human F(ab')2-HRP and TMB. The data (Figures 9A and B) show that S2P-FHA, S2P.1016L-FHA, and S2P.1016 / 20VI-FHA had approximately equal abilities to elicit antibody specificities that could block S2P-FHA binding by ACE2-Fc and NAbs directed against the ACE2 binding site (CB6, B38, and COVA2-15), NAbs directed against the NTD (COVA1-22), and COVA1-25, which binds to a non-RBD, non-NTD epitope in S. Sera from the three immunogen groups also showed blocking activity against CR3022, a non-neutralizing antibody directed against the base of the RBD that is hidden in the RBD-down conformation. The data show that the three immunogens have similar abilities to elicit neutralizing antibodies against the ACE2 binding site, the NTD, and undefined epitopes in the RBD and S outside the NTD. These data also show that the three immunogens have similar abilities to induce antibodies directed against epitopes that overlap with ACE2b, despite the results in the BLI experiments in which this site is considered to be less exposed in 1016L and 1016L / 20VI.

[0241] Example 7 - Preclinical evaluation Preclinical evaluation of the stabilized spike booster mRNA vaccine will be performed in guinea pigs. Three groups of 10 guinea pigs per group will initially receive two doses of mRNA encoding the ancestral spike at 3-week intervals to mimic the mRNA spike vaccine regime. Alternatively, three groups of 10 guinea pigs per group will receive two doses of adenovirus 5 encoding the ancestral spike at 12-week intervals to mimic the AstraZeneca vaccine regime. Guinea pigs will be boosted with mRNA encoding 1) the ancestral spike, 2) the novel stabilized spike, or 3) a placebo. Serum neutralization and spike protein binding (RBD and spike trimer) will be determined 14 and 28 days after boost. Serum neutralization assays will be performed using the spike-HIV luciferase reporter system (Jackson et al., 2020) and will include spike protein derived from the VOC. Spike binding will be determined by ELISA and spike protein derived from the VOC will be included in the analysis.

[0242] Example 8 - Phase I safety and immunogenicity trial of a universal booster mRNA vaccine Phase I clinical trials will be conducted to evaluate the safety and efficacy of the booster vaccine candidates (mRNA and adenoviral vector). Each vaccine candidate will be tested by a randomized, double-blind, placebo-controlled clinical trial. Trial participants will be 18-85 years old who have already received a full course of the current two-dose Pfizer (mRNA vaccine) or AstraZeneca (adenoviral vector vaccine) at least 6 months prior to the time of recruitment. Participants in the mRNA stabilized spike booster trial will be assigned to four groups: placebo, and 30ug of mRNA stabilized spike vaccine (a dose based on the current BioNTech-Pfizer vaccination regimen). The aim is to have 10 people in the placebo group and at least 20 people in the booster vaccine group (at least 60 people in total). The primary endpoints are: 1) local reactions, systemic events, and use of antipyretics or analgesics occurring within 7 days after receiving the booster vaccine or placebo, 2) undesirable and serious adverse events assessed up to 6 months after receiving the booster vaccine, 3) laboratory abnormalities assessed 1 and 7 days after receiving the booster vaccine, and 4) graded changes in laboratory assessments between baseline and 1 and 7 days after vaccine administration. Protocol-specified safety stopping rules will be in effect for all participants. Secondary endpoints are serum neutralization and spike protein binding (RBD and spike trimer) determined at 7, 14, 28, 180, and 365 days after the boost. Serum neutralization assays will be performed using the Spike-HIV luciferase reporter system (Jackson et al., 2020) and will include spike protein derived from VOCs. Spike binding will be determined by ELISA, and spike protein derived from VOCs will be included in the analysis.

[0243] Example 9 - S2P-FHA has enhanced responses against ancestral strains (ancestral strain Hu-1) and beta variants (B.1.351) compared to conventional mRNA and adenovirus vaccines Pseudovirus neutralization ID50 studies were performed to compare vaccine responses generated against the S2P-FHA1016 / 20VI spike protein with those generated in humans using conventional vaccines. Three doses of the S2P-FHA1016 / 20VI protein vaccine in guinea pigs were compared to two doses of mRNA or adenovirus vaccine given to humans. The S genotypes used in the S-HIV pseudotype assays are shown below the graph. Data are presented for 1016 / 20VI (Burnet VI on the left) compared to sera obtained 3-5 weeks after two doses of conventional vaccine in humans (exemplary conventional vaccine (conventional vaccine) on the right). Figure 14 shows neutralizing antibody responses against matched WT virus and highly resistant variants of concern (beta variants) from guinea pigs vaccinated with S2P-FHA protein trimers containing the ancestral and beta VOCs. Wilcoxon matched rank tests were used to determine whether differences in ID50 observed between groups were significant: ns is not significant, P > 0.05, **** is P < 0.0001. As shown in Figure 14, S2P-FHA1016 / 20VI showed higher neutralizing antibody responses against both ancestral and beta VOCs than conventional mRNA and adenovirus vaccines. The S2P-FHA1016 / 20VI antigen produces a desirable neutralizing antibody profile for both primary vaccine regimens (the first vaccine regimen used to generate an immune response in a subject against SARS-CoV-2) and booster vaccine regimens. Booster vaccines can be used, for example, in subjects receiving a primary vaccine regimen who have generated one or more of a response to ancestral strains, a low response to ancestral strains, a low immune response to VOCs, and a low immune response to beta VOCs.

[0244] Example 10 - Characterization of serum antibodies elicited by S2P-FHA, S2P.1016L-FHA, and S2P.1016 / 20VI-FHA trimer immunogens in experimental animals RBD binding titers of vaccine sera were determined using a capture ELISA format using plate-bound avidin to capture biotinylated Hu-1, delta, and omicron BA.1 RBD. Delta (B.1.617.2) VOC emerged in October 2020 and has become the dominant global variant. There are two notable mutations in the RBD: L452R and E484Q. Omicron (B.1.1.529), first identified in South Africa and Botswana (November 2021), has replaced the delta variant in most countries. Omicron consists of at least three genetically distinct sublineages (BA.1, BA.2, and BA.3) that emerged in 2021. Initially, BA.1 was the most common circulating variant, but the more infectious BA.2 variant is now becoming dominant. Thirty mutations occur in the spike, including 15 in the RBD and 8 in the NTD, which contain the major sites of neutralization. 5 ~1.36×10 5 Figure 15B shows that there are no significant differences between the immunogen groups with geometric mean binding titers ranging from 3.0×10 to 3.5×10. A small (approximately 3-fold) but significant reduction in binding titer was observed for Omicron BA.1 RBD for the three immunogen groups. Serum binding titers to Hu-1, Delta, and Omicron BA.1 S2P-FHA spike protein trimers bound directly to ELISA plates were then determined. Figure 15B reveals that there are no significant differences in binding titers to the three S2P-FHA trimer variants for the three immunogen groups. S2P-FHA binding titers were 3.0×10 to 3.5×10. 5 ~1.1×10 6 These results suggest that all immunogens generated similar antibody titers capable of binding to the S2P-FHA spike trimer.

[0245] Neutralizing activity in vaccine sera was then determined using S-pseudotyped HIV luciferase reporter virus and 293-ACE2 target cells as reported by Jackson et al. 2020. Pseudoviruses containing the S2P immunogen-matched Hu-1 spike glycoprotein or delta's showed potent neutralizing activity in S2P-FHA-, 1016L-, and 1016 / 20VI-immune sera, with mean IDs ranging from 3,900 to 5,100. 50 Serum neutralizing activity against Omicron BA.1 or betaspike-containing pseudoviruses was not significantly different compared to Hu-1 and delta pseudovirus neutralization, but IC for Omicron BA.1 pseudovirus was significantly higher than IC for Hu-1 and delta pseudovirus neutralization (Figure 16A). 50 There was a downward trend, with a <3-fold reduction in mean titers observed in the three groups.

[0246] We next tested neutralizing activity against authentic infectious SARS-CoV-2 viruses Hu-1, Delta, Omicron BA.1, and Beta using HAT-24 cells in the R-20 microneutralization assay developed by Aggarwal et al. 2021. Hu-1 and Delta viruses were potently neutralized by sera from the three immunogen groups, whereas Omicron BA.1 and Beta virus neutralization was approximately 1 log 10 The titers were only slightly reduced by 100% (Figure 16B). Figure 17 shows a summary of all geometric mean titers obtained in the ELISA (RBD and spike trimer), pseudotype virus neutralization, and microneutralization assays. Overall, the data show that three doses of S2P-FHA, S2P.1016L-FHA, and S2P.1016 / 20VI-FHA elicit high titer antibodies against the RBD and S trimer that were able to potently neutralize HIV pseudoviruses containing the matched ancestral Hu-1 spike, as well as pseudoviruses carrying spikes from delta, omicron BA.1, and beta VOC. High neutralization titers were also observed with authentic infectious SARS-CoV-2, but not with ID 50 Moderate at about 1 log 10 A reduction in was observed in omicron BA.1 and beta variants.

[0247] Example 11 - Assessing whether mutations in the alanine cavity have a general stabilizing effect when introduced into spike trimers derived from highly divergent SARS-CoV-2 sequences When compared to the ancestral sequence of Hu-1, 30 mutations have arisen in the Omicron BA.1 spike, with 8 in the N-terminal domain (NTD) and 15 in the RBD (Jung et al. 2022). The NTD and RBD contain the major neutralizing epitopes, and consistent with the presence of multiple mutations in these two domains, sera obtained from mRNA dual-vaccinated individuals retained very low neutralizing activity against Omicron BA.1 virus in vitro (Tada et al. 2022), corresponding to low vaccine efficacy against Omicron infection (Andrews et al. 2022).

[0248] The mutations present in the Omicron BA.1 spike (compared to the ancestral Hu-1) are: NTD: A67V; deletion of H69-V70; T95I; G142D; delV143-Y145; deletion of 211; L212I; insertion of EPE after residue 214; RBD:G339D;S371L;S373P;S375F;K417N;N440K;G446S;S477N;T478K;E484A;Q493R;G496S;Q498R;N501Y;Y505H; C-terminal region of S1: T547K; D614G; H655Y; N679K; P681H; S2:N764K;D796Y;N856K;Q954H;N969K;L981F

[0249] To determine whether mutations in the alanine cavity have a general stabilizing effect when introduced into spike trimers derived from highly branched VOC sequences, the omicronBA.1 versions of the S2P-FHA and S2P.1016 / 20VI-FHA constructs were prepared (sequences in Figures 24, 25), referred to here as S2P.omicron-FHA and S2P.omicron.VI-FHA, respectively. The S2P.omicron-FHA sequence contains a tissue plasminogen activator leader sequence upstream of S residues 16-1208, a furin cleavage site mutation, H681RRAR→P681GSAS, and diPro substitutions at positions 986 and 987. Foldon, octa-His, and avitag sequences were added to the C-terminus. The DNA sequences were cloned into a CMV promoter-driven expression vector, and the proteins were expressed in Expi293F cells by transfection.

[0250] Proteins were extracted from culture supernatants by divalent cation affinity chromatography and further purified by Superose6 SEC. S2P.omicron-FHA eluted as a major peak close to the position of thyroglobulin (669 kDa) (Figure 18A, top) with a profile nearly identical to that of S2P-FHA from the Hu-1 isolate (Figure 18A, bottom). The trimer was collected as a homogenous protein as shown by analytical SEC (Figure 18B). S2P.omicron.VI-FHA eluted as four species, however a prominent putative trimer peak was observed (Figure 18A, indicated by the dashed box in the middle). Fractions corresponding to the trimer (dashed box in Figure 18A) were pooled, concentrated, and rechromatographed on a Superose6 column to reveal a largely homogenous species corresponding to the trimer (Figure 18B, Figure 18C, top).

[0251] Thermofluorescence assays showed that S2P.omicron-FHA trimer had relatively high thermal stability with a melting temperature of 61° C. (FIG. 18C, top). S2P-FHA derived from Hu-1 had a melting temperature of 43° C. (FIG. 18C, bottom), consistent with FIG. 4C. No thermal unfolding was observed for S2P.omicron.VI-FHA (FIG. 18C, middle).

[0252] Purified S2P.omicron-FHA and S2P.omicron.VI-FHA trimers were further analyzed by SDS-PAGE under non-reducing and reducing (1% beta-mercaptoethanol) conditions without sample boiling (Figure 18D). S2P.omicron-FHA trimers were largely resolved to the expected monomer molecular weight of approximately 160 kDa in both the presence and absence of reducing agents. In contrast, the major S2P.omicron.VI-FHA species (indicated by arrow) was retained as a high molecular weight species migrating close to the top of the gel, with a minor species also observed at the monomer position. Figure 18E shows a repeat experiment in which samples were boiled for 5' prior to electrophoresis. Under non-reducing conditions, the results shown in Figure 18D were largely reproduced with the major S2P.omicron.VI-FHA species of high molecular weight again observed. However, upon boiling in the presence of reducing agents, this species was resolved to its monomer molecular weight. The data (FIG. 18E) suggest that the S2P.omicron.VI-FHA trimer is resistant to disruption by 0.8% w / v sodium dodecyl sulfate denaturant with and without 1% beta-mercaptoethanol at 25° C. The data further suggest that the S2P.omicron.VI-FHA trimer is resistant to disruption by 0.8% w / v sodium dodecyl sulfate denaturant after boiling. The S2P.omicron.VI-FHA trimer is only degraded to monomers after boiling in the presence of both 0.8% w / v sodium dodecyl sulfate denaturant and 1% beta-mercaptoethanol. The data suggest that the addition of the VI mutation into the alanine cavity of S2 confers ultrastability to the S2P.omicron.VI-FHA trimer, allowing the trimer structure to withstand greater denaturing forces.

[0253] Thyroglobulin was covalently coupled with bis(sulfosuccinimidyl) suberate to obtain an SDS-PAGE marker with a theoretical mol.wt of 669 kDa, close to that of the S trimer. Figure 18F shows that cross-linked thyroglobulin co-migrated with the SDS / beta-mercaptoethanol-resistant high molecular weight form S2P.omicron.VI-FHA after treatment with 0.8% SDS for 3 min at 25°C or 100°C, or with 0.8% SDS + 1% beta-mercaptoethanol for 3 min at 25°C prior to electrophoresis. Again, S2P.omicron.VI-FHA was resolved as a monomer after boiling in 0.8% SDS + 1% beta-mercaptoethanol. S2P.omicron-FHA and S2P-FHA proteins migrated as monomers after all treatments, except for some residual trimer S2P-FHA after treatment with 1% SDS at 25°C.

[0254] A capture ELISA using plate-bound streptavidin to capture biotinylated S2P.omicron-FHA or biotinylated S2P.omicron.VI-FHA trimers (FIG. 19, left and right panels, respectively) was used to determine whether these proteins could be recognized by conformation-dependent NAbs. The data show that RBD-directed NAbs COVOX222, S2H97, and to a lesser extent S2E12 and COVA2-17 bound to the two proteins with similar activity. Binding was also observed with NAbs against S2 CV3-25, COVA1-21 against NTD, and non-RBD-directed NAbs COVA1-25 and COVA1-22. HC33.1 is a Hepatitis C virus NAb that served as a negative control. The data show that S2P.omicron.VI-FHA trimers retain the conformation-dependent NAb epitopes also observed with S2P.omicron-FHA.

[0255] S2P-FHA, engineered using the ancestral Hu-1 SARS-CoV-2 sequence and the sequence of the variant of concern engineered here, contains a C-terminal trimerization module that stabilizes the trimer structure, similar to the soluble S trimers reported in the literature (Wrapp et al. 2020). This module is usually the trimer foldon domain of bacteriophage T4 fibritin (Figure 20A). NMR studies have shown that the native transmembrane domain is likely a three-stranded coiled coil embedded in the lipid bilayer. To determine the oligomerization state of the Hu-1 S ectodomain in the absence of the trimerization tag, Hu-1 S2P was truncated at the last residue Q1208 of the ectodomain to obtain S2P-1208.H6 (Figure 20A, see Figures 26 and 27 for sequence). The corresponding 1016 / 20VI mutant, S2P.VI-1208.H6, was also prepared. Both proteins have a 6His tag at the C-terminus to facilitate purification, designated H6. Proteins were expressed in 293FS cells and then extracted from the supernatant by divalent cation affinity chromatography. Superose6 SEC revealed that Hu-1 S2P-1208.H6 was eluted as a dimer in contrast to the trimeric structure of S2P-FHA (Figure 20B, top two panels). In contrast, for Hu-1 S2P.VI-1208.H6, trimers and dimers were observed in roughly equal amounts (Figure 20B, third panel). To enable purification of S2P.VI-1208.H6 trimers, proteins were expressed in Expi293F cells and eluted with an imidazole gradient from a HiTrap divalent cation-loaded column. Superose6 SEC showed a prominent trimer peak for S2P.VI-1208-H6 (Figure 20B, fourth panel).

[0256] Thermofluorescence assays showed that the Hu-1 S2P-FHA trimer and S2P-1208.H6 dimer had melting temperatures of 43° C., while S2P.VI-1208.H6 contained two species with melting temperatures of 43° C. and 58° C., respectively (FIG. 20C). SDS-PAGE under non-reducing and reducing conditions showed that the protein preparations were highly pure and denatured to monomers upon boiling with and without 0.8% SDS and 1% beta-mercaptanol, indicating that they were non-disulfide-linked trimers (FIG. 20D). These data indicate that the 1016 / 20VI mutations confer sufficient stability to the S2P trimer, eliminating the need for a trimerization tag, and conferring thermostability to purified S2P oligomers.

[0257] The effect of the 1016 / 20VI mutation in the context of full-length S2P, containing the native transmembrane domain and cytoplasmic tail, was examined. CMV-driven expression vectors containing codon-optimized genes encoding residues 1-1273 of the S glycoprotein from Hu-1, Delta, and OmicronBA.1 were prepared. The R / H681RRAR→P681GSAS mutation in the furin site and di-Pro "2P" substitutions at positions 986 and 987 were also included. See FIG. 21A for a schematic of S2P-1273 and FIGS. 28-33 for protein and DNA sequences.

[0258] To demonstrate expression of Hu-1, delta, and omicron BA.1 S2P-1273 glycoproteins and their 1016 / 20VI mutant form (S2P.VI-1273), DNA vectors were transfected into 293T cells. Cells were lysed and lysates were subjected to SDS-PAGE and Western blotting with rabbit anti-S1 polyclonal antibody. Figure 21B shows a single approx. 180 kDa protein band for each construct, consistent with that expected for uncleaved S monomers. Transfection with empty vector (pcDNA3) did not produce such a band.

[0259] Using flow cytometry, we demonstrated that Hu-1, delta, and omicron BA.1 S2P.1273 and S2P.VI-1273 glycoproteins are expressed on the cell surface and recognized by human monoclonal antibodies directed against key neutralizing epitopes. 293T cells were transfected with various S2P-1273 expression vectors and intact cells were stained with various human monoclonal NAbs and AlexaFluor-conjugated anti-human immunoglobulins. Cells were counterstained with propidium iodide to allow for the exclusion of dead cells from the analysis. The histograms in Figure 22 show that all expressed S2P-1273 glycoproteins bind to ACE2-Fc at similar levels and to the majority of the monoclonal NAbs tested. An isotype control HCV-specific antibody (HC33.1) showed no binding. The exception was CB6, which did not bind to the omicron spike, while COVA1-03 showed minimal binding to the delta spike. These data indicate that S2P-1273 glycoproteins are expressed on the cell surface, that they are capable of binding to the ACE2 receptor, and that they can carry diverse NAb epitopes.

[0260] The data shown in Figure 22 were further analyzed using gating to distinguish between non-fluorescent (nil), moderately (lo) fluorescent, and highly (hi) fluorescent cell populations (Figure 23A). Introduction of the VI mutation in the context of S2P-1273 did not alter ACE2 receptor binding capacity or exposure of broadly neutralizing epitopes in the RBD recognized by COVOX222, or epitopes in S2 recognized by CV3-25 (Figure 23B). However, VI increased exposure of epitopes recognized by broadly neutralizing antibodies S2H97, COVA2-17, and COVA1-21 in S2P-1273 derived from Hu-1 ancestral, delta, and omicron BA.1 isolates. Thus, the 1016 / 20VI mutation selectively enhances exposure of bNAb epitopes in an isolate-dependent manner.

[0261] Example 12 - Effect of the 1016 / 20VI mutation on the stability of full-length S2P-1273 and S2P-1208 derived from Hu-1 and OmicronBA.1 The effect of the 1016 / 20VI mutation on the stability of full-length S2P-1273 (containing the native transmembrane domain and cytoplasmic tail) derived from Hu-1 and OmicronBA.1 (S2P-1273 and S2P.omicron-1273, respectively) and expressed in 293T cells was examined. Cell lysates were adjusted to contain a final concentration of 1.2% (w / v) SDS and 0.25% (v / v) beta-mercaptoethanol and treated at the indicated temperatures before SDS-PAGE and Western blotting with rabbit anti-S1 polyclonal antibody. Purified S2P.omicron-FHA trimer forms AA and VI (as in FIG. 18F) treated with 0.67% SDS for 5 min at room temperature prior to electrophoresis were included to indicate the location of the monomer and trimer, respectively. FIG. 38 reveals prominent S2P.omicron-FHA monomer and the putative S2P.omicron.VI-FHA trimer bands in lanes 2 and 3, respectively. In the case of S2P-1273 (Figure 38, left panel), the predicted trimer is observed after treatment at 55°C, 70°C, and 85°C when the 1016 / 20VI mutation is present, but this band is absent in S2P-1273 containing Ala at positions 1016 and 1020. The trimer band is absent after treatment at 100°C, suggesting dissociation. S2P.omicron-1273 trimers appeared to be resistant to treatment at 55°C, 70°C, and 85°C, regardless of whether 1016 / 20VI was present, although slightly more trimers were observed with 1016 / 20VI (Figure 38, right panel). These data are consistent with the higher overall thermostability observed for S2P.omicron-FHA compared to S2P-FHA derived from Hu-1. The data suggest that the VI mutation confers stability to the S2P-1273 trimer, as observed with the soluble S2P-FHA trimer. The data also show that the S2P trimer derived from OmicronBA.1 exhibits greater overall stability than the Hu-1 S trimer.

[0262] Next, the effect of VI on the trimerization and stability of S2P-1208.H6, derived from the Omicron BA.1 sequence lacking the Foldon trimerization domain, was examined by adding S2P.omicron truncated at Q1208, the last residue of the ectodomain, to GSGS-H6 at the C-terminus to give S2P.omicron-1208.H6. The corresponding 1016 / 20 VI mutant, S2P.omicron.VI-1208.H6, was also prepared. Proteins were expressed in Expi293F cells and then extracted from the supernatant by divalent cation affinity chromatography. Superose6 SEC revealed that both proteins were predominantly eluted as trimers (Figure 39A). The purified trimers were reanalyzed by Superose6 SEC after freeze (-80°C)-thaw cycles, revealing that approximately 15% of the S2P.omicron-1208.H6 protein had dissociated into dimers. In contrast, the trimer structure of S2P.omicron.VI-1208.H6 was retained (Figure 39B). Thermofluorescence assay showed that the majority of purified S2P.omicron-1208.H6 had a melting temperature of 41°C, while the melting temperature of the S2P.omicron.VI-1208.H6 trimer was much higher at 64°C (Figure 39C). SDS-PAGE / Coomassie blue staining showed that the purity of the S2P.omicron-1208.H6 protein approached 100% (Figure 39D). These data indicated that the VI mutation in the context of the OmicronBA.1 ectodomain made it possible to obtain a highly stable soluble trimer, thereby eliminating the need for an exogenous trimerization domain to maintain the trimer structure.

[0263] Example 13. Effect of VI on exposure of epitopes recognized by broadly neutralizing human mAbs and ACE2-Fc in S2P-FHA trimers derived from Hu-1 and OmicronBA.1 Next, we used biolayer interferometry to compare the effect of VI on the exposure of epitopes recognized by broadly neutralizing human mAbs and ACE2-Fc in S2P-FHA trimers derived from Hu-1 and OmicronBA.1. ACE2-Fc and mAbs were attached to an anti-human IgG Fc capture biosensor while the S2P trimers were in the analyte phase. Comparison of sensograms showed an overall decreased binding of RBD-directed ligands (ACE2-Fc, S2E12, S2H97, and COVOX222) by S2P.VI-FHA derived from Hu-1 compared to S2P-FHA (Figure 40A). In contrast, this VI-dependent decrease in binding to RBD ligands was not evident for S2P.Omicron-FHA (Figure 40B). S2P-FHA proteins from Hu-1 and OmicronBA.1 bind equally to COVA1-25 with and without VI, recognizing an epitope outside the RBD. CV3-25 binds to an epitope within the S2 stem region and shows reduced binding to S2P.VI-FHA compared to other proteins, indicating that VI partially blocks the epitope in the context of Hu-1, but not OmicronBA.1. These data suggest that 1016 / 20VI promotes an RBD-down conformation in Hu-1 S2P-FHA trimers, but not OmicronBA.1-FHA trimers. Thus, VI stabilizes S2P.omicron trimers that also maintain exposure of major broad neutralizing epitopes within the RBD. Because Omicron strain mutations are likely to be widely represented in emerging strains, the results support the use of modified antigens disclosed herein to provide effective immunity to emerging strains.

[0264] Example 14 - Results Summary The coiled coil at the center of the SARS-CoV-2 and SARS-CoV prefusion trimers is formed by three arcuate helices that extend away from each other from a contact point mediated by inward-facing Ile1013 together with Leu1012. The remainder of the 3-4 repeats is composed mainly of polar residues that mediate few interhelical contacts. We hypothesized that this polar topology may be responsible for the relatively low thermostability of the prefusion trimers (observed up to 43.6 °C in the studies described herein). SARS-CoV is a respiratory pathogen, and the low body temperatures at these replication sites, ranging from 20.5 to 35.5 °C, may allow the spike trimers to maintain their structure and function in a sustained manner. However, exposure to higher body temperatures, as with intramuscular vaccination, may lead to destabilization and loss of conformation of the trimer spike over time, compromising immunogenicity.

[0265] Efforts to overcome the inherent instability of class I fusion proteins in their prefusion state through structure-based design have been widely adopted for HIV-1, Ebola, respiratory syncytial virus, Lassa virus, human metapneumovirus, and coronaviruses. Previous studies found that the introduction of two proline residues in the hinge-loop between the central coiled-coil forming helix CH1 and the heptad repeat 1 stabilized the S proteins of SARS-CoV and Middle East Respiratory Syndrome virus (MERS) in the prefusion trimer conformation without loss of receptor binding or antigenicity. In the case of SARS-CoV-2 spike, prefusion stabilization was achieved by the introduction of the S2-P mutation and the loss of the Arg682ArgAlaArg furin cleavage site (Wrapp et al., 2020), the latter of which is not present in either SARS-CoV or MERS spike. Current human SARS-CoV-2 vaccines contain either the parental S sequence (Oxford / AstraZeneca (Watanabe et al., 2021)) or S-2P / furin mutants (BioNTech / Pfizer, Johnson and Johnson (Bos et al., 2020; Vogel et al., 2021)) or S2P with an intact furin site (Moderna (Jackson et al., 2020)). The experiments described herein demonstrate that S2P We demonstrate that the S trimer can be further stabilized and exhibit enhanced stability and antigenic function by creating an artificial hydrophobic core in the center of the S2 coiled coil of the CH1 helix. In one embodiment, the artificial hydrophobic core can be generated by replacing Ala1016 and Ala1020 with bulkier hydrophobic residues to fill the cavity associated with these residues. Mutagenesis of Ala1016 (replacing the residue with a more hydrophobic residue), A1016L, shifted the melting temperature from 43.6°C to 58°C species, giving the highest proportion of 58°C species. In contrast, the 43.6°C species remained the predominant form with the substitution of Ala1020. All double substitutions were associated with more stable forms, except for 1016 / 20VV.Interestingly, increased thermostability was sometimes associated with reduced soluble S2P-FHA expression, with the lowest yields obtained with 1016 / 20II, 1016 / 20LL, 1016 / 20VF, 1016 / 20IF, and 1016 / 20FF. The 1016L and 1016 / 20VI mutants produced high yields suggesting that the bulk and / or shape of the side chains selected to fill the cavity can affect the folding of the S2P trimer, with some side chains having a more favorable effect on folding. In the case of Omicron BA.1 VOC, the 1016 / 20VI mutation stabilized the S2P-FHA trimer against harsh denaturing conditions such as boiling in the presence of 0.8% SDS or exposure to 0.8% SDS plus the reducing agent beta-mercaptoethanol at ambient temperature, consistent with an ultrastable S2P trimer. The data show that creating an artificial hydrophobic core in the center of the S2 CH coiled coil improves stability and expression. These mutations, in combination with known stabilizing mutations, such as S-2P and furin site mutations, can enhance the biophysical properties of coronavirus spike protein vaccines.

[0266] The stabilizing 1016L and 1016 / 20VI had measurable effects on the antigenicity of the Hu-1 SARS-CoV-2 spike trimer. Biolayer interferometry revealed that monoclonal antibodies blocking the ACE2 extracellular domain and the RBD-ACE2 interaction had reduced binding to the 1016L and 1016 / 20VI S2P trimer, suggesting that an RBD-down conformation was induced. Conversely, COVA1-25, directed against an epitope in S that is external to the RBD, showed improved binding to the 1016L and 1016 / 20VI S2P trimer, suggesting enhanced exposure of this domain in S. These effects of stabilizing mutations in the RBD orientation likely occur via allostery, whereby changes to the coiled-coil shape or conformation are transmitted to the distant RBD via the trimer structure. These changes to the RBD orientation in the context of the S2P trimer did not result in differences in the RBD-, S1-, or S2P trimer-directed antibody titers induced by the three immunogens studied here. Furthermore, S2P-FHA, 1016L, and 1016 / 20VI trimers generated comparable high NAb titers against strain-matched Hu-1 spike-copackaged pseudotypes after two vaccinations, which were further enhanced by three vaccinations. However, consistent with the altered antigenic landscape, 1016 / 20VI immune sera retained neutralizing capacity against beta / B.1.351 S variant pseudotypes, whereas this activity was significantly reduced in S2P-FHA- and 1016L-elicited sera. These data for 1016 / 20L indicate a 7.6- to 42-fold reduction in neutralizing titers against Beta / B.1.351 compared to those obtained with human vaccine sera from humans vaccinated with the BioNTech / Pfizer, Moderna, or Oxford / Astrazeneca vaccines ( Dejnirattisai et al., 2021 , Garcia-Beltran et al., 2021 ).Beta variants have been shown to exhibit the greatest resistance to NAbs elicited by natural infection or vaccination when compared to other VOCs, including alpha / B.1.1.7, gamma / P.1, and delta / B.1.617 (Dejnirattisai et al., 2021; Garcia-Beltran et al., 202118,21; Hoffmann et al., 2021). The differences in beta / B.1.351 variant neutralization capacity observed with S2P-FHA, 1016L, and 1016 / 20VI were not reflected in differences in binding to the Hu-1 RBD or RBD-NKY, which contains the beta / B.1.351 mutations K417N, E484K, and N501Y. These data indicate that the S2P vaccine elicits RBD-directed antibody specificity that does not target epitopes containing key residues mutated in the beta / B.1.351 VOC. Furthermore, serum neutralization capacity was not affected by the selected individual mutations present in the VOC, again suggesting that these antibodies target other than residues within the RBD that are mutated in the VOC. This observation is consistent with data obtained in serum-NAb cross-competition assays showing high titers of serum antibodies capable of blocking binding by ACE2 and NAbs directed against the RBD (CB6, B38, COVA2-15), COVA1-22 directed against the NTD, and COVA1-25 directed against an S1 epitope outside the RBD and NTD. Thus, all three immunogens elicited polyclonal responses against the S trimer, which may explain why the sera retain at least some capacity against the beta / B.1.351 variant. The mechanism by which 1016 / 20VI immune sera retain their neutralizing capacity against the Beta / B.1.351 variant was not explained by the serum-NAb competition assay, which may be due to the reliance of this assay on large IgG molecules present in serum that sterically block the binding of human IgG molecules to the S2P trimer. Overall, the three antigens appear to have similar abilities to induce antibodies directed against epitopes that overlap with the ACE2 binding site and the NTD, but the competitive ELISA is not sensitive enough to measure subtle differences in antibody binding modes.Thus, the RBD-down conformational propensity in 1016 / 20VI did not adversely affect its ability to elicit NAbs and appeared to correlate with superior NAb activity against neutralization-resistant VOCs.

[0267] Current SARS-COV-2 vaccines deliver the “ancestral” spike sequence derived from the original Hu-1 isolate, programming the vaccine recipient’s cells to produce the spike protein that elicits an antibody response. Successful vaccination of the world’s population has been complicated by high transmission rates in unvaccinated populations, allowing VOCs to evolve and spread. Breakthrough infections with VOCs in vaccinated individuals have raised major concerns that SARS-CoV-2 may overcome vaccine-induced immunity as it continues to replicate, evolve, and spread. Five major VOCs have spread in the population: alpha (UK origin), beta (South African origin), gamma (Brazilian origin), and delta (Indian origin). The enhanced transmission properties of the alpha and delta variants have allowed them to supplant ancestral Hu-1-derived viral lineages and spread worldwide. -omicron (B.1.1.529) is even more transmissible than delta and is currently the dominant VOC in most countries. Initially, BA.1 was the most common circulating type, but the more infectious BA.2 subvariant is now becoming predominant. Other subvariants, such as BA.4, BA.5, and BA.2.12.1, continue to emerge. Recent publications have shown that the efficacy of the AstraZeneca and Pfizer-BioNTech vaccines is slightly reduced against alpha and delta variants compared to the Hu-1 lineage, indicating that the immunity induced by these vaccines retains efficacy against these VOCs (Emary et al., 2021; Lopez et al., 2021). In contrast to alpha and delta, beta and gamma variants are more localized in distribution but exhibit properties that make vaccine-induced immunity less effective. For example, a clinical trial in South Africa revealed that the efficacy of the AstraZeneca vaccine against beta VOCs was only 10.4% (Madhi et al., 2021). This finding is particularly worrying given the theoretical emergence of variants with a combination of high transmissibility and vaccine-evasion properties that could render vaccinated populations more susceptible to COVID-19 infection.The extreme changes in the primary neutralization site of the omicron spike have significantly reduced the efficacy of a two-dose vaccination regimen using the ancestral sequence, necessitating the use of a booster to restore protective immune responses (Andrews et al. 2022, Magen et al. 2022). The data presented here show that the neutralizing capacity of vaccine sera raised against the stabilized S2P-FHA protein is only slightly reduced against beta and omicron VOCs, but the addition of a stable spike derived from a divergent VOC such as omicron to a conventional vaccine based on the ancestral sequence may broaden immunity for protection against emerging VOCs. Booster vaccines that broaden immunity against VOCs are essential for the long-term control of COVID-19 when they inevitably become endemic in the population. The 1016 / 20VI spike provides a strategy for developing a universal S booster vaccine that focuses antibody responses against highly conserved regions located outside the RBD in addition to the RBD.

[0268] The propensity for RBD-down conformation in S-based vaccines such as 1016 / 20VI may also be important with regard to the balance of neutralizing and infection-enhancing antibodies being elicited. Complicating the development of a SARS-CoV vaccine is evidence suggesting that S-directed antibodies may enhance viral entry and promote acute lung injury via Fc effector-mediated mechanisms. Antibody-dependent enhancement of infection may occur through receptor mimicry, where NAbs bind to the RBD and prime the S trimer for fusion activation. Viral internalization occurs through cell surface Fc gamma receptor interactions leading to viral fusion and entry. Studies of monoclonal IgG isolated from COVID-19 patients reveal that antibodies against one group of RBD epitopes can mediate ADE through an Fc gamma receptor-dependent mechanism. Alternatively, antibodies against the NTD can induce an up-RBD conformation, enhancing ACE2 binding and infectivity. Another study found that selected RBD NAbs and non-neutralizing NTD antibodies showed Fc gamma receptor-mediated enhancement of viral infection in vitro, but both types of antibodies protected against SARS-CoV-2 replication in monkeys and mice. These data imply that the observation of ADE in vitro does not necessarily predict this process in vivo. Nevertheless, locking the RBD in a face-down conformation in trimer spike vaccines such as 1016 / 20VI may favor the generation of neutralizing RBD-directed antibodies beyond ADE-antibody specificity.

[0269] While the unusual alanine cavity in the center of SARS-CoV strains such as SARS-CoV-2 S2 can be filled by substitution of hydrophobic residues at amino acid positions 1016 and 1020 as shown herein, it is possible that additional or alternative a and d residues in the CH coiled coil can be mutated to generate additional stabilizing mutations via structure-guided mutagenesis. Stabilizing the coiled coil has multiple advantages over existing diproline and furin mutagenesis approaches by increasing thermostability and locking the down-RBD to prevent potential generation of infection-enhancing antibody specificities. Furthermore, in SARS-CoV-2, it is shown herein that spike trimers containing alanine cavity mutations such as 1016 / 20VI induce improved neutralization breadth, strength, and longevity. The introduction of mutations such as 1016 / 20VI described herein provides a strategy to improve the nature, durability, and robustness of NAb responses against strains of SARS-CoV, including importantly new VOCs, to future tolerable vaccines for SARS-CoV-2. Indeed, the results show that the introduction of 1016 / 20VI into Omicron VOC has a stabilizing effect on the S2P trimer eliminating the need for a trimerization tag and confers thermal stability to the purified S2P oligomers. Thermal and denaturation stability are essential to meet the needs of the global vaccine supply chain while providing in vitro and in vivo benefits. In one embodiment, the modified antigen eliminates the need for a heterologous trimerization tag, reducing the risk of off-target reactivity.

[0270] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the invention as illustrated in the specific embodiments without departing from the spirit or scope of the invention as broadly described, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0271] This application claims priority to Australian Provisional Application No. 2021902500, entitled "Vaccine Antigen", filed on August 11, 2021, Australian Provisional Application No. 2021902530, entitled "Vaccine Antigen", filed on August 13, 2021, and PCT Application No. PCT / AU2022 / 050429, entitled "Vaccine Antigen", filed on May 6, 2022, the contents of which are incorporated by reference in their entireties herein.

[0272] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0273] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and is not to be construed as an admission that any or all of such matters form part of the prior art base or were common general knowledge in the art relevant to the invention as they existed before the priority date of each claim of this application.

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Claims

1. A coronavirus (CoV) vaccine antigen comprising a CoV S protein trimer, wherein the S protein trimer has been modified to include a structural modification that reduces the size of an alanine cavity within the coiled-coil region of the S protein trimer, and wherein the S protein trimer elicits a neutralizing antibody response.

2. 2. The CoV vaccine antigen of claim 1, wherein the structural modifications include one or more of the following features: i) within the coiled-coil; ii) creating an artificial hydrophobic core in the alanine cavity; and iii) one or more of amino acid substitution, disulfide bond, hydrogen bond, pi-stacking (π-π stacking), salt bridge, van der Waals interaction, or proline stabilization.

3. The CoV vaccine antigen of claim 1, comprising one or more of the following features: i) the thermal stability of the S protein trimer is increased compared to an S protein trimer without the structural modification; ii) the denaturant stability of the S protein trimer is increased compared to an S protein trimer without said structural modification; iii) the ACE2 receptor binding domain (RBD) of the S protein trimer is in a downward (non-ACE2 binding) orientation; and iv) lacking a transmembrane domain and / or a trimerization sequence.

4. 2. The CoV vaccine antigen of claim 1, wherein at least one amino acid in the coiled-coil region of an S protein monomer of the S protein trimer has been substituted with a more hydrophobic amino acid, and optionally the at least one amino acid or at least two amino acids are at positions a and / or d of a heptad repeat motif in the coiled-coil region of the S protein monomer.

5. the S protein monomer in the S protein trimer is i) VOC / VOI mutations: S13I, L18F, T19R, T20N, P26S, A67V, delH69-V70, D80A, T95I, D138Y, G142D, delY144, W152C, E154K, E156del, F157del, R158G, R190S, D215G, del242-245, D253G, R246I, K417N / T, N439K, L452R / Q, Y453F, S477N, T478K, E4 84K / Q, N501Y, F565L, A570D, D614G, H655Y, Q677H, P681H / R, I692V, A701V, T71 6I, F888L, D950N, S982A, T1027I, Q1071H, D1118H, A67V, del69-70, T95I, del 142-144, Y145D, del211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417 N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F; ii) the amino acid sequence of any one of SEQ ID NO:1 to SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:08, or a sequence at least 90% identical thereto; iii) the amino acid sequence of any one of SEQ ID NOs: 1 to 3, 6, and 8, comprising one or more of the mutations listed in iii); iv) S protein residues 1-1208 of SARS-CoV-2 variants of concern; v) S protein residues 1-1208 of SARS-CoV-2 notable variants; vi) S protein residues 1-1208 SARS-CoV-2 variants predicted to be highly damaging; vii) the amino acid sequence of SEQ ID NO: 25, or a sequence at least 90% identical thereto; and viii) the amino acid sequence of SEQ ID NO: 25, comprising one or more of the mutations listed in ii); The CoV vaccine antigen of claim 1, further comprising one or more of:

6. 5. The CoV vaccine antigen of claim 4, wherein the at least one amino acid in the coiled-coil region of the S protein monomer is A1016, or the at least one amino acid in the coiled-coil region of the S protein monomer is A1020.

7. 7. The CoV vaccine antigen of claim 6, wherein A1016 is substituted with valine.

8. The CoV vaccine antigen of claim 6, wherein A1020 is substituted with isoleucine.

9. 7. The CoV vaccine antigen of claim 6, wherein the at least one amino acid in the coiled-coil region of the S protein monomer is A1016 substituted with valine and A1020 is a substituted isoleucine.

10. The S protein trimer inhibits the neutralizing antibody response: a) a neutralizing antibody directed against said RBD; b) neutralizing antibodies directed against the N-terminal domain (NTD); c) neutralizing antibodies at undefined epitopes in S1 outside the RBD and NTD; and d) broad neutralization of epitopes outside the RBD (e.g., amino acids 16-329 and 522-1208 of SEQ ID NO:1, or amino acids 16-329 of SEQ ID NO:1) The CoV vaccine antigen of claim 1, which induces one or more of the following:

11. 2. The CoV vaccine antigen of claim 1, wherein the antigen has one or more of the following characteristics: i) is a soluble antigen; ii) stabilized in the prefusion conformation; and iii) comprises one or more additional modified regions that enhance stability and / or immunogenicity.

12. A vector or polynucleotide encoding the S protein monomer of the coronavirus (CoV) vaccine antigen of claim 1.

13. A lipid nanoparticle or a host cell comprising the vector or polynucleotide of claim 12.

14. 13. A method for producing a coronavirus (CoV) vaccine antigen according to claim 1, comprising culturing a host cell comprising the vector or polynucleotide of claim 12 in a culture medium.

15. A protein nanoparticle or virus-like particle comprising the coronavirus (CoV) vaccine antigen of claim 1.

16. A vaccine comprising the coronavirus (CoV) vaccine antigen of claim 1, the vector or polynucleotide encoding the S protein monomer of the coronavirus (CoV) vaccine antigen of claim 12, or the protein nanoparticle or virus-like particle of claim 15.

17. i) preventing or reducing the likelihood of CoV infection in a subject; ii) preventing or reducing the likelihood of severity of CoV symptoms in a subject; iii) reducing the severity and / or duration of a CoV infection in a subject; iv) preventing or reducing viral shedding in a subject; and v) treating a CoV infection in a subject.

18. A kit, device, surface, or strip comprising the coronavirus (CoV) vaccine antigen of claim 1.

19. i) preventing or reducing the likelihood of CoV infection in a subject; ii) preventing or reducing the likelihood of severity of CoV symptoms in a subject; iii) reducing the severity and / or duration of CoV infection in a subject; iv) preventing or reducing viral shedding in a subject; and v) Use of the coronavirus (CoV) vaccine antigen of claim 1 in the manufacture of a medicament for one or more of the following:

20. Use of said antigen or coding sequence in the manufacture of a preparation for the treatment, prevention or testing associated with coronavirus (CoV) infection in a population.

21. A method for producing a soluble S protein trimer lacking a heterologous trimerization sequence, wherein the S protein trimer is modified to include a structural modification that reduces the size of the alanine cavity within the coiled-coil region of the S protein trimer, and the S protein trimer elicits a neutralizing antibody response.

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