Stabilized SARS-COV-2 s antigens

Abstract

Provided herein are engineered protein comprising stabilized coronavirus S protein ectodomains, such as stabilized SARS-CoV-2 S2 domain-only ectodomains that exhibit improved conformational homogeneity and biophysical stability. The ectodomains are incorporated into nanoparticles. Methods are also provided for use of the stabilized coronavirus S protein ectodomains and / or nanoparticles as diagnostics, in screening platforms, and / or in vaccine compositions.

Description

DESCRIPTION STABILIZED SARS-COV-2 S ANTIGENS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of United States provisional application number 63 / 583,090, filed September 15, 2023, the entire contents of which are incorporated herein by reference. REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on September 12, 2024, is named UTFBP1348WO_ST26.xml and is 63,453 bytes in size. BACKGROUND 1. Field

[0003] The present disclosure relates generally to the fields of medicine, virology, immunology, and protein engineering. More particular, the disclosure relates to engineered protein comprising beta-coronavirus S protein ectodomains and the use thereof in drug design and vaccine formulation. 2. Description of Related Art

[0004] An outbreak of COVID-19, the disease caused by infection of the coronavirus SARS-CoV-2, began in December 2019 in China and has resulted in millions of infections and more than 100 thousand deaths. Like the virus that caused the SARS outbreak several years prior, SARS-CoV, the SARS-CoV-2 viruses use their spike proteins to bind host cellular receptor angiotensin-converting enzyme 2 (ACE2) through an interaction between the receptor binding domain (RBD) of the spike glycoprotein and the human ACE2 protein. Although the sequence and structure of the SARS-CoV-2 spike protein are known (see, e.g., Wrapp et al. 2020), there remains a need for stabilized S proteins that could be used for identifying drug candidates and for stimulating an effective immune response to the S protein. 1 4893-1038-0739, v.1SUMMARY

[0005] Provided herein are engineered proteins comprising an engineered coronavirus S2 protein ectodomain that comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of positions 697-1159 of SEQ ID NO: 1 or 2, wherein the engineered coronavirus S2 protein ectodomain comprises the following substitution(s) relative to the sequence of SEQ ID NO: 1 or 2: V991W and / or T998W.

[0006] The engineered coronavirus S2 protein ectodomain may further comprise the following substitutions relative to the sequence of SEQ ID NO: 1 or 2: K986P and V987P. The engineered coronavirus S2 protein ectodomain may further comprise the following substitutions relative to the sequence of SEQ ID NO: 1 or 2: F817P, A892P, A899P, A942P, K986P, and V987P.

[0007] The engineered coronavirus S2 protein ectodomain may comprise the following substitutions relative to the sequence of SEQ ID NO: 1 or 2: F817P, A892P, A899P, A942P, K986P, V987P, V991W, and T998W.

[0008] The engineered coronavirus S2 protein ectodomain may further comprise the following substitutions relative to the sequence of SEQ ID NO: 1 or 2: S704C and K790C. The engineered coronavirus S2 protein ectodomain may further comprise the following substitutions relative to the sequence of SEQ ID NO: 1 or 2: Q957E.

[0009] The engineered coronavirus S2 protein ectodomain may comprise the following substitutions relative to the sequence of SEQ ID NO: 1 or 2: S704C, K790C, F817P, A892P, A899P, A942P, Q957E, K986P, V987P, V991W, and T998W.

[0010] The engineered coronavirus S2 protein ectodomain may further comprise an additional engineered disulfide bond, an additional cavity filling substitution, and / or a substitution that provides an electrostatic or polar interaction. For example, the engineered coronavirus S2 protein ectodomain may further comprise an engineered disulfide bond comprising paired cysteine substitutions at positions corresponding to: Y707C / P792C, Y707C / T883C, S704C / K790C, A713C / L894C, Q755C / N969C, G757C / S968C, G891C / P1069C, S1030C / D1041C, G1035C / V1040C, S735C / T859C, I770C / A1015C, L727C / S1021C, V911C / N1108C, A672C / A694C, A1080C / I1132C, S659C / S698C, 24893-1038-0739, v. 1V722C / A930C, A903C / Q913C, S974C / D979C, P728C / V951C, V736C / L858C, S884C / A893C, P807C / S875C, T791C / A879C, G799C / A924C, A570C / V963C, T874C / S1055C, V729C / A1022C, L822C / A1056C, Q965C / S1003C, A972C / Q992C, I980C / Q992C, A1078C / V1133C, H1088C / T1120C, I870C / S1055C, T1117C / D1139C, T1116C / Y1138C, I896C / Q901C, G885C / Q901C, F1103C / P1112C, G889C / L1034C, E819C / S1055C, A972C / I980C, I1081C / N1135C, or E819C / Q1054C.

[0011] The engineered coronavirus S2 protein ectodomain may further comprise a cavity filling substitution selected from: T724M, I1013F, H1058W, Q901M, S875F, H1088W, L1141F, V1040F, T778L, L938F, V963L, R1039F, V826L, A899F, Q779M, L894F, H1058F, H1058Y, V1040Y, H1088Y, V1104I, R1000Y, R1000W, A944F, T724I, A944Y, S730L, A890V, D1118F, or S1003V. The engineered coronavirus S2 protein ectodomain may further comprise a proline substitution selected from: F817P, L865P, T866P, A892P, A899P, T912P, A893P, Q895P, K921P, L922P, N978P, A942P, G946P, or S975P. The engineered coronavirus S2 protein ectodomain may further comprise an electrostatic interaction substitution selected from: T961D, T961E, L966E, L966D, T859K, T752K, T912R, L828K, L828R, S730R, T961D, A766E, P862E, T859K, Q957E, G769K, G769E, T778Q, A713S, or I1130Y.

[0012] The engineered coronavirus S2 protein ectodomain may comprise a sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to positions 697-1159 of SEQ ID NO: 1. The engineered coronavirus S2 protein ectodomain may comprise a sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to positions 697-1159 of SEQ ID NO: 2. The engineered coronavirus S2 protein ectodomain may comprise an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 9. The engineered coronavirus S2 protein ectodomain may comprise the amino acid sequence of SEQ ID NO: 9.

[0013] The C-terminal residue of the engineered coronavirus S2 protein ectodomain may be linked to a protein nanoparticle subunit by a peptide linker, or may be directly linked to the protein nanoparticle subunit. The protein nanoparticle subunit may be a self- assembling protein nanoparticle subunit. The self-assembling protein nanoparticle subunit 34893-1038-0739, v. 1may be a ferritin nanoparticle subunit, encapsulin nanoparticle subunit, Sulfur Oxygenase Reductase (SOR) nanoparticle subunit, or lumazine synthase nanoparticle subunit. The protein nanoparticle subunit may be a ferritin molecule derived from the insect Trichoplusia ni.

[0014] The engineered protein may comprise an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to positions 18-658 of the amino acid sequence of SEQ ID NO: 3. The engineered protein may comprise the amino acid sequence of positions 18-658 of SEQ ID NO: 3. The engineered protein may comprise an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to positions 18-668 of the amino acid sequence of SEQ ID NO: 4. The engineered protein may comprise the amino acid sequence of positions 18-668 of SEQ ID NO: 4.

[0015] The engineered protein may comprise an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 3 or 4. The engineered protein may comprise the amino acid sequence of SEQ ID NO: 3 or 4.

[0016] Provided herein are protein nanoparticles, comprising the engineered proteins described herein. The protein nanoparticle may be a self-assembling protein nanoparticle. The self-assembling protein nanoparticle may be a ferritin nanoparticles, encapsulin nanoparticles, Sulfur Oxygenase Reductase (SOR) nanoparticles, and lumazine synthase nanoparticles.

[0017] The nanoparticles may comprise an additional antigen. The engineered protein and the additional antigen may both be engineered proteins described herein. The engineered protein and the additional antigen may share a sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0018] The additional antigen may be an engineered protein comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27 or 28, of which the C-terminal residue is linked to a protein 44893-1038-0739, v. 1nanoparticle subunit by a peptide linker, or is directly linked to the protein nanoparticle subunit.

[0019] The additional antigen may be an engineered protein comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of positions 18-658 or 18-668 of SEQ ID NO: 23 or 24, respectively. As such, the engineered protein in the nanoparticle may comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of positions 18-658 of SEQ ID NO: 3, and the additional antigen may be an engineered protein comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of positions 18-668 of SEQ ID NO: 24. Equally so, the engineered protein in the nanoparticle may comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of positions 18-668 of SEQ ID NO: 4, and the additional antigen may be an engineered protein comprising an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of positions 18-658 of SEQ ID NO: 23.

[0020] The engineered coronavirus S2 protein ectodomain is fused or conjugated to a trimerization domain. The engineered coronavirus S2 protein ectodomain may be fused to a trimerization domain. The trimerization domain may be positioned C-terminally relative to the engineered coronavirus S2 protein ectodomain. The trimerization domain may comprise a T4 fibritin trimerization domain.

[0021] The engineered coronavirus S2 protein ectodomain may be fused or conjugated to a transmembrane domain. The engineered coronavirus S2 protein ectodomain may be fused to a transmembrane domain. The transmembrane domain may comprise a beta- coronavirus spike protein transmembrane domain. The transmembrane domain may comprise a homologous beta-coronavirus spike protein transmembrane domain. The transmembrane domain may comprise a SARS-CoV-2 transmembrane domain. 54893-1038-0739, v. 1

[0022] The engineered protein may further comprise a coronavirus S1 protein ectodomain. The engineered protein may comprise an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of positions 16-1208 of SEQ ID NO: 1 or 2. The engineered protein may comprise an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of positions 319-1208 of SEQ ID NO: 1 or 2.

[0023] Provided herein are engineered coronavirus trimers comprising at least one subunit comprising an engineered coronavirus S2 protein ectodomain described herein. The trimer may be stabilized in a prefusion conformation relative to a trimer of wildtype S protein subunits. The trimer may comprise at least one engineered disulfide bond between subunits. The at least one engineered disulfide bond between subunits may be selected from: Y707C and P792C; Y707C and T883C; S704C and K790C; A713C and L394C; Q755C and N969C; G757C and S968C; G891C and P1069C; S1030C and D1041C; and G1035C and V1040C. The at least one engineered disulfide bond between subunits may be selected from: V705C and A983C; T547C and N968C; T961C and S758C; and / or T961C and Q762C.

[0024] Provided herein are nucleic acid molecules comprising a nucleotide sequence that encodes an amino acid sequence of an engineered coronavirus S2 protein ectodomain or an engineered protein described herein. The nucleic acids may comprise a DNA expression vector. The nucleic acids may comprise an mRNA.

[0025] Provided herein are immunogenic compositions comprising an engineered coronavirus S2 protein ectodomain, an engineered protein, a protein nanoparticle, an engineered trimer, or a nucleic acid molecule described herein, and a pharmaceutically acceptable carrier. The composition may further comprise an adjuvant. The immunogenic composition may be for use in inducing an immune response to a SARS-CoV-2 S ectodomain in a subject.

[0026] Provided herein are methods of preventing a coronavirus infection or a disease associated with a coronavirus infection in a subject, where the methods comprise administering to the subject an effective amount of an immunogenic composition described 64893-1038-0739, v. 1herein. The methods may elicit a beta-coronavirus cross-reactive immune response. The methods may elicit the production of pan-coronavirus antibodies in the subject.

[0027] Provided herein are uses of an engineered coronavirus S2 protein ectodomain, an engineered protein, a protein nanoparticle, an engineered trimer, or a nucleic acid molecule described herein in the manufacture of an immunogenic composition for inducing an immune response to a SARS-CoV-2 S ectodomain.

[0028] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0029] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0030] FIG. 1. Antigenicity of SARS-CoV-2 stabilized spike stem-nanoparticles (SSS-nps) as determined by biolayer interferometry (BLI).

[0031] FIG. 2. Size exclusion chromatography (SEC) profiles of the SARS-CoV-2 SSS-nps. SARS-CoV-2 SSS-WW-np has the higher peak.

[0032] FIG.3. Negative stain electron microscopy images of SARS-CoV-2 SSS-nps.

[0033] FIG.4. Serum antibody titers in mice immunized with SARS-CoV-2 SSS-nps.

[0034] FIG. 5. Serum neutralizing antibody titers in mice immunized with SARS- CoV-2 SSS-nps.

[0035] FIG. 6. Betacoronavirus cross-reactive serum antibody titers in mice immunized with SARS-CoV-2 SSS-nps. 74893-1038-0739, v. 1

[0036] FIG. 7. FPLC profiles of the SARS-CoV and combo NPs. SARS-CoV- 2(HC) / SARS-CoV(LC) SSS-WW-np has the highest peak. SARS-CoV SSS-WW-np has the lowest peak.

[0037] FIG. 8. Negative stain EM of SARS-CoV and SARS-CoV-2 / SARS-CoV combo SSS-WW-nps.

[0038] FIG.9. HexaPro-SS-∆stalk S2-only trimer and contect within the SARS-CoV- 2 spike protein. The model of the HexaPro-SS-Δstalk S2-only trimer in the closed prefusion conformation is depicted with a cartoon representation where the protomers are highlighted with different shades. The spike’s S1 subunit, here shown with a gray transparent surface only to provide context, is not present in this construct. N-glycans linked to S2 are shown as dark gray sticks. Glycans linked to S1 are omitted for clarity. HexaPro-SS-Δstalk is an S2- only construct derived from the prefusion-stabilized SARS-CoV-2 HexaPro spike (Hsieh et al., 2024). The substitutions (S704C, K790C, Q957E) incorporated in HexaPro-SS-Δstalk (Hsieh et al., 2024) are highlighted in the right panels. They stabilize S2 in a prefusion trimer assembly, preventing protomer dissociation via formation of interprotomer salt bridges and disulfide bonds.

[0039] FIG. 10a-b. Probability distribution of progress coordinates in the HexaPro- SS-2W WE simulations. a, Probability distributions of WE progress coordinates from WE iterations preceding reweighting, in between 1stand 2ndreweighting, and after 2ndreweighting are shown for HexaPro-SS-2W. The probability distribution is plotted on an inverted natural log scale [i.e., -lnP(x)], corresponding to the free energy in 1 / kBT unit. From left to right, the probability is shown for the two progress coordinates (x-axis: P987Cα triangle area; y-axis: RMSDCH) as an average, for the P987Cαtriangle area-only as a time (i.e., WE iterations) evolution, and for the RMSDCH-only as time (i.e., WE iterations) evolution, respectively. b, 1-D probability distributions for P987Cαtriangle area (left panel) and RMSDCH(right panel). Different shades are used to plot the average probability calculated from WE iterations preceding reweighting, in between 1stand 2ndreweighting, and after 2ndreweighting, respectively. Dashed lines demarcate closed and open states. An arrow indicates the closed- to-open transition.

[0040] FIG. 11a-b. Conformations from cMD and GaMD simulations. a, Top-down view of SARS-CoV-2 spike HexaPro-SS-∆stalk in the open prefusion conformation and 84893-1038-0739, v. 1select conformations from all-atom conventional MD and GaMD simulations. The open crystal structure is shown in gray, with the central helices highlighted as fully opaque. The central helices of alternative simulation methods are shown in color; all-atom conventional MD is shown, and GaMD at the high (E=2) and low (E=1) acceleration level is. The conformations from all-atom MD and GaMD simulations were selected from the frame with the greatest distance between P987Cα-P987Cαat the top of the central helices, indicating a departure from the closed state en route to a partially open conformation. b, Comparative analysis of Root Mean Square Deviation (RMSD) values for three different MD simulation conditions: GaMD (E1), GaMD (E2), and all-atom conventional MD (Unbiased MD). The RMSD values, measured in Angstroms (Å), provide insights into the structural differences in the trajectories over simulation steps.

[0041] FIG. 12a-b. Probability distribution of progress coordinates in the HexaPro- SS-V991W WE simulations. a, Probability distributions of WE progress coordinates from WE iterations preceding reweighting, in between 1stand 2ndreweighting, and after 2ndreweighting are shown for HexaPro-SS-V991W. The probability distribution is plotted on an inverted natural log scale [i.e., -lnP(x)], corresponding to the free energy in 1 / kBT unit. From left to right, the probability is shown for the two progress coordinates (x-axis: P987Cα triangle area; y-axis: RMSDCH) as an average, for the P987Cαtriangle area-only as a time (i.e., WE iterations) evolution, and for the RMSDCH-only as time (i.e., WE iterations) evolution, respectively. b, 1-D probability distributions for P987Cα triangle area (left panel) and RMSDCH(right panel). Different shades are used to plot the average probability calculated from WE iterations preceding reweighting, in between 1stand 2ndreweighting, and after 2ndreweighting, respectively. Dashed lines demarcate closed and open states. An arrow indicates the closed-to-open transition.

[0042] FIG. 13a-e. HexaPro-SS-Δstalk S2 trimer opening. a, Progress coordinates used in the WE simulation of HexaPro-SS-Δstalk, namely P987Cα triangle area and RMSDCH, are highlighted with shapes (triangle and cylinders, respectively) drawn on top of the molecular structures. The CHs as in the closed conformation of the simulated construct are shown with shades and are overlayed on the open conformation as in the crystal structure (colored in gray, PDB ID: 8U1G (Hsieh et al., 2024)). b, Distribution of the conformations sampled in the opening pathways obtained from the WE simulation. Each black point represents a conformation sampled along an opening pathway. The RMSD of the CH to 94893-1038-0739, v. 1CHOpen-crystal (y-axis) is plotted against the area of the triangle formed by the P987Cα at the CH apex (x-axis). The hexagonal bin color is scaled to the mean interprotomer distance of the black data points within the respective bin. A trace of one of the successful pathways is shown as a black line with white points corresponding to the closed, partially open, and open conformations. c-e, Molecular representation of closed (c), partially open (d), and open (e) conformations as highlighted in (b). Chain-A is depicted in light gray, chain-B in gray, and chain-C in dark gray. The CHs and upper helices (UHs, residues 945-977) are illustrated with a cartoon representation, whereas the rest of the chain is shown as an opaque surface. Glycans are shown as gray sticks.

[0043] FIG. 14a-b. Probability distribution of progress coordinates in the HexaPro- SS-Δstalk WE simulation. a, Probability distributions of WE progress coordinates from WE iterations preceding reweighting, in between 1stand 2ndreweighting, and after 2ndreweighting are shown for HexaPro-SS-Δstalk. The probability distribution is plotted on an inverted natural log scale [i.e., -lnP(x)], corresponding to the free energy in 1 / kBT unit. From left to right, the probability is shown for the two progress coordinates (x-axis: P987Cα triangle area; y-axis: RMSDCH) as an average, for the P987Cαtriangle area-only as a time (i.e., WE iterations) evolution, and for the RMSDCH-only as time (i.e., WE iterations) evolution, respectively. b, 1-D probability distributions for P987Cαtriangle area (left panel) and RMSDCH (right panel). Different shades of gray are used to plot the average probability calculated from WE iterations preceding reweighting, in between 1stand 2ndreweighting, and after 2ndreweighting, respectively. Dashed lines demarcate closed and open states. An arrow indicates the closed-to-open transition.

[0044] FIG. 15a-b. Probability distribution of progress coordinates in the HexaPro- SS-T998W WE simulations. a, Probability distributions of WE progress coordinates from WE iterations preceding reweighting, in between 1stand 2ndreweighting, and after 2ndreweighting are shown for HexaPro-SS-T998W. The probability distribution is plotted on an inverted natural log scale [i.e., -lnP(x)], corresponding to the free energy in 1 / kBT unit. From left to right, the probability is shown for the two progress coordinates (x-axis: P987Cα triangle area; y-axis: RMSDCH) as an average, for the P987Cα triangle area-only as a time (i.e., WE iterations) evolution, and for the RMSDCH-only as time (i.e., WE iterations) evolution, respectively. b, 1-D probability distributions for P987Cαtriangle area (left panel) and RMSDCH (right panel). Different shades are used to plot the average probability calculated 104893-1038-0739, v. 1from WE iterations preceding reweighting, in between 1stand 2ndreweighting, and after 2ndreweighting, respectively. Dashed lines demarcate closed and open states. An arrow indicates the closed-to-open transition.

[0045] FIG. 16a-b. Overlay of the open S2 trimer crystal structure and open WE model. The HexaPro-SS-Δstalk S2-only trimer in the open prefusion conformation is depicted with a cartoon representation from side (a) and top-down (b) perspectives. The open WE model shows protomers highlighted with different shades and the open crystal structure (PDB ID: 8U1G (Hsieh et al., 2024)) is shown in gray. The RMSD of the C-alpha atoms of the S2 central helices (residues 987-1033) is 1.64 Å. The RMSD of the C-alpha, considering all the residues of the open S2 crystal structure, is 3.76 Å.

[0046] FIG. 17. Additional open conformation sampled during WE simulations. Beyond the closed-to-open transitions, WE simulations allowed us to sample further open conformations, that we renamed as Super Open, to Split Asymmetric, Split Skewed, and Super Split Asymmetric. Selected pathways leading to each one of these states are indicated with a line (black to white) on top of the 2D probability distribution plot for the two progress coordinates used in the WE simulations of HexPro-SS-Δstalk (x-axis: P987Cαtriangle area; y- axis: RMSDCH). Respective molecular representations for these additional states are reported around the plot. A top-down viewpoint is used, with the protomers colored with different shades. Although Split Asymmetric and Super Split Asymmetric states are located on the left- hand side region of the plot, denoted by low P987Cαtriangle area values, they should instead be located on the right-end side of the probability plot with larger values of P987Cα triangle area. This discrepancy occurred because one apex separated significantly from the other two (>100 Å), extending beyond the central box of the simulation. Consequently, it entered the adjacent periodic image, interacting with the other two apexes from the opposite direction. This caused Amber’s cpptraj program to see the tree apexes almost as in the closed conformation with low values of P987Cα triangle area, thus explaining all the conformations residing above the closed state in the probability distribution plot. An attempt was made to prevent this inconvenience by setting an unusually large simulation box (we added a 25 Å layer of water molecules from the solute edge in each direction), but such an exceedingly large opening (> 100 Å P987–P987 distance) could not be foreseen.

[0047] FIG. 18. Contact heatmaps for central helix (CH) residues. CH contact score heat maps for the HexaPro-SS-Δstalk base construct and HexaPro-SS-Δstalk tryptophan 114893-1038-0739, v. 1mutants in the closed, partially open, and open conformations. A star indicates a position mutated to tryptophan. Positions 991 and 998 are underlined for each S2 construct. The scale ranges from 0 (weak or transient contacts) to 1 (persistent or extensive contacts), and individual contact scores are normalized to the 95thpercentile value of the HexaPro-SS- Δstalk contact scores.

[0048] FIG. 19. Contact heatmaps for upper helix (UH) residues. UH contact score heat maps for the HexaPro-SS-Δstalk base construct and HexaPro-SS-Δstalk tryptophan mutants in the closed, partially open, and open conformations.

[0049] FIG. 20. Contact heatmaps for heptad repeat 1 (HR1) residues. HR1 contact score heat maps for the HexaPro-SS-Δstalk base construct and HexaPro-SS-Δstalk tryptophan mutants in the closed, partially open, and open conformations.

[0050] FIG. 21. Contacts between residues in the central helices during S2 opening. a-c, HexaPro-SS-Δstalk CHs in the closed conformation from a top-down view (a), side view in the context of the S2 trimer (gray) (b), and side view (c). CHs are highlighted. The sidechains of the residue pointing toward the interior of the CHs are shown as sticks with the C atoms colored by the corresponding residue contact score as in the closed conformation of the HexaPro-SS-Δstalk. d,e, Contact score heat maps for the HexaPro-SS-Δstalk base construct (d) and HexaPro-SS-Δstalk tryptophan substitutions (e) in the closed, partially open, and open states. The scale ranges from 0 (weak or transient contacts) to 1 (persistent or extensive contacts, corresponding to the 95thpercentile value of the HexaPro-SS-Δstalk contact scores). The star indicates the substituted residue.

[0051] FIG. 22. Probability distribution of progress coordinates within the region-of- interest in the HexaPro-SS-∆stalk, HexaPro-SS-V991W, HexaPro-SS-T998W, and HexaPro- SS-2W WE simulations. Probability distributions of WE progress coordinates from WE iterations after the 2ndreweighting are shown for HexaPro-SS-∆stalk, HexaPro-SS-V991W, HexaPro-SS-T998W, and HexaPro-SS-2W. The probability distribution is plotted on an inverted natural log scale [i.e., -lnP(x)], corresponding to the free energy in 1 / kBT unit. The probability is shown for the two progress coordinates (x-axis: P987Cα triangle area; y-axis: RMSDCH) as an average. The closed state is marked with a “C” and the open state is marked with an “O.” 124893-1038-0739, v. 1

[0052] FIG 23a-d. Conformational space sampled successful pathways obtained from WE simulations. Distribution of the conformations sampled in the opening pathways obtained from the WE simulation of (a) HexaPro-SS-Δstalk (b) HexaPro-SS-V991W, (c) HexaPro-SS- T998W, (d) HexaPro-SS-2W. Each black point represents a conformation sampled along an opening pathway. The RMSD of the CH to CHOpen-crystal (y-axis) is plotted against the area of the triangle formed by the P987Cαat the CH apex (x-axis). In the left-side panels, the hexagonal bin color is scaled to the mean interprotomer distance of the black data points within the respective bin, whereas on the right-side panels, the hexagon bin color is scaled according to the standard deviation of the black data points within the respective bin. For (a), a trace of one of the successful pathways is shown as a black line with white points corresponding to the closed, partially open, and open conformations.

[0053] FIG. 24a-b. Aggregate molecular time to first opening and distribution of molecular time to opening. a, Aggregate molecular time to the first opening event observed in the WE simulations of HexaPro-SS-Δstalk, HexaPro-SS-V991W, HexaPro-SS-T998W, and HexaPro-SS-2W. The number in µs is reported on top of each respective bar. b, Distribution of molecular time to opening for all the successful pathways observed in the WE simulations of HexaPro-SS-Δstalk, HexaPro-SS-V991W, HexaPro-SS-T998W, and HexaPro-SS-2W, respectively. The distributions are shown as kernel densities and do not take into account the weights of successful pathways. Differently from the opening event duration (FIG. 23), the molecular time to opening event (indicated in units of ns) takes into account the time spent in the closed state. A dashed line indicates the peak of each distribution.

[0054] FIG 25a-d. Probability of S2 opening event duration. Probability of S2 trimer opening event duration as calculated from the opening pathways observed in the WE simulations of (a) HexaPro-SS-Δstalk, (b) HexaPro-SS-V991W, (c) HexaPro-SS-T998W, and (d) HexaPro-SS-2W. The probability considers the weight of the successful pathways calculated from WE simulations. Differently from the molecular time to opening event (FIG. 22), the duration of the opening event does not take into account the time spent in the closed state, but rather the effective molecular time required by the system to reach the open state after leaving the closed state. A solid black line indicates the most likely event duration.

[0055] FIG. 26a-d. Rate constants of S2 trimer closed-to-open (opening) transition. a- d, Time (i.e., iteration #) evolution of rate constant estimates (kopening) in units of s-1of S2 trimer closed-to-open (opening) transition as calculated from WE simulations of HexaPro- 134893-1038-0739, v. 1SS-Δstalk, HexaPro-SS-V991W, HexaPro-SS-T998W, and HexaPro-SS-2W. The uncertainty in the rate constants is illustrated with a semi-transparent shaded region that extends from the lower to the upper bounds of the confidence interval (95%) for the observable. Solid black circles are marked at the iterations where reweighting took place. a, Time (i.e., iteration #) evolution of rate constant estimates (kopening) in units of s-1of S2 trimer closed-to-open (opening) assessed as a rolling average calculated across all WE iterations over 1-iteration windows with 95% confidence interval. b, Magnified view of the rate constant evolution after iteration #200 as calculated in panel a. c, Time (i.e., iteration #) evolution of rate constant estimates (kopening) in units of s-1of S2 trimer closed-to-open (opening) assessed as a rolling average calculated across all WE iterations over iteration windows of width corresponding to the 10% of the total number of iterations for the respective WE simulation with 95% confidence interval. d, Time (i.e., iteration #) evolution of rate constant estimates (kopening) in units of s-1of S2 trimer closed-to-open (opening) assessed as a rolling average calculated for all WE iterations following first reweighting over 1-iteration windows with 95% confidence interval.

[0056] FIG. 27. Schematic representation of a thermodynamic cycle used to calculate changes in protein folding free energy upon mutation (∆∆G mutation folding). The left column depicts the folding process of a wild-type protein, with the associated folding free energy as ΔG wild type folding. The same folding process is presented on the right column, but in the case of a mutated protein, with the associated folding free energy ΔG mutant folding. The reaction shown in the top row corresponds to the alchemical transformation of the folded wild type protein into the folded mutant, resulting in the free energy difference between the two forms as ΔG mutation folded. The process depicted in the bottom row represents the same alchemical transformation (wild type into mutant), but in the case of the protein in the unfolded state, with the associated free energy difference ΔG mutation unfolded. While computing the free energy differences for the vertical processes is computationally demanding, those for the horizontal transformations are more accessible. Therefore, ΔΔG mutation folding can be calculated by taking the difference between ΔG mutation folded and ΔG mutation unfolded.

[0057] FIG. 28a-d. Alchemical non-equilibrium free energy calculations. The output of PMX (Gapsys et al., 2015) analyze script for non-equilibrium MD simulations performed for (a) HexaPro-SS-V991W (folded state), (b) HexaPro-SS-T998W (folded state), and (c) 144893-1038-0739, v. 1HexaPro-SS-2W (folded state), (d) G-V-G / G-T-G tripeptides (unfolded state). In (a) and (b), the left, central, and right plots account for the V991W or T998W mutation introduced in Chains A, A+B, A+B+C, respectively. In (c), the left, central, and right plots account for the V991W mutation introduced in Chains A, A+B, A+B+C, respectively, on top of a base construct already incorporating T998W in all three Chains. In each plot, the left side displays the work values corresponding to both the forward and reverse transitions for each starting structure. 1000 non-equilibrium MD simulations were run in the forward direction (wild type-to-mutant, highlighted in light gray) and 1000 non-equilibrium MD simulations in the reverse direction (mutant-to-wild type, highlighted in dark gray) for both the folded and the unfolded state. During these calculations, one amino acid is gradually morphed into the mutated amino acid (forward) or vice-versa (reverse). On the right side of the plots, histograms illustrate the distributions of these work values, and their overlap allows the determination of the free energy difference.

[0058] FIG. 29a-e. V991W and T998W stabilize HexaPro-SS-Δstalk in the closed conformation. a, Thermodynamic stabilization of the closed prefusion conformation as imparted by each tryptophan variant relative to the base system (HexaPro-SS-Δstalk). Values of ∆∆Gmutation-folding are calculated via alchemical non-equilibrium calculations and reported in kcal / mol. Error bars denote uncertainties calculated via bootstrapping. b, Distribution of the number of interprotomer contacts established per frame by residues at positions 991+998 from the ensemble of closed conformations extracted from respective WE simulations. Distributions are shown as kernel densities. c, Relative frequency of occurrence (%) of the top six residue–residue interprotomer contacts occurring in the closed conformations observed in HexaPro-SS-Δstalk and HexaPro-SS-2W WE simulations. Relative frequencies are calculated with respect to the total number of closed conformations extracted from the respective WE simulation. d,e, Molecular representation of the most important interprotomer contacts in HexaPro-SS-2W in (d) the closed conformation and (e) the partially open conformation. Chain-A is depicted in light gray, chain-B in gray, chain-C in dark gray, and interacting residues are highlighted with sticks. Solid lines connect residues that form hydrophobic (π–π) interactions, whereas dashed lines indicate electrostatic (cation–π and salt bridge) interactions. A representative closed conformation of HexaPro-SS-2W was selected for this purpose upon clustering of closed conformations retrieved from the respective WE simulation (FIG.30). 154893-1038-0739, v. 1

[0059] FIG. 30. Cluster analysis of the closed conformations extracted from WE simulation of HexaPro-SS-2W. All the closed conformations sampled during the WE simulations of HexaPro-SS-2W were clustered using k-means clustering based on the number of interprotomer contacts made by CH, UH, and HR1 helices (x-axis) and the P987TRIANGLE-AREA observables. The three identified clusters are highlighted with different shades. Centroids for each cluster are marked with solid black circles. Dashed lines indicate the values of the observables corresponding to the identified centroids. The population density is emphasized through contour lines illustrated with a plasma color palette, with the area encircled by the contour representing the highest population density. Individual population densities for each cluster are shown on the top x-axis for the number of interprotomer contacts and on the right y-axis for the P987TRIANGLE-AREA.

[0060] FIG. 31a-d. Contact analysis of the closed conformations extracted from WE simulations. The bar plots show the relative frequency of occurrence (x-axis, % to the total number of frames × 3protomers) of residue pairs within CH, UH, HR1 involved in interprotomer contacts (y-axis) across all the closed conformations extracted from the WE simulations of (a) HexaPro-SS-Δstalk, (b) HexaPro-SS-V991W, (c) HexaPro-SS-T998W, (d) and HexaPro- SS-2W. Only contacts with a relative frequency larger than 50% are shown. Contacts are estimated based on a distance cutoff of 4.5 Å between heavy atoms only.

[0061] FIG. 32a-d. Contact analysis of the closed conformations extracted from WE simulations (residues 991 and 998 only). The bar plots show the relative frequency of occurrence (x-axis, % to the total number of frames × 3protomers) of residue pairs that involve residues at position 991 and 998 forming interprotomer contacts (y-axis) across all the closed conformations extracted from the WE simulations of (a) HexaPro-SS-Δstalk, (b) HexaPro- SS-V991W, (c) HexaPro-SS-T998W, (d) and HexaPro-SS-2W. Only the contacts with a relative frequency larger than 1% are shown. Contacts are estimated based on a distance cutoff of 4.5 Å between heavy atoms only.

[0062] FIG. 33. Number of interprotomer residue contacts in the closed conformations from WE simulations. The distribution of the number of interprotomer contacts per frame involving residues within CH, UH, and HR1 helices is plotted for HexaPro-SS-Δstalk (left-most peak), HexaPro-SS-V991W (second-to-left-most peak), HexaPro-SS-T998W (second-to-right-most peak), and HexaPro-SS-2W (right-most peak) using kernel density curves. The contacts are assessed exclusively from the closed 164893-1038-0739, v. 1conformations extracted from their respective WE simulation and are calculated based on a distance cutoff of 4.5 Å considering only the heavy atoms. Dashed lines are drawn from the respective distribution peak.

[0063] FIG. 34a-g. Cellular expression, thermostability, and structural characterization of HexaPro-SS-2W. a, SDS-PAGE of purified S2 constructs (HexaPro-SS- ∆stalk, HexaPro-SS-V991W, HexaPro-SS-T998W, and HexaPro-SS-2W). b, Size-exclusion chromatography (SEC) of purified S2 constructs. Both tryptophan substitutions increase protein expression yield, with V991W+T998W and T998W resulting in a greater increase. c, Differential scanning fluorimetry of S2 constructs, including the original HexaPro (S1+S2). HexaPro-SS-2W exhibits superior thermal stability to all HexaPro constructs, with a ~16 °C increase in Tm relative to HexaPro. d, Cryo-EM map (Resolution: 2.8 Å) of closed prefusion state HexaPro-SS-2W (∆stalk) from side and top-down perspectives. e-g, Top-down perspective of HexaPro-SS-2W structure highlighting V991W and T998W packing within the S2 interior; the tryptophan sidechains self-associate between chains in offset edge-to-edge and edge-to-face π–π stacking orientations. The EM map is shown as a transparent gray volume.

[0064] FIG. 35. Cryo-EM data processing workflow for HexaPro-SS-2W. The following steps were performed in cryoSPARC (Punjani et al., 2017) v4.0.2 to process the cryo-EM dataset: motion correction, CTF estimation, 2D classification, ab-initio reconstruction, heterogeneous refinement, and homogeneous refinement (C3 symmetry).

[0065] FIG. 36. Hydrophobic packing in the cryo-EM structure of HexaPro-SS-2W. Sidechains of residues contributing to forming hydrophobic packing and D994–R995 salt bridge at the S2 trimer apex are illustrated as sticks colored in varying tones according to the respective chain (A being the lightest, C being the darkest). For these residues, a transparent sphere representation is also overlaid. The rest of the protein is shown as transparent cartoons.

[0066] FIG. 37a-c. Immunogenicity of HexaPro-SS and HexaPro-SS-2W constructs. a,b, Sera from mice immunized with HexaPro-SS-2W can neutralize rVSV-CoV-2 variants. a, 6–8-week-old female C57BL / 6J mice (n = 6) were primed (week 0) and boosted (week 3) with 10 µg of immunogens HexaPro-SS, HexaPro-SS-2W, or PBS. b, Sera was isolated at week 7 and evaluated for neutralization capacity against rVSVs bearing the spike proteins of 174893-1038-0739, v. 1SARS-CoV-2 Wuhan-1 or SARS-CoV-2 Omicron BA.1. Lower area under the curve (AUC) values correspond to better sera neutralizing capability. AUCs were compared across groups by ordinary one-way ANOVA with Tukey’s multiple comparisons test (** p <0.01; n.s. not significant). c, Biolayer interferometry sensorgrams showing binding responses (colored lines indicate 1:1 binding fit, black lines indicate reference subtracted response) of previously reported neutralizing or non-neutralizing antibodies to epitopes on the S2 subunit in HexaPro- SS (top) and HexaPro-SS-2W (bottom) constructs.

[0067] FIG. 38. Individual replicates of rVSV-SARS-CoV-2 neutralization assays. Female C57BL / 6J mice (n = 6) were immunized with HexaPro-SS, HexaPro-SS-2W (V991W+T998W), or PBS. Serial 3-fold dilutions were made for each serum sample and assayed for neutralizing ability against rVSV-SARS-CoV-2 (Wuhan-1), on the left, or (Omicron BA.1), on the right, in singlicate. One mouse immunized with HexaPro-SS needed to be euthanized for humane reasons before sufficient blood could be collected. As a result, the corresponding neutralization assay against rVSV-SARS-CoV-2 (Omicron BA.1) for that immunization group only had n = 5. Infectivity curves were fitted with nonlinear regression (variable slope; four parameters; GraphPad Prism 9.3.1). Area under the curve values were calculated using GraphPad Prism 9.3.1.

[0068] FIG. 39. Biolayer interferometry sensorgrams showing binding of previously reported neutralizing or non-neutralizing antibodies to epitopes on the S2 subunit in the SARS-CoV-2 HexaPro-SS-T998W construct (including stalk region). Colored lines indicate 1:1 binding fit, black lines indicate reference subtracted response.

[0069] FIG. 40. Antibody response in Syrian hamsters immunized with SARS-CoV-2 SSS-np and SSS-WW-np. Male Syrian hamsters (n = 15) were immunized with adjuvant alone, SARS-CoV-2 S2P, SARS-CoV-2 SSS-np, or SARS-CoV-2 SSS-WW-np intramuscularly thrice at weeks 0, 4, and 8. All protein and nanoparticle vaccines were given at 10 µg with AddaVax adjuvant. Serum samples were collected at week 10 and ELISA binding antibodies were measured against SARS-CoV-2 S6P (ancestral strain) and SARS- CoV-2 S2P of EG.5.1 subvariant. Lower and upper limit of detection are indicated as dash lines.

[0070] FIG. 41. Protection of Syrian hamsters immunized with SARS-CoV-2 SSS-np and SSS-WW-np. Male Syrian hamsters (n = 15) were immunized with adjuvant alone, 184893-1038-0739, v. 1SARS-CoV-2 S2P, SARS-CoV-2 SSS-np, or SARS-CoV-2 SSS-WW-np intramuscularly thrice at weeks 0, 4, and 8. All protein and nanoparticle vaccines were given at 10 µg with AddaVax adjuvant. All animals were intranasally challenged at week 12 with SARS-CoV-2 EG.5.1 subvariant (10,000 PFU). Five animals from each group were euthanized on days 2, 4, and 7 for virological readout. DETAILED DESCRIPTION

[0071] SARS-CoV-2 has been evolving since its emergence in late 2019, and it poses challenges to control the virus by targeting the most vulnerable yet hypervariable receptor- binding domain on its spike envelope protein. In contrast, the stem domain of spike (S2) is more conserved and targeted by several broadly cross-reactive human monoclonal antibodies, suggesting that the S2 can be a viable target for broadly protective vaccines.

[0072] Provided herein are stabilized S2 trimers derived from SARS-CoV-2 and / or SARS-CoV. Conformation of the S2 trimers was improved by incorporating two cavity filling mutations identified through molecular simulations, resulting in fully closed stabilized S2 trimers for both SARS-CoV-2 and SARS-CoV.

[0073] These stabilized S2 trimers can be used, for example, to elicit broadly cross- protective immunity against sarbecoviruses and potentially other beta-coronaviruses. These stabilized S2 trimers can also be used to detect S protein binding antibodies in a sample. Thus, the engineered proteins provided herein allow for more effective method for vaccination against coronavirus as well as enabling new assay method for detecting anti- coronavirus antibodies, e.g., biological samples.

[0074] Self-assembling nanoparticles have been demonstrated as useful vaccine tools to greatly enhance vaccine immunogenicity and tested in many clinical trials as well as a licensed product against SARS-CoV-2. One of such nanoparticle platforms is insect ferritin- based system, which is a spontaneously self-assembling two-component nanoparticle, with distinct heavy and light chain subunits that each can display trimeric antigens on the exterior. Thus, four copies each of two different trimers can be displayed on a single insect ferritin nanoparticle.

[0075] Provided herein are nanoparticle immunogens that display either one or two types of stabilized S2 trimers derived from SARS-CoV-2 and / or SARS-CoV on their exterior 194893-1038-0739, v. 1as vaccine candidates to elicit broadly cross-protective immunity against sarbecoviruses and potentially other beta-coronaviruses. Upon immunization, the nanoparticle immunogens displaying the SARS-CoV-2 stabilized S2 trimers elicited strong antibody responses in mice that were highly reactive to homologous SARS-CoV-2 ancestral spike as well as antigenically distant Omicron lineage spike. In addition, the immune sera had weak but detectable neutralizing activity against SARS-CoV-2 pseudovirus. Cross-reactivity to spike proteins derived from other betacoronaviruses including SARS-CoV, OC43, HKU1, and MERS-CoV was also detected in the immune sera from mice immunized with the stabilized S2-nanoparticles, suggesting that the S2-based approaches can offer not only the variant- proof SARS-CoV-2 vaccine solution but also potential pan-betacoronavirus vaccine solution to combat against this diverse and hypervariable family of viruses. I. Coronavirus Spike Protein and Stabilized Stem S2

[0076] The spike protein of SARS-CoV-2 plays an essential role in virus entry into host cells and thus a primary target by neutralizing antibodies. The spike protein comprises an N-terminal S1 subunit and a C-terminal S2 subunit, which are responsible for receptor binding and membrane fusion. The S1 subunit, which directly interacts with the host receptor, is composed of an N-terminal domain (NTD), a receptor-binding domain (RBD) and subdomains (SDs). For SARS-CoV-2, transient hinging of the RBD into an ‘up’ conformation allows for host-cell receptor engagement. In the S2 subunit, a helix-loop region spanning from the fusion peptide (FP) to heptad repeat 1 (HR1), constitutes a metastable structure that transitions to a long stable α-helix in the postfusion conformation during viral entry. The FP, HR1, central helix (CH) and connector domain (CD) comprise the globular head of the S2 subunit. In contrast, the helical stalk region, connecting the globular head to the viral membrane, is elongated and highly flexible (Ke et al., 2020; Turoňová et al., 2020). The stalk region can be further divided into a hip, knee, and ankle, wherein the knee functions as a hinge, providing a considerable range in flexibility and movement for the spikes on the viral surface.

[0077] The spike binds to a cellular receptor through its RBD, which triggers a conformational change of the spike. The activated spike is cleaved by a protease (such as TMPRSS2 for SARS-CoV and SARS-CoV-2) at S1 / S2 site to release the S1 subunit and expose the FP on S2 subunit. The HR1 and HR2 refold to the post-fusion conformation to drive membrane fusion. As the S1 is responsible for interacting directly with the host 204893-1038-0739, v. 1receptor, the majority of vaccine-induced antibodies and antibodies elicited upon natural infection target this subunit, most notably the RBD (Corbett et al., 2020b, 2020a; Jackson et al., 2020). In addition, a portion of neutralizing monoclonal antibodies (mAbs) isolated from convalescent COVID-19 patients recognize the NTD (Chi et al., 2020; Liu et al., 2020). Amino acid changes are concentrated in the S1 subunit among the majority of SARS-CoV-2 variants described thus far. Additionally, recent data suggest that antibodies in convalescent sera that target neutralization-sensitive epitopes in the S1 domain induce a selective pressure that yields escape mutations in these epitopes (Andreano et al., 2020).

[0078] Provided herein are rationally engineered CoV S ectodomains (see exemplary substitutions provided in Table 1, each of which comprises V991W and / or T998W substitutions) as stabilized immunogens to elicit antibodies cross-reactive with all three epidemic β-HCoVs. Using a stabilized stem construct, the production of antibodies was elicited which enabled the identification of a novel site of vulnerability in the S2 subunit, which will inform development of next-generation CoV vaccines and fortify CoV pandemic preparedness.

[0079] Any of the substitutions described herein may engineered into any known coronavirus S protein variant, including, but not limited to, a coronavirus S protein having any one or more of the following modifications (see SEQ ID NO: 1): L5F, S13I, L18F, T19R, T20N, P26S, Q52R, A67V, H69del, V70del, V70I, D80A, T95I, D138Y, Y144del, Y144V, W152C, E154K, R190S, D215G, L242del, A243del, L244del, D253G, W258L, K417N, K417T, L452R, S477N, T478K, E484K, E484Q, E484K, N501Y, A570D, D614G, H655Y, Q677H, P681R, P681H, A701V, T716I, F888L, D950N, S982A, T1027I, D1118H, and V1176F. Exemplary combinations of such modifications are provided in Table 2. Table 1. Exemplary S ectodomain substitutions and mutations Mutations (relative to SEQ ID NO: 1 or 2)Mutations (relative to SEQ ID NO: 1 or 2) F817P / A892P / A899P / A942P / K986P / V987P / S704C / K790C / T998WTable 2. SARS-CoV-2 Variant Classification and Definitions Variant Name Spike Protein Substitutions (see SEQ ID NO: 4) (Pango lineage) I, Y, G, el, N, R, K,II. Definitions

[0080] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise. Also, the use of the term “portion” can include part of a moiety or the entire moiety. 22 4893-1038-0739, v. 1

[0081] As used herein, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0082] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the disclosed subject matter. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0083] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0084] The term “antibody” refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes, for instance, chimeric, humanized, fully human, and bispecific antibodies. An “antibody” is a species of an antigen binding protein. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but 234893-1038-0739, v. 1in some instances can include fewer chains such as antibodies naturally occurring in camelids which can comprise only heavy chains. Antibodies can be derived solely from a single source, or can be “chimeric,” that is, different portions of the antibody can be derived from two different antibodies as described further below. The antigen binding proteins, antibodies, or binding fragments can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

[0085] Naturally occurring antibody structural units typically comprise a tetramer. Each such tetramer typically is composed of two identical pairs of polypeptide chains, each pair having one full-length “light” (in certain embodiments, about 25 kDa) and one full- length “heavy” chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that can be responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgA1 and IgA2. Within full-length light and heavy chains, typically, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See, e.g., Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light / heavy chain pair typically form the antigen binding site. 244893-1038-0739, v. 1

[0086] The term “variable region” or “variable domain” refers to a portion of the light and / or heavy chains of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino terminal amino acids in the light chain. In certain embodiments, variable regions of different antibodies differ extensively in amino acid sequence even among antibodies of the same species. The variable region of an antibody typically determines specificity of a particular antibody for its target.

[0087] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which can enable binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987) or Chothia et al., Nature, 342:878-883 (1989).

[0088] In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions.

[0089] In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and / or solving the structure of the antibody-ligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition and the contact definition. 254893-1038-0739, v. 1

[0090] The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g., Chothia et al., J. Mol. Biol., 196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989). The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); “AbM™, A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). The contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[0091] By convention, the CDR regions in the heavy chain are typically referred to as H1, H2, and H3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus. The CDR regions in the light chain are typically referred to as L1, L2, and L3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus.

[0092] The term “light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0093] The term “heavy chain” includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide, and the CH domains are at the carboxyl-terminus, with the CH3 being closest to the carboxy- terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including 264893-1038-0739, v. 1IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.

[0094] A bispecific or bifunctional antibody typically is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai et al., Clin. Exp. Immunol., 79: 315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

[0095] The term “antigen” refers to a substance capable of inducing adaptive immune responses. Specifically, an antigen is a substance which serves as a target for the receptors of an adaptive immune response. Typically, an antigen is a molecule that binds to antigen- specific receptors but cannot induce an immune response in the body by itself. Antigens are usually proteins and polysaccharides, less frequently also lipids. As used herein, antigens also include immunogens and haptens.

[0096] An “Fc” region comprises two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0097] The “Fv region” comprises the variable regions from both the heavy and light chains but lacks the constant regions.

[0098] An antibody that “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. For example, the Coronavirus S protein specific antibodies of the present invention are specific to Coronavirus S protein. In some embodiments, the antibody that binds to Coronavirus S protein has a dissociation constant (Kd) of ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10−8M or less, e.g., from 10−8M to 10−13M, e.g., from 10−9M to 10−13M).

[0099] The term “compete” when used in the context of antigen binding proteins (e.g., atnibody or antigen-binding fragment thereof) that compete for the same epitope means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or antigen-binding fragment thereof) being tested prevents or 274893-1038-0739, v. 1inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand, or a reference antibody) to a common antigen (e.g., Coronavirus S protein or a fragment thereof). Numerous types of competitive binding assays can be used to determine if one antigen binding protein competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242- 253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol.137:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol.25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol.32:77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabelled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assay (competing antigen binding proteins) include antigen binding proteins binding to the same epitope as the reference antigen binding proteins and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur. Additional details regarding methods for determining competitive binding are provided in the examples herein. Usually, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of a reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65- 70%, 70-75% or 75% or more. In some instances, binding is inhibited by at least 80-85%, 85- 90%, 90-95%, 95-97%, or 97% or more.

[0100] The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. The epitope can be either linear epitope or a conformational epitope. A linear epitope is formed by a continuous sequence of amino acids from the antigen and interacts with an antibody based on their primary structure. A conformational epitope, on the other hand, is composed of discontinuous sections of the antigen’s amino acid sequence and interacts with the antibody based on the 3D structure of 284893-1038-0739, v. 1the antigen. In general, an epitope is approximately five or six amino acid in length. Two antibodies may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.

[0101] The term “host cell” means a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.

[0102] The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm”). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math.48:1073.

[0103] In calculating percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res.12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm). A gap opening penalty (which is calculated as 3× the average diagonal, wherein the “average diagonal” is 294893-1038-0739, v. 1the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1 / 10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A.89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

[0104] Examples of parameters that can be employed in determining percent identity for polypeptides or nucleotide sequences using the GAP program can be found in Needleman et al., 1970, J. Mol. Biol.48:443-453.

[0105] Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted if so desired to result in an alignment that spans at least 50 or other number of contiguous amino acids of the target polypeptide.

[0106] The term “link” as used herein refers to the association via intramolecular interaction, e.g., covalent bonds, metallic bonds, and / or ionic bonding, or inter-molecular interaction, e.g., hydrogen bond or noncovalent bonds.

[0107] The term “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given signal peptide that is operably linked to a polypeptide directs the secretion of the polypeptide from a cell. In the case of a promoter, a promoter that is operably linked to a coding sequence will direct the expression of the coding sequence. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

[0108] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, 304893-1038-0739, v. 1although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0109] The term “polynucleotide” or “nucleic acid” includes both single- stranded and double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2′,3′-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

[0110] The terms “polypeptide” or “protein” means a macromolecule having the amino acid sequence of a native protein, that is, a protein produced by a naturally- occurring and non-recombinant cell; or it is produced by a genetically-engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally occurring amino acid and polymers. The terms “polypeptide” and “protein” specifically encompass Coronavirus S protein binding proteins, antibodies, or sequences that have deletions from, additions to, and / or substitutions of one or more amino acid of antigen-binding protein. The term “polypeptide fragment” refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion as compared with the full-length native protein. Such fragments can also contain modified amino acids as compared with the native protein. In certain embodiments, fragments are about five to 500 amino acids long. For example, fragments can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of a CORONAVIRUS S PROTEIN-binding antibody, useful fragments include but are not limited to a CDR region, a variable domain of a heavy and / or light chain, a portion of an antibody chain or just its variable region including two CDRs, and the like.

[0111] “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to induce an immune response when administered to a subject. In some embodiments, a 314893-1038-0739, v. 1disclosed insect ferritin heavy or light chain comprises from 1-10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10) conservative substitutions compared to a corresponding native insect ferritin heavy or light chain sequence, respectively. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Thus, a conservative substitution does not alter the basic function of a protein of interest. Non-conservative substitutions are those that reduce an activity or function of the protein, such as the ability to induce an immune response when administered to a subject. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity.

[0112] Ferritin nanoparticle: A multi-subunit, globular shaped protein complex. In nature, native ferritin proteins self-assemble into a globular structure that stores iron and releases it in a controlled fashion. Production and expression of ferritin nanoparticles based on monomeric ferritin subunits that are linked to influenza HA ectodomains have been previously described (see, e.g., Kanekiyo et al., Nature 499, 102-106, 2013 and Zhang, Y. Int. J. Mol. Sci., 12:5406-5421, 2011, which are incorporated by reference herein).

[0113] Fusion Protein: A single polypeptide chain including the sequence of two or more heterologous proteins, often linked by a peptide linker.

[0114] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an “antigen-specific response”). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies.

[0115] As used herein, reference to “at least 90% identity” refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.

[0116] The pharmaceutically acceptable carriers useful in this invention are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and 324893-1038-0739, v. 1physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), 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 to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0117] As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0118] The term “therapeutically effective amount” or “effective dosage” as used herein refers to the dosage or concentration of a drug effective to treat a disease or condition. For example, with regard to the use of the monoclonal antibodies or antigen- binding fragments thereof disclosed herein to treat viral infection.

[0119] “Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.

[0120] As used herein, a “vector” refers to a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication. A vector may also include one or more therapeutic genes and / or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and / or proteins other than 334893-1038-0739, v. 1those native to the cell. A vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like. III. Pharmaceutical Formulations

[0121] The present disclosure provides pharmaceutical compositions comprising an engineered, stabilized beta-coronavirus S2 domain-only protein; a protein nanoparticle that includes an engineered, stabilized beta-coronavirus S2 domain-only protein; a nucleic acid molecule encoding an engineered, stabilized beta-coronavirus S2 domain-only protein; and viral vector comprising an engineered, stabilized beta-coronavirus S2 domain- only protein and / or encoding the engineered, stabilized beta-coronavirus S2 domain-only protein in its genomic material. Such compositions can be used for stimulating an immune response, such as part of a vaccine formulation. 1. Nanoparticles

[0122] Provided herein are protein nanoparticles (such as self-assembling protein nanoparticles) that include a recombinant SARS-CoV-2 S2 domain-only ectodomain trimer displayed on its surface. Non-limiting examples of self-assembling protein nanoparticles include ferritin nanoparticles, encapsulin nanoparticles, Sulfur Oxygenase Reductase (SOR) nanoparticles, and lumazine synthase nanoparticles, which are comprised of an assembly of monomeric subunits including ferritin proteins, encapsulin proteins, SOR proteins, and lumazine synthase, respectively. Additional protein nanoparticle structures are described by Heinze et al., J Phys Chem B., 120(26):5945-52, 2016; Hsia et al., Nature, 535(7610):136-9, 2016; and King et al., Nature, 510(7503):103-8, 2014; each of which is incorporated by reference herein.

[0123] To construct such protein nanoparticles, nucleic acid encoding a protomer of the SARS-CoV-2 S2 domain-only ectodomain trimer can be fused to nucleic acid encoding a subunit of the protein nanoparticle (such as a ferritin protein, an encapsulin protein, a SOR protein, or a lumazine synthase protein) and expressed in cells under appropriate conditions. The fusion protein self-assembles into a nanoparticle and can be purified. Alternatively, a purified SARS-CoV-2 S2 domain-only ectodomain trimer can be linked (for example, via bioconjugation) to subunits of a purified self-assembling protein nanoparticle (such as a ferritin protein, an encapsulin protein, a SOR protein, or a lumazine synthase protein) and the resulting nanoparticle / S trimer purified. 344893-1038-0739, v. 1

[0124] The SARS-CoV-2 S2 domain-only ectodomain trimer may be included in a self-assembling protein nanocage that directs its own release from cells inside small vesicles in a manner that resembles viruses, for example, as described in Votteler et al., “Designed proteins induce the formation of nanocage-containing extracellular vesicles,” Nature 540, 292-29, 2016. This hybrid biomaterial can fuse its membranes with target cells and deliver its contents, thereby transferring cargoes from one cell to another.

[0125] A protomer of a disclosed recombinant SARS-CoV-2 S2 domain-only ectodomain trimer can be linked to a ferritin subunit to construct a ferritin nanoparticle. Ferritin nanoparticles and their use for immunization purposes (e.g., for immunization against SARS-CoV-2 antigens) have been disclosed in the art (see, e.g., Kanekiyo et al., Nature, 499:102-106, 2013, incorporated by reference herein in its entirety). Ferritin is a globular protein that is found in all animals, bacteria, and plants, and which acts primarily to control the rate and location of polynuclear Fe(III)2O3 formation through the transportation of hydrated iron ions and protons to and from a mineralized core. The globular form of the ferritin nanoparticle is made up of monomeric subunits, which are polypeptides having a molecule weight of approximately 17-20 kDa. An example of the amino acid sequence of one such monomeric ferritin subunit is represented by: ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKK LIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFN FLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO: 11)

[0126] Each monomeric subunit has the topology of a helix bundle which includes a four antiparallel helix motif, with a fifth shorter helix (the c-terminal helix) lying roughly perpendicular to the long axis of the 4 helix bundle. According to convention, the helices are labeled ‘A, B, C, D & E’ from the N-terminus respectively. The N-terminal sequence lies adjacent to the capsid three-fold axis and extends to the surface, while the E helices pack together at the four-fold axis with the C-terminus extending into the capsid core. The consequence of this packing creates two pores on the capsid surface. It is expected that one or both of these pores represent the point by which the hydrated iron diffuses into and out of the capsid. Following production, these monomeric subunit proteins self-assemble into the globular ferritin protein. Thus, the globular form of ferritin comprises 24 monomeric, subunit proteins, and has a capsid-like structure having 432 symmetry. Methods of constructing ferritin nanoparticles are known to the person of ordinary skill in the art and are further 354893-1038-0739, v. 1described herein (see, e.g., Zhang, Int. J. Mol. Sci., 12:5406-5421, 2011, which is incorporated herein by reference in its entirety).

[0127] The ferritin polypeptide may be insect ferritin, E. coli ferritin, Helicobacter pylori ferritin, human light chain ferritin, bullfrog ferritin or a hybrid thereof, such as E. coli-human hybrid ferritin, E. coli-bullfrog hybrid ferritin, or human-bullfrog hybrid ferritin. Exemplary amino acid sequences of ferritin polypeptides and nucleic acid sequences encoding ferritin polypeptides for use to make a ferritin nanoparticle including a recombinant SARS-CoV-2 S2 domain-only ectodomain can be found in GENBANK®, for example at accession numbers ZP_03085328, ZP_06990637, EJB64322.1, AAA35832, NP_000137 AAA49532, AAA49525, AAA49524 and AAA49523, which are specifically incorporated by reference herein in their entirety as available Apr.10, 2015.

[0128] Provided herein are recombinant SARS-CoV-2 S2 domain-only ectodomain ferritin nanoparticles that are based on insect ferritin proteins, and that have been engineered for display of two different trimeric antigens at an equal ratio. Unlike bacterial ferritin, insect ferritin includes twelve copies of two different subunits (termed heavy and light chains; 24 subunits total). The insect ferritin heavy chains trimerize and the insect ferritin light chains trimerize (forming four trimers of heavy chains and four trimers of light chains) and self-assemble into the globular nanoparticle. In the disclosed recombinant insect ferritin nanoparticles, each insect ferritin heavy chain includes an N-terminal fusion to a first protein, and each insect ferritin light chain includes an N-terminal fusion to a second protein. In some cases, the first protein and the second protein may be the same. In some cases, the first protein and the second protein may share 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. As the insect ferritin heavy and light chains trimerize and self-assemble into the globular nanoparticle, the corresponding first and second proteins also self-assemble into trimers of the first protein and trimers of the second protein. Display of two, optionally diverse, antigen trimers on the same ferritin nanoparticle allows B cells to simultaneously recognize both antigens, thus leading to improved breadth of recognition.

[0129] Typically, the recombinant insect ferritin nanoparticle, with the heavy and light chain ferritin proteins, forms a globular nanoparticle shape with the trimers of the first and second proteins extending in a radially outward direction from the outer surface of the globular nanoparticle. In this shape, the trimers of the first and second protein are readily presented to a subject’s immune system when the recombinant insect ferritin nanoparticle is 364893-1038-0739, v. 1administered to the subject. Accordingly, in several embodiments, the recombinant insect ferritin nanoparticles described herein can be used as a platform for multimerized display of trimeric antigens such as the SARS-CoV-2 S2 domain-only ectodomain.

[0130] In several embodiments, the insect ferritin heavy and light chains can be from the Lepidoptera order of insects, such as ferritin heavy and light chains from Trichoplusia (such as Trichoplusia ni), or ferritin heavy and light chains from manduca. Exemplary ferritin heavy and light chain amino acid sequences for Trichoplusia ni and manduca proteins are provided below:

[0131] Trichoplusia ni ferritin heavy chain (Acc. No., PDB 1Z6O; SEQ ID NO: 12) TQCNVNPVQIPKDWITMHRSCRNSMRQQIQMEVGASLQYLAMGAHFSKDVVNRPGFAQLFFD AASEEREHAMKLIEYLLMRGELTNDVSSLLQVRPPTRSSWKGGVEALEHALSMESDVTKSIR NVIKACEDDSEFNDYHLVDYLTGDFLEEQYKGQRDLAGKASTLKKLMDRHEALGEFIFDKKL LGIDV

[0132] Trichoplusia ni ferritin light chain (Acc. No. AAX94729.1; SEQ ID NO: 13) GITSNSLALPRCNAVYGEYGSHGNVATELQAYAKLHLERSYDYLLSAAYFNNYQTNRAGFSK LFKKLSDEAWSKTIDIIKHVTKRGDKMNFDQHSTMKTERKNYTAENHELEALAKALDTQKEL AERAFYIHREATRNSQHLHDPEIAQYLEEEFIEDHAEKIRTLAGHTSDLKKFITANNGHDLS LALYVFDEYLQKTV

[0133] Manduca ferritin heavy chain (Acc. No. AAK39636.1; SEQ ID NO: 14) MKAILLSVAGLLAVLAPAIATQCHVNPVNIQREWITMHRSCRDSMRRQIQMEVGASLQYLAM GAHFSKDKINRPGFAKLFFDAAGEEREHAMKLIEYLLMRGELTNDVTSLIQVRAPQRNKWEG GVDALEHALKMESDVTKSIRTVIKACEDDPEFNDYHLVDYLTGEFLEEQYKGQRDLAGKAST LKKMLDRNSALGEFIFDKKLMGMDI

[0134] Manduca ferritin light chain (Acc. No. AAF44717.1; SEQ ID NO: 15) MNPITFFVACLLALCGAVAADTCYQDVSLDCSQVSNSLTLPNCNAVYAEYGHHGNVAKEMQA YAALHLERSYEYLLSSSYFNNYQTNRAGFSKLFRKLSDDAWEKTIDLIKHITMRGDEMNFAQ RSTQKSVDRKNYTVELHELESLAKALDTQKELAERAFFIHREATRNSQHLHDPEVAQYLEEE FIEDHAKTIRNLAGHTTDLKRFVSGDNGQDLSLALYVFDEYLQKTV

[0135] Two different viral strains can be attached in an equal configuration (equal fraction of one strain vs. the other); however, the geometry of the native insect ferritin 374893-1038-0739, v. 1particle does not allow the attachment of viral envelope protein antigens that assemble as trimers. Accordingly, to properly position the “base” of the trimers formed by the first and second proteins, the insect ferritin heavy and light chains include N-terminal truncations. The truncations position the portion of the first and second proteins that is proximal to the nanoparticle surface in an orientation similar to that found in the native trimer of each of these proteins. For example, in embodiments where the first and second proteins are viral envelope protein ectodomains that co-assemble into trimers on the recombinant insect ferritin nanoparticle, the heavy and light chain N-terminal truncations allow for positioning of the portion of the first and second proteins that is proximal to the nanoparticle surface in an orientation similar to that of the native trimer found on the viral surface.

[0136] In some embodiments, the recombinant insect ferritin nanoparticle includes recombinant ferritin heavy chains comprising 172 to 174 amino acids from the C- terminus of an insect ferritin heavy chain and a deletion of the remaining N-terminal amino acids. In additional embodiments, the recombinant insect ferritin nanoparticle comprises recombinant ferritin light chains comprising 182 to 184 amino acids from the C-terminus of an insect ferritin light chain and a deletion of the remaining N-terminal amino acids. In more embodiments, the recombinant insect ferritin nanoparticle comprises recombinant ferritin heavy chains comprising 172 to 174 amino acids from the C-terminus of an insect ferritin heavy chain and a deletion of the remaining N-terminal amino acids, and recombinant ferritin light chains comprise 182 to 184 amino acids from the C-terminus of an insect ferritin light chain and a deletion of the remaining N-terminal amino acids. The N-terminal amino acid of each recombinant heavy and light chain in the recombinant insect ferritin nanoparticle can be fused to the C-terminal amino acid of the first or second protein, respectively (or to a peptide linker that can be fused the C-terminal amino acid of the first or second protein).

[0137] In some embodiments, the insect ferritin heavy chain can be a Trichoplusia ni ferritin heavy chain with an 18 amino acid N-terminal truncation. For example, the insect ferritin heavy chain comprises an amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence at least 90% identical thereto. In some embodiments, the insect ferritin light chain can be a Trichoplusia ni ferritin light chain with a 17 amino acid N-terminal truncation. For example, the insect ferritin light chain comprises an amino acid sequence set forth as SEQ ID NO: 8, or an amino acid sequence at least 90% identical thereto. In some embodiments, the insect ferritin heavy chain comprises an amino acid 384893-1038-0739, v. 1sequence at least 90% identical to SEQ ID NO: 7, and the insect ferritin light chain comprises an amino acid sequence at least 90% identical SEQ ID NO: 8. In some embodiments, the insect ferritin heavy chain comprises an amino acid sequence set forth as SEQ ID NO: 7, and the insect ferritin light chain comprises an amino acid sequence set forth as SEQ ID NO: 8.

[0138] In some embodiments, the insect ferritin heavy chain can be a manduca ferritin heavy chain with a 38 amino acid N-terminal truncation. For example, the insect ferritin heavy chain comprises an amino acid sequence set forth as SEQ ID NO: 16, or an amino acid sequence at least 90% identical thereto. In some embodiments, the insect ferritin light chain can be a manduca ferritin light chain with a 48 amino acid N-terminal truncation. For example, the insect ferritin light chain comprises an amino acid sequence set forth as SEQ ID NO: 17, or an amino acid sequence at least 90% identical thereto. In some embodiments, the insect ferritin heavy chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 16, and the insect ferritin light chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 17. In some embodiments, the insect ferritin heavy chain comprises an amino acid sequence set forth as SEQ ID NO: 16, and the insect ferritin light chain comprises an amino acid sequence set forth as SEQ ID NO: 17.

[0139] Exemplary insect ferritin heavy and light chain sequences with N- terminal truncations are set forth below:

[0140] Trichoplusia ni ferritin heavy chain with 18-aa N-terminal truncation (SEQ ID NO: 7) RSCRNSMRQQIQMEVGASLQYLAMGAHFSKDVVNRPGFAQLFFDAASEEREHAMKLIEYLLM RGELTNDVSSLLQVRPPTRSSWKGGVEALEHALSMESDVTKSIRNVIKACEDDSEFNDYHLV DYLTGDFLEEQYKGQRDLAGKASTLKKLMDRHEALGEFIFDKKLLGIDV

[0141] Trichoplusia ni ferritin light chain with 17-aa N-terminal truncation (SEQ ID NO: 8) EYGSHGNVATELQAYAKLHLERSYDYLLSAAYFNNYQTNRAGFSKLFKKLSDEAWSKTIDII KHVTKRGDKMNFDQHSTMKTERKNYTAENHELEALAKALDTQKELAERAFYIHREATRNSQH LHDPEIAQYLEEEFIEDHAEKIRTLAGHTSDLKKFITANNGHDLSLALYVFDEYLQKTV

[0142] Manduca ferritin heavy chain with 38-aa truncation (SEQ ID NO: 16) RSCRDSMRRQIQMEVGASLQYLAMGAHFSKDKINRPGFAKLFFDAAGEEREHAMKLIEYLLM RGELTNDVTSLIQVRAPQRNKWEGGVDALEHALKMESDVTKSIRTVIKACEDDPEFNDYHLV DYLTGEFLEEQYKGQRDLAGKASTLKKMLDRNSALGEFIFDKKLMGMDI 394893-1038-0739, v. 1

[0143] Manduca ferritin light chain with 48-aa N-terminal truncation (SEQ ID NO: 17) EYGHHGNVAKEMQAYAALHLERSYEYLLSSSYFNNYQTNRAGFSKLFRKLSDDAWEKTIDLI KHITMRGDEMNFAQRSTQKSVDRKNYTVELHELESLAKALDTQKELAERAFFIHREATRNSQ HLHDPEVAQYLEEEFIEDHAKTIRNLAGHTTDLKRFVSGDNGQDLSLALYVFDEYLQKTV

[0144] In additional embodiments, the recombinant insect ferritin heavy and light chain proteins can comprise modifications of the ferritin sequences, such as amino acid substitutions, deletions or insertions, glycosylation and / or covalent linkage to unrelated proteins (e.g., a protein tag), as long as the recombinant ferritin protein retains the ability to form a globular ferritin nanoparticle with co-assembled first and second protein trimers. These variations in sequence can be naturally occurring variations or they can be engineered through the use of genetic engineering techniques. In some embodiments, the recombinant insect ferritin heavy chain comprises a L113Y substitution. In some embodiments, the recombinant insect ferritin light chain comprises L123E and 1189K substitutions. In additional embodiments, the recombinant insect ferritin heavy chain comprises a L113Y substitution, and the recombinant insect ferritin light chain comprises L123E and 1189K substitutions.

[0145] In certain embodiments, the recombinant insect ferritin nanoparticle provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular 404893-1038-0739, v. 1properties or functions of the immunogen to be improved or altered, whether the immunogen derivative will be used in a therapy under defined conditions, etc.

[0146] The recombinant insect ferritin nanoparticle can be derivatized or linked to another molecule (such as another peptide or protein). In general, the recombinant insect ferritin nanoparticle is derivatized such that the binding of antibodies to the trimeric antigens included on the recombinant insect ferritin nanoparticle is not affected adversely by the derivatization or labeling. For example, the recombinant insect ferritin nanoparticle can be functionally linked (by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as an antibody or protein or detection tag.

[0147] For production purposes, the insect ferritin heavy and light chain fusion proteins can include an N-terminal signal peptide that is cleaved during cellular processing. In some embodiments, the insect ferritin heavy and light chain fusion proteins can include a native signal peptide corresponding to a viral envelope protein ectodomain included on the fusion protein. In additional embodiments, the insect ferritin heavy and light chain fusion proteins can include a signal peptide comprising the amino acid sequence set forth as MDSKGSSQKGSRLLLLLVVSNLLLPQGVLA (bPRL(LA) signal peptide, SEQ ID NO: 18) or MPMGSLQPLATLYLLGMLVASVLA (hCD5 signal peptide, SEQ ID NO: 19) or MKAKLLVLLCTFTATYA (SEQ ID NO: 20).

[0148] Fusion of the first and second protein to the recombinant insect ferritin heavy and light chains is preferably done such that the first and second proteins do not interfere with self-assembly of the ferritin heavy and light chains into the globular insect ferritin nanoparticle structure, and that the ferritin heavy and light chains do not interfere with the trimerization of the first proteins or the trimerization of the second proteins. In some embodiments, the insect ferritin heavy and light chains can be fused directly to the first and second proteins, respectively, without affecting ferritin self-assembly or antigen trimerization. In other embodiments, the insect ferritin heavy and light chains can be fused indirectly to the first and second proteins, respectively, using a peptide linker. The peptide linker can position the ferritin heavy and light chains and the first and second protein, respectively, with regard to one another, such that the fusion protein maintains the ability to self-assemble into nanoparticles including trimerized first and second proteins. Preferable amino acids for the peptide linker include those having small side chains and / or those which 414893-1038-0739, v. 1are not charged. Such amino acids are less likely to interfere with proper folding and activity of the fusion protein. Accordingly, preferred amino acids to use in linker sequences, either alone or in combination are serine, glycine and alanine (such as a glycine linker, a serine linker, or a glycine-serine linker). Non-limiting exemplary linker sequences include SGG, GGSGG (SEQ ID NO: 21), and AGGSGG (SEQ ID NO: 22).

[0149] Sequences of exemplary insect ferritin heavy and light chain fusion proteins including heavy or light chain ferritin subunits fused to SARS-CoV-2 S2 domain- only ectodomains are provided below:

[0150] SARS-CoV-2 SSS-WW-np HC (SEQ ID NO: 3), where amino acids 1- 17 are the signal peptide, amino acids 18-480 are the SARS-CoV-2 S2 domain-only ectodomain, amino acid 481-485 are a peptide linker, and amino acids 486-658 are iFerr HC: MKAKLLVLLCTFTATYAMSLGAENCVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMY ICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYCTPPIKDFGGFNFS QILPDPSKPSKRSPIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLL TDEMIAQYTSALLAGTITSGWTFGAGPALQIPFPMQMAYRFNGIGVTQNVLYENQKLIANQF NSAIGKIQDSLSSTPSALGKLQDVVNQNAEALNTLVKQLSSNFGAISSVLNDILSRLDPPEA EWQIDRLIWGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMS FPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQ IITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHGGSGGRSCRNSMRQQI QMEVGASLQYLAMGAHFSKDVVNRPGFAQLFFDAASEEREHAMKLIEYLLMRGELTNDVSSL LQVRPPTRSSWKGGVEALEHALSMESDVTKSIRNVIKACEDDSEFNDYHLVDYLTGDFLEEQ YKGQRDLAGKASTLKKLMDRHEALGEFIFDKKLLGIDV

[0151] SARS-CoV-2 SSS-WW-np LC (SEQ ID NO: 4), where amino acids 1- 17 are the signal peptide, amino acids 18-480 are the SARS-CoV-2 S2 domain-only ectodomain, amino acid 481-485 are a peptide linker, and amino acids 486-668 are iFerr LC: MKAKLLVLLCTFTATYAMSLGAENCVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMY ICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYCTPPIKDFGGFNFS QILPDPSKPSKRSPIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLL TDEMIAQYTSALLAGTITSGWTFGAGPALQIPFPMQMAYRFNGIGVTQNVLYENQKLIANQF NSAIGKIQDSLSSTPSALGKLQDVVNQNAEALNTLVKQLSSNFGAISSVLNDILSRLDPPEA EWQIDRLIWGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMS FPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQ IITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHGGSGGEYGSHGNVATE LQAYAKLHLERSYDYLLSAAYFNNYQTNRAGFSKLFKKLSDEAWSKTIDIIKHVTKRGDKMN FDQHSTMKTERKNYTAENHELEALAKALDTQKELAERAFYIHREATRNSQHLHDPEIAQYLE EEFIEDHAEKIRTLAGHTSDLKKFITANNGHDLSLALYVFDEYLQKTV 424893-1038-0739, v. 1

[0152] SARS-CoV-2 SSS-np HC (SEQ ID NO: 5), where amino acids 1-17 are the signal peptide, amino acids 18-480 are the SARS-CoV-2 S2 domain-only ectodomain, amino acid 481-485 are a peptide linker, and amino acids 486-658 are iFerr HC: MKAKLLVLLCTFTATYAMSLGAENCVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMY ICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYCTPPIKDFGGFNFS QILPDPSKPSKRSPIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLL TDEMIAQYTSALLAGTITSGWTFGAGPALQIPFPMQMAYRFNGIGVTQNVLYENQKLIANQF NSAIGKIQDSLSSTPSALGKLQDVVNQNAEALNTLVKQLSSNFGAISSVLNDILSRLDPPEA EVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMS FPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQ IITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHGGSGGRSCRNSMRQQI QMEVGASLQYLAMGAHFSKDVVNRPGFAQLFFDAASEEREHAMKLIEYLLMRGELTNDVSSL LQVRPPTRSSWKGGVEALEHALSMESDVTKSIRNVIKACEDDSEFNDYHLVDYLTGDFLEEQ YKGQRDLAGKASTLKKLMDRHEALGEFIFDKKLLGIDV

[0153] SARS-CoV-2 SSS-np LC (SEQ ID NO: 6), where amino acids 1-17 are the signal peptide, amino acids 18-480 are the SARS-CoV-2 S2 domain-only ectodomain, amino acid 481-485 are a peptide linker, and amino acids 486-668 are iFerr LC: MKAKLLVLLCTFTATYAMSLGAENCVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMY ICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYCTPPIKDFGGFNFS QILPDPSKPSKRSPIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLL TDEMIAQYTSALLAGTITSGWTFGAGPALQIPFPMQMAYRFNGIGVTQNVLYENQKLIANQF NSAIGKIQDSLSSTPSALGKLQDVVNQNAEALNTLVKQLSSNFGAISSVLNDILSRLDPPEA EVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMS FPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQ IITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHGGSGGEYGSHGNVATE LQAYAKLHLERSYDYLLSAAYFNNYQTNRAGFSKLFKKLSDEAWSKTIDIIKHVTKRGDKMN FDQHSTMKTERKNYTAENHELEALAKALDTQKELAERAFYIHREATRNSQHLHDPEIAQYLE EEFIEDHAEKIRTLAGHTSDLKKFITANNGHDLSLALYVFDEYLQKTV

[0154] Sequences of exemplary insect ferritin heavy and light chain fusion proteins including heavy or light chain ferritin subunits fused to SARS-CoV S2 domain-only ectodomains are provided below:

[0155] SARS-CoV SSS-WW-np HC (SEQ ID NO: 23), where amino acids 1- 17 are the signal peptide, amino acids 18-480 are the SARS-CoV S2 domain-only ectodomain, amino acid 481-485 are a peptide linker, and amino acids 486-658 are iFerr HC: MKAKLLVLLCTFTATYAMSLGADSCIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMY ICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQMYCTPTLKYFGGFNFS QILPDPLKPTKRSPIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLL TDDMIAAYTAALVSGTATAGWTFGAGPALQIPFPMQMAYRFNGIGVTQNVLYENQKQIANQF NKAISQIQESLTTTPTALGKLQDVVNQNAEALNTLVKQLSSNFGAISSVLNDILSRLDPPEA 434893-1038-0739, v. 1EWQIDRLIWGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMS FPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQ IITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHGGSGGRSCRNSMRQQI QMEVGASLQYLAMGAHFSKDVVNRPGFAQLFFDAASEEREHAMKLIEYLLMRGELTNDVSSL LQVRPPTRSSWKGGVEALEHALSMESDVTKSIRNVIKACEDDSEFNDYHLVDYLTGDFLEEQ YKGQRDLAGKASTLKKLMDRHEALGEFIFDKKLLGIDV

[0156] SARS-CoV SSS-WW-np LC (SEQ ID NO: 24), where amino acids 1- 17 are the signal peptide, amino acids 18-480 are the SARS-CoV-2 S2 domain-only ectodomain, amino acid 481-485 are a peptide linker, and amino acids 486-668 are iFerr LC: MKAKLLVLLCTFTATYAMSLGADSCIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMY ICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQMYCTPTLKYFGGFNFS QILPDPLKPTKRSPIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLL TDDMIAAYTAALVSGTATAGWTFGAGPALQIPFPMQMAYRFNGIGVTQNVLYENQKQIANQF NKAISQIQESLTTTPTALGKLQDVVNQNAEALNTLVKQLSSNFGAISSVLNDILSRLDPPEA EWQIDRLIWGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMS FPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQ IITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHGGSGGEYGSHGNVATE LQAYAKLHLERSYDYLLSAAYFNNYQTNRAGFSKLFKKLSDEAWSKTIDIIKHVTKRGDKMN FDQHSTMKTERKNYTAENHELEALAKALDTQKELAERAFYIHREATRNSQHLHDPEIAQYLE EEFIEDHAEKIRTLAGHTSDLKKFITANNGHDLSLALYVFDEYLQKTV

[0157] SARS-CoV SSS-np HC (SEQ ID NO: 25), where amino acids 1-17 are the signal peptide, amino acids 18-480 are the SARS-CoV-2 S2 domain-only ectodomain, amino acid 481-485 are a peptide linker, and amino acids 486-658 are iFerr HC: MKAKLLVLLCTFTATYAMSLGADSCIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMY ICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQMYCTPTLKYFGGFNFS QILPDPLKPTKRSPIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLL TDDMIAAYTAALVSGTATAGWTFGAGPALQIPFPMQMAYRFNGIGVTQNVLYENQKQIANQF NKAISQIQESLTTTPTALGKLQDVVNQNAEALNTLVKQLSSNFGAISSVLNDILSRLDPPEA EVQIDRLIGGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMS FPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQ IITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHGGSGGRSCRNSMRQQI QMEVGASLQYLAMGAHFSKDVVNRPGFAQLFFDAASEEREHAMKLIEYLLMRGELTNDVSSL LQVRPPTRSSWKGGVEALEHALSMESDVTKSIRNVIKACEDDSEFNDYHLVDYLTGDFLEEQ YKGQRDLAGKASTLKKLMDRHEALGEFIFDKKLLGIDV

[0158] SARS-CoV SSS-np LC (SEQ ID NO: 26), where amino acids 1-17 are the signal peptide, amino acids 18-480 are the SARS-CoV-2 S2 domain-only ectodomain, amino acid 481-485 are a peptide linker, and amino acids 486-668 are iFerr LC: MKAKLLVLLCTFTATYAMSLGADSCIAYSNNTIAIPTNFSISITTEVMPVSMAKTSVDCNMY ICGDSTECANLLLQYGSFCTQLNRALSGIAAEQDRNTREVFAQVKQMYCTPTLKYFGGFNFS QILPDPLKPTKRSPIEDLLFNKVTLADAGFMKQYGECLGDINARDLICAQKFNGLTVLPPLL TDDMIAAYTAALVSGTATAGWTFGAGPALQIPFPMQMAYRFNGIGVTQNVLYENQKQIANQF 444893-1038-0739, v. 1NKAISQIQESLTTTPTALGKLQDVVNQNAEALNTLVKQLSSNFGAISSVLNDILSRLDPPEA EVQIDRLIGGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMS FPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFSPQ IITTDNTFVSGNCDVVIGIINNTVYDPLQPELDSFKEELDKYFKNHGGSGGEYGSHGNVATE LQAYAKLHLERSYDYLLSAAYFNNYQTNRAGFSKLFKKLSDEAWSKTIDIIKHVTKRGDKMN FDQHSTMKTERKNYTAENHELEALAKALDTQKELAERAFYIHREATRNSQHLHDPEIAQYLE EEFIEDHAEKIRTLAGHTSDLKKFITANNGHDLSLALYVFDEYLQKTV

[0159] The protein nanoparticles can be expressed in appropriate cells (e.g., HEK 293 Freestyle cells) and fusion proteins are secreted from the cells self-assembled into nanoparticles. The nanoparticles can be purified using known techniques, for example by a few different chromatography procedures, e.g. Mono Q (anion exchange) followed by size exclusion (SUPEROSE® 6) chromatography.

[0160] Several embodiments include a monomeric subunit of a ferritin, encapsulin, SOR, or lumazine synthase protein, or any portion thereof which is capable of directing self-assembly of monomeric subunits into the globular form of the protein Amino acid sequences from monomeric subunits of any known ferritin, encapsulin, SOR, or lumazine synthase protein can be used to produce fusion proteins with the recombinant SARS-CoV-2 S2 domain-only ectodomain or immunogenic fragment thereof, so long as the monomeric subunit is capable of self-assembling into a nanoparticle displaying the recombinant SARS-CoV-2 S2 domain-only ectodomain or immunogenic fragment thereof on its surface.

[0161] The fusion proteins need not comprise the full-length sequence of a monomeric subunit polypeptide of a ferritin, encapsulin, SOR, or lumazine synthase protein. Portions, or regions, of the monomeric subunit polypeptide can be utilized so long as the portion comprises amino acid sequences that direct self-assembly of monomeric subunits into the globular form of the protein. 2. Nucleic Acids

[0162] In the case that a nucleic acid molecule encoding an engineered, stabilized beta-coronavirus S2 domain-only protein is used in a pharmaceutical composition, the nucleic acid molecule may comprise or consist of deoxyribonucleotides and / or ribonucleotides, or analogs thereof, covalently linked together. A nucleic acid molecule as described herein generally contains phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkage, e.g., phosphoramidate, 454893-1038-0739, v. 1phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be made; alternatively, mixtures of different polynucleotide analogs, and mixtures of naturally occurring polynucleotides and analogs may be made. A nucleic acid molecule may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double- and single- stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A nucleic acid molecule is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “nucleic acid sequence” is the alphabetical representation of a nucleic acid molecule. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0163] In some embodiments, the nucleic acids of the present disclosure comprise one or more modified nucleosides comprising a modified sugar moiety. Such compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to an oligonucleotide comprising only nucleosides comprising naturally occurring sugar moieties. In some embodiments, modified sugar moieties are substituted sugar moieties. In some embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.

[0164] In some embodiments, modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not limited to 464893-1038-0739, v. 1substituents at the 2' and / or 5' positions. Examples of sugar substituents suitable for the 2'- position, include, but are not limited to: 2'-F, 2'-OCH3("OMe" or "O-methyl"), and 2'- O(CH2)2OCH3 ("MOE"). In certain embodiments, sugar substituents at the 2' position are selected from allyl, amino, azido, thio, O-allyl, O--C1-C10alkyl, O--C1-C10substituted alkyl; OCF3, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn), and O--CH2--C(=O)--N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10alkyl. Examples of sugar substituents at the 5'-position, include, but are not limited to: 5'-methyl (R or S); 5'- vinyl, and 5'-methoxy. In some embodiments, substituted sugars comprise more than one non-bridging sugar substituent, for example, T-F-5'-methyl sugar moieties (see, e.g., PCT International Application WO 2008 / 101157, for additional 5',2'-bis substituted sugar moieties and nucleosides).

[0165] Nucleosides comprising 2'-substituted sugar moieties are referred to as 2'-substituted nucleosides. In some embodiments, a 2'-substituted nucleoside comprises a 2'- substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn) or O--CH2- -C(=O)--N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl. These 2'-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

[0166] In some embodiments, a 2'-substituted nucleoside comprises a 2'- substituent group selected from F, NH2, N3, OCF3, O--CH3, O(CH2)3NH2, CH2— CH=CH2, O--CH2—CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O--(CH2)2--O-- N(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (O--CH2--C(=O)-- N(Rm)(Rn) where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl.

[0167] In some embodiments, a 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from F, OCF3, O--CH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2--O--N(CH3)2, --O(CH2)2O(CH2)2N(CH3)2, and O--CH2-- C(=O)--N(H)CH3. 474893-1038-0739, v. 1

[0168] In some embodiments, a 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from F, O--CH3, and OCH2CH2OCH3.

[0169] In some embodiments, nucleosides of the present disclosure comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present disclosure comprise one or more modified nucleobases.

[0170] In some embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4- b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H- pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3- d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Patent 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al., 1991; and those disclosed by Sanghvi, Y. S., 1993.

[0171] Representative United States Patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases 484893-1038-0739, v. 1include without limitation, U.S. Patents 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, each of which is herein incorporated by reference in its entirety.

[0172] Additional modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. For example, one additional modification of the ligand conjugated oligonucleotides of the present disclosure involves chemically linking to the oligonucleotide one or more additional non-ligand moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., 1989), cholic acid (Manoharan et al., 1994), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., 1992; Manoharan et al., 1993), a thiocholesterol (Oberhauser et al., 1992), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., 1991; Kabanov et al., 1990; Svinarchuk et al., 1993), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., 1995; Shea et al., 1990), a polyamine or a polyethylene glycol chain (Manoharan et al., 1995), or adamantane acetic acid (Manoharan et al., 1995), a palmityl moiety (Mishra et al., 1995), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., 1996). For some aspects, a nucleic acid molecule encoding an engineered Coronavirus S protein is a modified RNA, such as, for example, a modified mRNA. Modified (m)RNA contemplates certain chemical modifications that confer increased stability and low immunogenicity to mRNAs, thereby facilitating expression of therapeutically important proteins. For instance, N1-methyl- pseudouridine (N1mΨ) outperforms several other nucleoside modifications and their combinations in terms of translation capacity. In some embodiments, the (m)RNA molecules used herein may have the uracils replaced with psuedouracils such as 1-methyl-3′- pseudouridylyl bases. In some embodiments, some of the uracils are replaced, but in other embodiments, all of the uracils have been replaced. The (m)RNA may comprise a 5' cap, a 5' UTR element, an optionally codon optimized open reading frame, a 3' UTR element, and a poly(A) sequence and / or a polyadenylation signal. 494893-1038-0739, v. 1

[0173] The nucleic acid molecule, whether native or modified, may be delivered as a naked nucleic acid molecule or in a delivery vehicle, such as a lipid nanoparticle. A lipid nanoparticle may comprise one or more nucleic acids present in a weight ratio to the lipid nanoparticles from about 5:1 to about 1:100. In some embodiments, the weight ratio of nucleic acid to lipid nanoparticles is from about 5:1, 2.5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100, or any value derivable therein.

[0174] In some embodiments, the lipid nanoparticles used herein may contain one, two, three, four, five, six, seven, eight, nine, or ten lipids. These lipids may include triglycerides, phospholipids, steroids or sterols, PEGylated lipids, or a group with an ionizable group such as an alkyl amine and one or more hydrophobic groups such as C6 or greater alkyl groups.

[0175] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more steroid or a steroid derivative. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term “steroid” is a class of compounds with a four ring 17 carbon cyclic structure which can further comprises one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or a double bond between two or more carbon atoms.

[0176] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more PEGylated lipids (or PEG lipid). In some embodiments, the present disclosure comprises using any lipid to which a PEG group has been attached. In some embodiments, the PEG lipid is a diglyceride which also comprises a PEG chain attached to the glycerol group. In other embodiments, the PEG lipid is a compound which contains one or more C6-C24 long chain alkyl or alkenyl group or a C6-C24 fatty acid group attached to a linker group with a PEG chain. Some non-limiting examples of a PEG lipid includes a PEG modified phosphatidylethanolamine and phosphatidic acid, a PEG ceramide conjugated, PEG modified dialkylamines and PEG modified 1,2-diacyloxypropan-3-amines, PEG modified diacylglycerols and dialkylglycerols. In some embodiments, PEG modified diastearoylphosphatidylethanolamine or PEG modified dimyristoyl-sn-glycerol. In some embodiments, the PEG modification is measured by the molecular weight of PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight from about 504893-1038-0739, v. 1100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000. The molecular weight of the PEG modification is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, to about 15,000. Some non-limiting examples of lipids that may be used in the present disclosure are taught by U.S. Patent 5,820,873, WO 2010 / 141069, or U.S. Patent 8,450,298, which is incorporated herein by reference.

[0177] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more phospholipids. In some embodiments, any lipid which also comprises a phosphate group. In some embodiments, the phospholipid is a structure which contains one or two long chain C6-C24 alkyl or alkenyl groups, a glycerol or a sphingosine, one or two phosphate groups, and, optionally, a small organic molecule. In some embodiments, the small organic molecule is an amino acid, a sugar, or an amino substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipid defines lipid and lipid-like molecules with both a positive charge and a negative charge.

[0178] In some aspects of the present disclosure, lipid nanoparticle containing compounds containing lipophilic and cationic components, wherein the cationic component is ionizable, are provided. In some embodiments, the cationic ionizable lipids contain one or more groups that are protonated at physiological pH but may be deprotonated and have no charge at a pH above 8, 9, 10, 11, or 12. The ionizable cationic group may contain one or more protonatable amines which are able to form a cationic group at physiological pH. The cationic ionizable lipid compound may also further comprise one or more lipid components such as two or more fatty acids with C6-C24 alkyl or alkenyl carbon groups. These lipid groups may be attached through an ester linkage or may be further added through a Michael addition to a sulfur atom. In some embodiments, these compounds may be a dendrimer, a dendron, a polymer, or a combination thereof.

[0179] In some aspects of the present disclosure, composition containing compounds containing lipophilic and cationic components, wherein the cationic component is 514893-1038-0739, v. 1ionizable, are provided. In some embodiments, ionizable cationic lipids refer to lipid and lipid-like molecules with nitrogen atoms that can acquire charge (pKa). These lipids may be known in the literature as cationic lipids. These molecules with amino groups typically have between 2 and 6 hydrophobic chains, often alkyl or alkenyl such as C6-C24 alkyl or alkenyl groups, but may have at least 1 or more than 6 tails.

[0180] In some embodiments, the amount of the lipid nanoparticle with the nucleic acid molecule encapsulated in the pharmaceutical composition is from about 0.1% w / w to about 50% w / w, from about 0.25% w / w to about 25% w / w, from about 0.5% w / w to about 20% w / w, from about 1% w / w to about 15% w / w, from about 2% w / w to about 10% w / w, from about 2% w / w to about 5% w / w, or from about 6% w / w to about 10% w / w. In some embodiments, the amount of the lipid nanoparticle with the nucleic acid molecule encapsulated in the pharmaceutical composition is from about 0.1% w / w, 0.25% w / w, 0.5% w / w, 1% w / w, 2.5% w / w, 5% w / w, 7.5% w / w, 10% w / w, 15% w / w, 20% w / w, 25% w / w, 30% w / w, 35% w / w, 40% w / w, 45% w / w, 50% w / w, 55% w / w, 60% w / w, 65% w / w, 70% w / w, 75% w / w, 80% w / w, 85% w / w, 90% w / w, to about 95% w / w, or any range derivable therein.

[0181] In some aspects, the present disclosure comprises one or more sugars formulated into pharmaceutical compositions. In some embodiments, the sugars used herein are saccharides. These saccharides may be used to act as a lyoprotectant that protects the pharmaceutical composition from destabilization during the drying process. These water- soluble excipients include carbohydrates or saccharides such as disaccharides such as sucrose, trehalose, or lactose, a trisaccharide such as fructose, glucose, galactose comprising raffinose, polysaccharides such as starches or cellulose, or a sugar alcohol such as xylitol, sorbitol, or mannitol. In some embodiments, these excipients are solid at room temperature. Some non-limiting examples of sugar alcohols include erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotritol, maltotetraitol, or a polyglycitol.

[0182] In some embodiments, the amount of the sugar in the pharmaceutical composition is from about 25% w / w to about 98% w / w, 40% w / w to about 95% w / w, 50% w / w to about 90% w / w, 50% w / w to about 70% w / w, or from about 80% w / w to about 90% w / w. In some embodiments, the amount of the sugar in the pharmaceutical composition is from about 10% w / w, 15% w / w, 20% w / w, 25% w / w, 30% w / w, 35% w / w, 40% w / w, 45% 524893-1038-0739, v. 1w / w, 50% w / w, 52.5% w / w, 55% w / w, 57.5% w / w, 60% w / w, 62.5% w / w, 65% w / w, 67.5% w / w, 70% w / w, 75% w / w, 80% w / w, 82.5% w / w, 85% w / w, 87.5% w / w, 90% w / w, to about 95% w / w, or any range derivable therein.

[0183] In some embodiments, the pharmaceutically acceptable polymer is a copolymer. The pharmaceutically acceptable polymer may further comprise one, two, three, four, five, or six subunits of discrete different types of polymer subunits. These polymer subunits may include polyoxypropylene, polyoxyethylene, or a similar subunit. In particular, the pharmaceutically acceptable polymer may comprise at least one hydrophobic subunit and at least one hydrophilic subunit. In particular, the copolymer may have hydrophilic subunits on each side of a hydrophobic unit. The copolymer may have a hydrophilic subunit that is polyoxyethylene and a hydrophobic subunit that is polyoxypropylene.

[0184] In some embodiments, expression cassettes are employed to express an engineered, stabilized beta-coronavirus S2 domain-only protein, either for subsequent purification and delivery to a cell / subject, or for use directly in a viral-based delivery approach. Provided herein are expression vectors which contain one or more nucleic acids encoding an engineered, stabilized beta-coronavirus S2 domain-only protein.

[0185] Expression requires that appropriate signals be provided in the vectors and include various regulatory elements such as enhancers / promoters from both viral and mammalian sources that drive expression of the engineered, stabilized beta-coronavirus S2 domain-only protein in cells. Throughout this application, the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i.e., is under the control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. An “expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites. 534893-1038-0739, v. 1

[0186] The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0187] At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0188] Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0189] In certain embodiments, viral promotes such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well- known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a 544893-1038-0739, v. 1promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.

[0190] Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

[0191] Below is a list of promoters / enhancers and inducible promoters / enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter / enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

[0192] The promoter and / or enhancer may be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and / or DQ β, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, β-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, α-fetoprotein, t-globin, β-globin, c-fos, c-HA- ras, insulin, neural cell adhesion molecule (NCAM), α1-antitrypain, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), SV40, polyoma, retroviruses, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon ape leukemia virus.

[0193] Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. Any 554893-1038-0739, v. 1polyadenylation sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

[0194] There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals.

[0195] One method for in vivo delivery involves the use of an adenovirus expression vector. “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express engineered, stabilized beta-coronavirus S2 domain-only protein that has been cloned therein. In this context, expression does not require that the gene product be synthesized.

[0196] The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double- stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB. In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.

[0197] Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and 564893-1038-0739, v. 1high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5’-tripartite leader (TPL) sequence which makes them preferred mRNAs for translation. In one system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.

[0198] Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses E1 proteins. Since the E3 region is dispensable from the adenovirus genome, the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the E1, the D3 or both regions. In nature, adenovirus can package approximately 105% of the wild-type genome, providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the E1 and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the E1-deleted virus is incomplete.

[0199] Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells 574893-1038-0739, v. 1of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.

[0200] The adenoviruses of the disclosure are replication defective, or at least conditionally replication defective. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is one exemplary starting material that may be used to obtain the conditional replication-defective adenovirus vector for use in the present disclosure.

[0201] Other viral vectors may be employed as expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus, adeno-associated virus (AAV) and herpesviruses may be employed. They offer several attractive features for various mammalian cells.

[0202] In embodiments, particular embodiments, the vector is an AAV vector. AAV is a small virus that infects humans and some other primate species. AAV is not currently known to cause disease. The virus causes a very mild immune response, lending further support to its apparent lack of pathogenicity. In many cases, AAV vectors integrate into the host cell genome, which can be important for certain applications, but can also have unwanted consequences. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell, although in the native virus some integration of virally carried genes into the host genome does occur. These features make AAV a very attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models. Recent human clinical trials using AAV for gene therapy in the retina have shown promise. AAV belongs to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. The virus is a small (20 nm) replication-defective, nonenveloped virus.

[0203] Wild-type AAV has attracted considerable interest from gene therapy researchers due to a number of features. Chief amongst these is the virus's apparent lack of pathogenicity. It can also infect non-dividing cells and has the ability to stably integrate into the host cell genome at a specific site (designated AAVS1) in the human chromosome 19. This feature makes it somewhat more predictable than retroviruses, which present the threat of a random insertion and of mutagenesis, which is sometimes followed by development of a 584893-1038-0739, v. 1cancer. The AAV genome integrates most frequently into the site mentioned, while random incorporations into the genome take place with a negligible frequency. Development of AAVs as gene therapy vectors, however, has eliminated this integrative capacity by removal of the rep and cap from the DNA of the vector. The desired gene together with a promoter to drive transcription of the gene is inserted between the inverted terminal repeats (ITR) that aid in concatemer formation in the nucleus after the single-stranded vector DNA is converted by host cell DNA polymerase complexes into double-stranded DNA. AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact for the life of the host cell. In dividing cells, AAV DNA is lost through cell division, since the episomal DNA is not replicated along with the host cell DNA. Random integration of AAV DNA into the host genome is detectable but occurs at very low frequency. AAVs also present very low immunogenicity, seemingly restricted to generation of neutralizing antibodies, while they induce no clearly defined cytotoxic response. This feature, along with the ability to infect quiescent cells present their dominance over adenoviruses as vectors for human gene therapy.

[0204] The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed, which is about 4.7 kilobase long. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry.

[0205] The Inverted Terminal Repeat (ITR) sequences comprise 145 bases each. They were named so because of their symmetry, which was shown to be required for efficient multiplication of the AAV genome. The feature of these sequences that gives them this property is their ability to form a hairpin, which contributes to so-called self-priming that allows primase-independent synthesis of the second DNA strand. The ITRs were also shown to be required for both integration of the AAV DNA into the host cell genome (19th chromosome in humans) and rescue from it, as well as for efficient encapsidation of the AAV DNA combined with generation of a fully assembled, deoxyribonuclease-resistant AAV particles. 594893-1038-0739, v. 1

[0206] With regard to gene therapy, ITRs seem to be the only sequences required in cis next to the therapeutic gene: structural (cap) and packaging (rep) proteins can be delivered in trans. With this assumption many methods were established for efficient production of recombinant AAV (rAAV) vectors containing a reporter or therapeutic gene. However, it was also published that the ITRs are not the only elements required in cis for the effective replication and encapsidation. A few research groups have identified a sequence designated cis-acting Rep-dependent element (CARE) inside the coding sequence of the rep gene. CARE was shown to augment the replication and encapsidation when present in cis.

[0207] In some aspects, the present disclosure provides pharmaceutical compositions that contain one or more salts. The salts may be an inorganic potassium or sodium salt such as potassium chloride, sodium chloride, potassium phosphate dibasic, potassium phosphate monobasic, sodium phosphate dibasic, or sodium phosphate monobasic. The pharmaceutical composition may comprise one or more phosphate salts, so as to generate a phosphate buffer solution. The phosphate buffer solution may comprise each of the phosphates to buffer a solution to a pH from about 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or any range derivable therein.

[0208] In some aspects, the present disclosure comprises one or more excipients formulated into pharmaceutical compositions. An “excipient” refers to pharmaceutically acceptable carriers that are relatively inert substances used to facilitate administration or delivery of an API into a subject or used to facilitate processing of an API into drug formulations that can be used pharmaceutically for delivery to the site of action in a subject. Furthermore, these compounds may be used as diluents in order to obtain a dosage that can be readily measured or administered to a patient. Non-limiting examples of excipients include polymers, stabilizing agents, surfactants, surface modifiers, solubility enhancers, buffers, encapsulating agents, antioxidants, preservatives, nonionic wetting or clarifying agents, viscosity increasing agents, and absorption-enhancing agents.

[0209] In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and can preferably include an adjuvant. Water is a particular carrier when the 604893-1038-0739, v. 1pharmaceutical composition is administered by injections, such an intramuscular injection. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0210] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.

[0211] Engineered proteins or nucleic acids encoding engineered proteins of the present disclosure, as described herein, can be formulated for parenteral administration, e.g., formulated for injection via the intradermal, intravenous, intramuscular, subcutaneous, intra-tumoral or even intraperitoneal routes. The formulation could alternatively be administered by a topical route directly to the mucosa, for example by nasal drops, inhalation, or by nebulizer. Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0212] Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an 614893-1038-0739, v. 1ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0213] The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0214] Dosage can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or in a booster immunization schedule. In a multiple dose schedule the various doses may be given by the same or different routes. Multiple doses will typically be administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

[0215] The compositions disclosed herein may be used to treat both children and adults. Thus a human subject may be less than 1 year old, 1-5 years old, 5-16 years old, 16-55 years old, 55-65 years old, or at least 65 years old.

[0216] Preferred routes of administration include, but are not limited to, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, and intraoccular injection. Particularly preferred routes of administration include intramuscular, intradermal and subcutaneous injection. IV. Immunogenic Compositions

[0217] Immunogenic compositions comprising a disclosed recombinant insect ferritin nanoparticle and a pharmaceutically acceptable carrier are also provided. Such compositions can be administered to a subject by a variety of modes, for example, by an intranasal route. Standard methods for preparing administrable immunogenic compositions are described, for example, in such publications as Remingtons Pharmaceutical Sciences, 19th Ed., Mack Publishing Company, Easton, Pa., 1995. 624893-1038-0739, v. 1

[0218] The immunogenic compositions can be formulated for administration to a subject by a variety of administration modes, including mucosal administration modes such as by oral, rectal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to other surfaces, and non-mucosal routes, including by intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes.

[0219] Potential carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffer saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents. The resulting aqueous solutions may be packaged for use as is or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple dosing.

[0220] The immunogenic composition can contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually 1% w / v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben. A bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component.

[0221] The immunogenic composition can contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

[0222] In certain embodiments, the recombinant insect ferritin nanoparticles can be administered in a time-release formulation, for example in an immunogenic composition that includes a slow release polymer. These compositions can be prepared with vehicles that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Prolonged delivery in various compositions of the disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminum monostearate hydrogels and gelatin. 634893-1038-0739, v. 1When controlled release formulations are desired, controlled release binders suitable for use in accordance with the disclosure include any biocompatible controlled release material which is inert to the active agent and which is capable of incorporating the vaccine and / or other biologically active agent. Numerous such materials are known in the art. Useful controlled-release binders are materials that are metabolized slowly under physiological conditions following their delivery (for example, at a mucosal surface, or in the presence of bodily fluids). Appropriate binders include, but are not limited to, biocompatible polymers and copolymers well known in the art for use in sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues, and do not trigger significant adverse side effects, such as nasal irritation, immune response, inflammation, or the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body.

[0223] The immunogenic composition may optionally include an adjuvant to enhance the immune response of the host. Suitable adjuvants are, for example, toll-like receptor agonists, alum, AlPO4, alhydrogel, Lipid-A and derivatives or variants thereof, oil- emulsions, saponins, neutral liposomes, liposomes containing the recombinant virus, and cytokines, non-ionic block copolymers, and chemokines. Non-ionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), among many other suitable adjuvants well known in the art, may be used as an adjuvant (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants have the advantage in that they help to stimulate the immune system in a non-specific way, thus enhancing the immune response to a pharmaceutical product.

[0224] In some embodiments, the immunogenic composition can be provided in unit dosage form for use to induce an immune response in a subject, for example, to prevent or inhibit SARS-CoV-2 infection in the subject. A unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or more preselected unit dosages, and / or a metering mechanism for administering the unit dose or multiples thereof.

[0225] The immunogenic composition typically contains an effective amount of disclosed recombinant insect ferritin nanoparticles, and can be prepared by conventional 644893-1038-0739, v. 1techniques. Preparation of immunogenic compositions, including those for administration to human subjects, is generally described in Pharmaceutical Biotechnology, Vol. 61 Vaccine Design—the subunit and adjuvant approach, edited by Powell and Newman, Plenum Press, 1995. New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., U.S.A. 1978. Typically, the amount of antigen in each dose of the immunogenic composition is selected as an amount which induces an immune response without significant adverse side effects.

[0226] The amount of the disclosed recombinant insect ferritin nanoparticles included in the therapeutic composition can vary depending upon the specific antigen employed, the route and protocol of administration, and the target population, for example. For protein therapeutics, typically, each human dose will comprise 1-1000 μg of protein, such as from about 1 μg to about 100 μg, for example, from about 1 μg to about 50 μg, such as about 1 μg, about 2 μg, about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 40 μg, or about 50 μg. The amount utilized in an immunogenic composition is selected based on the subject population (e.g., infant or elderly). An optimal amount for a particular composition can be ascertained by standard studies involving observation of antibody titers and other responses in subjects. It is understood that a therapeutically effective amount of a disclosed recombinant insect ferritin nanoparticle comprises an amount that is ineffective at eliciting an immune response by administration of a single dose, but that is effective upon administration of multiple dosages, for example in a prime-boost administration protocol. V. Methods of Eliciting an Immune Response

[0227] The disclosed recombinant insect ferritin nanoparticles can be administered to a subject to elicit an immune response to the trimeric antigens included on the recombinant insect ferritin nanoparticle in the subject. Upon immunization, the subject responds by producing antibodies specific for the two different trimeric antigens on the recombinant insect ferritin nanoparticle. In addition, innate and cell-mediated immune responses are induced, which can provide antiviral effectors as well as regulating the immune response. The immune response can be a protective immune response, for example a response that prevents or reduces subsequent infection with a virus including the trimeric antigen. The immune response can be a therapeutic immune response, for example a response 654893-1038-0739, v. 1that treats or inhibits current infection with a virus including the trimeric antigen and illnesses associated therewith.

[0228] Typical subjects intended for treatment with the compositions and methods of the present disclosure include humans, as well as non-human primates and other animals. To identify subjects for prophylaxis or treatment according to the methods of the disclosure, accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease of condition, or to determine the status of an existing disease or condition in a subject. These screening methods include, for example, conventional work-ups to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods, such as various ELISA and other immunoassay methods. These and other routine methods allow the clinician to select patients in need of therapy using the methods and immunogenic compositions of the disclosure. In accordance with these methods and principles, a recombinant insect ferritin nanoparticle and / or other biologically active agent can be administered according to the teachings herein as an independent prophylaxis or treatment program, or as a follow-up, adjunct or coordinate treatment regimen to other treatments.

[0229] In some embodiments, a subject is selected for treatment that has, or is at risk for developing, a SARS-CoV-2 infection, for example because of exposure or the possibility of exposure to SARS-CoV-2. Such a subject can then be administered an effective amount of a recombinant insect ferritin nanoparticle including trimeric antigens that are trimeric SARS-CoV-2 S2-domain only ectodomains to induce an immune response to SARS- CoV-2 S protein. The immune response can neutralize autologous virus (e.g., the strain for the trimeric antigens on the nanoparticle) or heterologous virus (e.g., strains other than the strain of the trimeric antigen on the nanoparticle). In some such embodiments, the immune response inhibits subsequent SARS-CoV-2 infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by SARS-CoV-2) by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (prevention of detectable SARS-CoV-2 infection), as compared to a suitable control.

[0230] In some embodiments, when used to prevent or treat a SARS-CoV-2 infection, the recombinant insect ferritin nanoparticle comprises trimeric antigens two 664893-1038-0739, v. 1different SARS-CoV-2 S2 domain-only ectodomain trimers, and administration of an effective amount the recombinant insect ferritin nanoparticle to a subject induces an immune response in the subject that neutralizes the relevant virus.

[0231] The immunogenic composition may be administered by any suitable method, including but not limited to, via injection, aerosol delivery, nasal spray, nasal droplets, oral inoculation, or topical application.

[0232] An effective amount of the recombinant insect ferritin nanoparticle and / or other biologically active agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and / or ameliorate a selected disease or condition or one or more symptom(s) thereof.

[0233] The recombinant insect ferritin nanoparticle can be administered to the subject in a single bolus delivery, via continuous delivery (for example, continuous transdermal, mucosal or intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol). The effective amount of the recombinant insect ferritin nanoparticle can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms or detectable conditions associated with a targeted disease or condition as set forth herein.

[0234] The administration of an effective amount of a recombinant insect ferritin nanoparticle of the disclosure can be for either prophylactic or therapeutic purpose. When provided prophylactically, the recombinant insect ferritin nanoparticle is provided in advance of any symptom, for example in advance of infection, such as in the form of a yearly flu shot. The prophylactic administration of the recombinant insect ferritin nanoparticle serves to prevent or ameliorate any subsequent infection. When provided therapeutically, the recombinant insect ferritin nanoparticle is provided at (or shortly after) the onset of a symptom of disease or infection. Thus, when used to prevent or treat a viral infection (such as SARS-CoV-2 infection), the recombinant insect ferritin nanoparticle of the disclosure can be provided prior to the anticipated exposure to virus so as to attenuate the anticipated severity, duration or extent of an infection and / or associated disease symptoms, after exposure or suspected exposure to the virus, or after the actual initiation of an infection. 674893-1038-0739, v. 1

[0235] Determination of effective dosages is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject. Suitable models in this regard include, for example, murine, rat, porcine, feline, ferret, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models (for example, immunologic and histopathologic assays). Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the recombinant insect ferritin nanoparticle (for example, amounts that are effective to elicit a desired immune response or alleviate one or more symptoms of a targeted disease).

[0236] The actual dosage of the recombinant insect ferritin nanoparticle will vary according to factors such as the disease indication and particular status of the subject (for example, the subject's age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the vaccine for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. As described above in the forgoing listing of terms, a therapeutically effective amount is also one in which any toxic or detrimental side effects of the recombinant insect ferritin nanoparticle and / or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects. A non-limiting range for a therapeutically effective amount of a recombinant insect ferritin nanoparticle and / or other biologically active agent within the methods and formulations of the disclosure is about 0.01 mg / kg body weight to about 10 mg / kg body weight, such as about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, or about 10 mg / kg, for example 0.01 mg / kg to about 1 mg / kg body weight, about 0.05 mg / kg to about 5 mg / kg body weight, about 0.2 mg / kg to about 2 mg / kg body weight, or about 1.0 mg / kg to about 10 mg / kg body weight. In some embodiments, the dosage includes a set amount of a disclosed recombinant insect ferritin nanoparticle, such as from about 1-300 μg, for example, a dosage of about 10-300 μg, 684893-1038-0739, v. 1about 60 μg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 μg. As used herein with reference to a concentration or amount, “about” refers to + / −5%. Therefore, “about 100 μg” refers to 95-105 μg.

[0237] Upon administration of an effective amount of a disclosed recombinant insect ferritin nanoparticle (for example, via injection, aerosol, oral, topical or other route), the immune system of the subject typically responds to the recombinant insect ferritin nanoparticle by producing antibodies specific for the trimeric antigens on the recombinant insect ferritin nanoparticle. Such a response signifies that an effective amount of the recombinant insect ferritin nanoparticle was delivered. An effective amount can be achieved by single or multiple administrations (including, for example, multiple administrations per day), daily, or weekly administrations. For each particular subject, specific dosage regimens can be evaluated and adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of the vaccine. In some embodiments, the antibody response of a subject administered the compositions of the disclosure will be determined in the context of evaluating effective dosages / immunization protocols. In most instances it will be sufficient to assess the antibody titer in serum or plasma obtained from the subject. Decisions as to whether to administer booster inoculations and / or to change the amount of the composition administered to the individual can be at least partially based on the antibody titer level. The antibody titer level can be based on, for example, an immunobinding assay which measures the concentration of antibodies in the serum which bind to a specific antigen, for example, SARS-CoV-2 S protein.

[0238] An immunogenic composition including one or more of the disclosed recombinant insect ferritin nanoparticle can be used in coordinate (or prime-boost) vaccination protocols or combinatorial formulations. In certain embodiments, novel combinatorial immunogenic compositions and coordinate immunization protocols employ separate recombinant insect ferritin nanoparticle or formulations, each directed toward eliciting an anti-viral immune response, such as an immune response to SARS-CoV-2 S proteins. Separate immunogenic compositions that elicit the anti-viral immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic compositions) in a coordinate (or prime-boost) immunization protocol. 694893-1038-0739, v. 1

[0239] There can be several boosts, and each boost can be a different disclosed immunogen. In some examples that the boost may be the same immunogen as another boost, or the prime. The prime and boost can be administered as a single dose or multiple doses, for example two doses, three doses, four doses, five doses, six doses or more can be administered to a subject over days, weeks or months. Multiple boosts can also be given, such one to five (e.g., 1, 2, 3, 4 or 5 boosts), or more. Different dosages can be used in a series of sequential immunizations. For example a relatively large dose in a primary immunization and then a boost with relatively smaller doses.

[0240] In some embodiments, the boost can be administered about two, about three to eight, or about four, weeks following the prime, or about several months after the prime. In some embodiments, the boost can be administered about 5, about 6, about 7, about 8, about 10, about 12, about 18, about 24, months after the prime, or more or less time after the prime. Periodic additional boosts can also be used at appropriate time points to enhance the subject's “immune memory.”

[0241] The adequacy of the vaccination parameters chosen, e.g., formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers and / or neutralizing activity during the course of the immunization program. To assess neutralization activity, following immunization of a subject, serum can be collected from the subject at appropriate time points, frozen, and stored for neutralization testing. Methods to assay for neutralization activity are known and are further described herein, and include, but are not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry based assays, single-cycle infection assays. In some embodiments, the serum neutralization activity can be assayed using a panel of pseudoviruses. In addition, the clinical condition of the subject can be monitored for the desired effect. If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of immunogenic composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response. Thus, for example, the dose of the disclosed immunogen can be increased or the route of administration can be changed. 704893-1038-0739, v. 1VI. Immunodetection Methods

[0242] In still further embodiments, the present disclosure concerns immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting Coronavirus S protein. While such methods can be applied in a traditional sense, another use will be in quality control and monitoring of vaccine stocks, where antibodies according to the present disclosure can be used to assess the amount or integrity (i.e., long term stability) of antigens. Alternatively, the methods may be used to screen various antibodies for appropriate / desired reactivity profiles.

[0243] Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In particular, a competitive assay for the detection and quantitation of Coronavirus S protein also is provided. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). In general, the immunobinding methods include obtaining a sample suspected of containing Coronavirus S protein, and contacting the sample with a first antibody in accordance with the present disclosure, as the case may be, under conditions effective to allow the formation of immunocomplexes.

[0244] These methods include methods for detecting or purifying Coronavirus S protein or Coronavirus S protein from a sample. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the Coronavirus S protein will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the Coronavirus S protein-expressing cells immunocomplexed to the immobilized antibody, which is then collected by removing the organism or antigen from the column.

[0245] The immunobinding methods also include methods for detecting and quantifying the amount of Coronavirus S protein or related components in a sample and the detection and quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing Coronavirus S protein and contact the sample with an antibody that binds Coronavirus S protein or components thereof, 714893-1038-0739, v. 1followed by detecting and quantifying the amount of immune complexes formed under the specific conditions. In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing Coronavirus S protein, such as a tissue section or specimen, a homogenized tissue extract, a biological fluid (e.g., a nasal swab), including blood and serum, or a secretion, such as feces or urine.

[0246] Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to Coronavirus S protein. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

[0247] In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and / or a biotin / avidin ligand binding arrangement, as is known in the art.

[0248] The antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then 724893-1038-0739, v. 1generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0249] Further methods include the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.

[0250] One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.

[0251] Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA / biotin / streptavidin / antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous 734893-1038-0739, v. 1amplification capability and specificity of PCR can be utilized to detect a single antigen molecule. A. ELISAs

[0252] Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.

[0253] In one exemplary ELISA, the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the Coronavirus S protein is added to the wells. After binding and washing to remove non- specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti-Coronavirus S protein antibody that is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection may also be achieved by the addition of a second anti-Coronavirus S protein antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[0254] In another exemplary ELISA, the samples suspected of containing the Coronavirus S protein (e.g., potentially infected cells) are immobilized onto the well surface and then contacted with the anti- Coronavirus S protein antibodies of the disclosure. After binding and washing to remove non-specifically bound immune complexes, the bound anti- Coronavirus S protein antibodies are detected. Where the initial anti-Coronavirus S protein antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-Coronavirus S protein antibody, with the second antibody being linked to a detectable label.

[0255] Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below. 744893-1038-0739, v. 1

[0256] In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[0257] In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.

[0258] “Under conditions effective to allow immune complex (antigen / antibody) formation” means that the conditions preferably include diluting the antigens and / or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS) / Tween. These added agents also tend to assist in the reduction of nonspecific background.

[0259] The “suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so.

[0260] Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS / Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined. 754893-1038-0739, v. 1

[0261] To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

[0262] After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer. B. Western Blot

[0263] The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native / non- denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

[0264] Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.

[0265] The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a 764893-1038-0739, v. 1combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.

[0266] In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e., binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies. C. Immunohistochemistry

[0267] The antibodies of the present disclosure may also be used in conjunction with both fresh-frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990). 774893-1038-0739, v. 1

[0268] Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen “pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and / or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and / or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and / or cutting 25-50 serial sections from the capsule. Alternatively, whole frozen tissue samples may be used for serial section cuttings.

[0269] Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing / pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue / agar block from the tube; infiltrating and / or embedding the block in paraffin; and / or cutting up to 50 serial permanent sections. Again, whole tissue samples may be substituted. D. Immunodetection Kits

[0270] In still further embodiments, the present disclosure concerns immunodetection kits for use with the immunodetection methods described above. As the antibodies may be used to detect Coronavirus S protein, the antibodies may be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to an Coronavirus S protein, and optionally an immunodetection reagent.

[0271] In certain embodiments, the antibody may be pre-bound to a solid support, such as a column matrix and / or well of a microtitre plate. The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

[0272] Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a 784893-1038-0739, v. 1number of exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure.

[0273] The kits may further comprise a suitably aliquoted composition of Coronavirus S protein, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.

[0274] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow- molded plastic containers into which the desired vials are retained. E. Flow Cytometry and FACS

[0275] The antibodies of the present disclosure may also be used in flow cytometry or FACS. Flow cytometry is a laser- or impedance-based technology employed in many detection assays, including cell counting, cell sorting, biomarker detection and protein engineering. The technology suspends cells in a stream of fluid and passing them through an electronic detection apparatus, which allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis disorders, especially blood cancers, but has many other applications in basic research, clinical practice and clinical trials.

[0276] Fluorescence-activated cell sorting (FACS) is a specialized type of cytometry. It provides a method for sorting a heterogenous mixture of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescent characteristics of each cell. In general, the technology involves a cell suspension entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the 794893-1038-0739, v. 1fluorescence of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based immediately prior to fluorescence intensity being measured, and the opposite charge is trapped on the droplet as it breaks form the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge.

[0277] In certain embodiments, to be used in flow cytometry or FACS, the antibodies of the present disclosure are labeled with fluorophores and then allowed to bind to the cells of interest, which are analyzed in a flow cytometer or sorted by a FACS machine. VII. Examples

[0278] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1

[0279] To increase the stability of S2 subunit trimers in the prefusion conformation, two mutations were engineered into the central helices of a SARS-CoV-2 S2 antigen having background substitutions of S704C, K790C, F817P, A892P, A899P, A942P, Q957E, K986P, and V987P (with positions relative to SEQ ID NO: 1) as follows: V991W and T998W. These mutations stabilize S2 in the closed conformation, producing an S2 construct with superior thermal stability and increased protein expression relative to all current SARS-CoV-2 spike constructs.

[0280] The background substitutions come from the introduction of two consecutive prolines within the HR1 / CH hinge point (K986P and V987P) in the S2 subunit of the engineered SARS-CoV-2 ‘S-2P’ spike because these substitution may be a successful strategy to prevent progression to the postfusion state (Wrapp, D. et al 2020; Pallesen, J. et al 2017). The background substitutions also include substitutions that may further stabilize the 804893-1038-0739, v. 1prefusion conformation, along with improved cellular expression, with the incorporation of four additional prolines in the S2 subunit (F817P, A892P, A899P, A942P), resulting in the SARS-CoV-2 ‘HexaPro’ spike (Hsieh, C.-L. et al 2020). The three S1 subunits may sit atop and seal together the HexaPro S2 subunits nestled underneath. Interestingly, previous HDX- MS experiments showed that the SARS-CoV-2 HexaPro spike can also sample an open conformation where the S2 trimer interfaces become solvent accessible (Costello, S. M. et al 2022). This may suggest that, in the absence of S1, the HexaPro S2-only trimer would most likely dissociate into individual monomers. To stabilize the HexaPro S2-only trimer in the prefusion conformation, the background substitutions further comprised the introduction of an interprotomer disulfide bond via two cysteine substitutions (S704C and K790C) and an interprotomer salt bridge with R765 upon Q957E substitution (FIG. 9) (Hsieh, C.-L. et al 2024). However, the apex of the resulting construct (without the stalk), termed ‘HexaPro-SS- Δstalk’, was shown to splay apart, adopting semi-open and open conformations (Hsieh, C. -L. et al 2024). The pronounced conformational plasticity exhibited by the HexaPro-SS-Δstalk S2 prefusion trimer underscores the potential for improving its stability by inducing a shift in the equilibrium towards a closed conformation. FIG. 9 shows the model of the HexaPro-SS- Δstalk S2-only trimer in the closed prefusion conformation with a cartoon representation where the protomers are highlighted with different shades. The spike’s S1 subunit, shown in FIG.9 with a gray transparent surface only to provide context, is not present in this construct. N-glycans linked to S2 are shown as dark gray sticks in FIG. 9. Glycans linked to S1 are omitted for clarity in FIG. 9. HexaPro-SS-Δstalk is an S2-only construct derived from the prefusion-stabilized SARS-CoV-2 HexaPro spike (Hsieh, C. -L. et al 2024). The substitutions (S704C, K790C, Q957E) incorporated in HexaPro-SS-Δstalk are highlighted in the right panels of FIG. 9. They may stabilize S2 in a prefusion trimer assembly, which may prevent protomer dissociation via formation of interprotomer salt bridges and disulfide bonds.

[0281] Prefusion-stabilized S2 asymmetrically transitions to a splayed open conformation. To probe the stability of the prefusion-stabilized HexaPro-SS-Δstalk S2-only base construct, the conformational plasticity of the closed conformation within the nanosecond timescale was characterized by performing all-atom conventional MD simulations. A model of HexaPro-SS-Δstalk in the closed conformation was built from the S2 domain of the cryo-EM structure of the SARS-CoV-2 HexaPro spike (PDB ID: 6XKL32) integrated with the fusion peptide’s residue coordinates grafted from the SARS-CoV-23Q-2P spike cryo-EM structure (PDB ID: 7JJI42). The distinctive substitutions engineered in 814893-1038-0739, v. 1HexaPro-SS-Δstalk33 (i.e., S704C, K790C, Q957E) were then incorporated using Visual Molecular Dynamics (VMD) (Humphrey, W. et al 1996), and S2’s N-linked glycans were added as described by Casalino et al. The final system described in FIG. 9, comprising ~274,000 atoms with explicit water molecules and ions, was subjected to four, ~600 ns-long MD simulation replicates (Table 3). In these simulations, one protomer’s apex may have been transiently swinging away from the other two protomers (FIG. 11); however, the trimer did not adopt an open conformation where the three protomers are splayed apart. Panel A of FIG. 11 shows a Top-down view of SARS-CoV-2 spike HexaPro-SS-∆stalk in the open prefusion conformation and select conformations from all-atom conventional MD and GaMD simulations. The open crystal structure is shown in gray, with the central helices highlighted as fully opaque. The conformations from all-atom MD and GaMD simulations were selected from the frame with the greatest distance between P987Cα-P987Cα at the top of the central helices, indicating a departure from the closed state en route to a partially open conformation. Panel B of FIG. 11 shows a comparative analysis of Root Mean Square Deviation (RMSD) values for three different MD simulation conditions: GaMD (E1), GaMD (E2), and all-atom conventional MD (Unbiased MD). The RMSD values, measured in Angstroms (Å), provide insights into the structural differences in the trajectories over simulation steps. Notably, the slight detachment of one protomer from the others was observed only once throughout a combined sampling of 2.6 µs, suggesting that trimer opening may be a rare (or infrequent) event when investigated by means of conventional MD. Thus, to elucidate the complete transition of HexaPro-SS-Δstalk from a closed conformation to an open conformation, an enhanced sampling method may have been necessary.

[0282] Exploration of a system’s conformational space, such as the one conducted with conventional MD, may be limited by high free energy barriers that separate distinct minima in which a system may remain trapped. As large conformational transitions occurring in the millisecond-to-second timescale are computationally prohibitive via conventional MD, enhanced sampling methods may be employed instead (Zuckerman, D. M. et al 2017; Bernardi, R. C. et al 2015; Wang, J. et al 2021). Therefore, it was first attempted to inquire into the conformational transition of HexaPro-SS-Δstalk from the closed to the open conformation using Gaussian accelerated MD (GaMD). GaMD is a method that applies a harmonic boost potential to smooth energy barriers on the potential energy surface, expanding the accessibility of long-timescale dynamic motions (Wang, J. et al 2021). However, the applied boost potential at the highest acceleration level may have been 824893-1038-0739, v. 1insufficient to induce sampling of open conformations (Table 3, FIG. 11, and Table 4). The opening of the HexaPro-SS-Δstalk S2 trimer was investigated using the WE path-sampling method (Zuckerman, D. M. et al 2017; Huber, G. A. et al 1996). By running many short (0.1 ns) stochastic MD simulations in parallel, and iteratively replicating trajectories that have explored new regions of configurational space defined by appropriately binned progress coordinates, the WE strategy facilitates atomically detailed sampling of large conformational changes without alteration of the system’s free energy (Zhang, B. W. et al 2010).

[0283] To enhance sampling of open conformations of the initially closed HexaPro-SS-Δstalk construct, two progress coordinates were defined (FIG. 13a,b): i) the area of the triangle formed by the Cα atoms of the three P987 residues (one for each protomer) residing at the apex of the CHs, and ii) the RMSD of the CHs with respect to the CHs as in the open conformation resolved in the crystal structure (PDB ID: 8U1G33). WE simulations of HexaPro-SS-Δstalk were performed with WESTPA 2.049,50 and Amber20,51 generating 180 continuous opening pathways across 45 µs of aggregate sampling time (Table 3, FIG.14, and Table 5). The weighted ensemble (WE) methodology is an enhanced sampling strategy for studying rare events in complex biological systems (Greaney, A. J. et al 2021). WE strategy involves running numerous MD simulations in parallel generating as many trajectories, also called ‘walkers,” of length ^, each one carrying a statistically determined weight. At regular time intervals, ^, a resampling process is implemented to enhance the abundance of ‘promising’ trajectories that have made progress toward the target state. Promising trajectories are determined based on one (or more) progress coordinates chosen between the starting and desired states (such as closed and open), which are divided into bins. A cycle of MD simulation and resampling defines a WE ‘iteration.’ It is important to note that the WE strategy relies on stochastic dynamics (Greaney, A. J. et al 2021). Therefore, the use of a stochastic thermostat to couple MD simulations with, such as the Langevin thermostat, where pseudo-random values are generated using a random seed based on the current date and time to re-initialize velocities before each simulation (i.e., walker), is suitable (Greaney, A. J. et al 2021). Moreover, trajectory weights are adjusted using rigorous statistical principles to ensure that no bias is introduced into the system dynamics. This implied that all the initial trajectories carry equal statistical weights, whose sum is equal to one. Then, all the trajectories are closely monitored to ensure that the sum of weights remains one throughout the simulation, thereby maintaining unbiased dynamics (Dacon, C. et al 2023). At each resampling step, trajectories that move to empty bins are replicated, and their 834893-1038-0739, v. 1weights are evenly distributed among the resulting offspring trajectories. Trajectories that fail to make progress are occasionally terminated, and their weights are combined with other trajectories that will be continued. This strategy allows for the computation of rate constants for reaching any desired state, provided that enough sampling has been carried out. WE simulations can be performed under non-equilibrium steady-state or equilibrium conditions (Halfmann, P. J. et al 2022; Pinto, D. et al 2021). At odds with non-equilibrium steady-state WE simulations, equilibrium WE simulations do not require the definition of a target state in advance. Moreover, in non-equilibrium steady-state WE simulations trajectories reaching the target state are ‘recycled’ back to the initial state with the same weights to ensure that non- equilibrium steady-state conditions are maintained (Halfmann, P. J. et al 2022; Pinto, D. et al 2021). Equilibrium WE simulations may have been performed to investigate the opening mechanism of the HexaPro-SS-∆stalk construct. Table 3: Summary of the systems that were simulated in this work. List of abbreviations: conventional MD simulations (cMD), Gaussian accelerated MD simulations (GaMD), weighted ensemble MD simulations (WE), alchemical non-equilibrium free energy calculations (FEC). Total a Salt Aggregate Method SystemBox dimensionstoms ÅÅ ÅConcentration Sampling Time844893-1038-0739, v. 1Unfolded 50 Å x 50 Å x (G-T-G)50 Å5,946 0.15 0.60a oos poe a sa s cs o s ua os pe o e a ow a g acceleration (E=2). Boost Boost Parameter Low Acceleration High Acceleration σ0P(kcal / mol) 175.0 350.085 4893-1038-0739, v.1rof g) sni loeta etfelor f t )n .s s s sm n n n n edw uolm / glerpmoroer nve.e.8.0.1.0.ll acofcgk(gaoc eodi rt g s 4 5 4 5± ± ± ± ± - ni s leota arnin1 5 7 9l ngat du nae.7.9.8.4boi at tniardlot d,er )inautDepsom(31316151eehtiof5q±T2.s En oi .er tsngsy)rn WtatGIFyeavenia n web 089478591oi udf ge htm1 1 1 117mehort efhtidmas 1clga dnn metisuoto ,sut pn soitn )l116243icrabrtstceerroal go.u Goitni 0.0.0± ilixenapu e) tssnerc)s mi Δ s Δatudlm / ofla- ± ± c6 1 8k7 9 1. qg-o hty EM(.2-.8-21nnio nc iawd(- nepetdo ar s sW6n ngonT 82 9 4 3n k a( eoit itiraludP) Fsm20009241E 1h-tsalu u(.0.0.0.0mdeM W m oclisvrmnirfac E es ]g 4 5 3 3orfst dybnit01010101dnaettset gW alnalre ro uneof s gnihg )x) x) x) x - ) eyaniwep e1 5 5 1 3w- uoccl emihtko e s(.1.0.0.2cletacerrft a t± ± ± ± acsar)l: smla p s 5go.1.4.0.p4 ( 1 ( 3 ( 7 (eit tihm(uiwntgni ar erl i [mbn ilatiotep ] 4 4 2 3o n0101010uqotiTu,s foit 1x x x x eltf eqe-n o b noir gaelgae ninalu1 ))6)5)0)8ssotnal bep- .m0.1. .ko miss( 3 0vrem sborafPlu tacmu l isnl l± u6± .7± ± .6.3.o,ifsraf[ 1 ( 3 ( 9 ( 2 (omeiFme E tWotytrsnhcaysaela,mso nkl Wo1W iats9 89Wmdr meutMmf to )sal tn Δ9- V92-T-1-SL.v, Sal,:ugrfµu (maS.ir S-SSSSS- orAT935n 7emi it dhet gisn a a Vor- o - rorPaO0T- 8ls galniblE ePaP P30h ieupWmxe a axe 1- acTaewcelme eracashthtHxexeH39H H84 5

[0284] The wealth of atomic-level information provided by these simulations allowed for characterizing the mechanism by which the HexaPro-SS-Δstalk S2-only immunogen adopts a splayed open conformation. Starting from a closed conformation (FIG. 13c), the initial detachment of chain-A leads to an asymmetric, partially open conformation (FIG. 13d) until chain-B and chain-C separate to form the open conformation (FIG. 13e). Notably, asymmetric breathing was also recently reported for influenza HA (Casalino, L. et al 2022). In the open conformation, the distances between the P987 residues increase from values close to ~20 Å (as in the closed conformation) to values in the range of ~35 to ~40 Å. Remarkably, the open conformation obtained through WE simulations closely aligns with the open conformation resolved using X-ray crystallography (FIG. 16) (Hsieh, C. -L. et al 2024). Interestingly, further open conformations where at least one of the trimer’s protomers kept moving further away (FIG. 17). The opening pathways were used for analyzing the contacts at the interfacial helices in the closed, partially open, and open conformations (FIG. 21a-c, FIG. 18, FIG. 19, and FIG. 20). Using PyContact52, interprotomer residue-residue interactions involving the CHs (residues 978-1030), upper helices (UHs, residues 738-783), and HR1 helices (residues 945-977) were examined. S2 trimer opening occurs upon a progressive breakage of interprotomer residue-residue interactions within the CHs from apex to base, following a trend from hydrophilic to hydrophobic (FIG.21c). A salt bridge between R995 and D994 breaks before the hydrogen bond network consisting of Q1002, Q1005, T1006, and T1009 is broken until the loss of the hydrophobic interactions between L1012 and I1013 leads to the separation of chain-A. The helices in chain-A did not form strong interactions in the partially open conformations, and interactions throughout the trimer were almost null in the open conformation (FIG. 21a-c, FIG. 18, FIG. 19, and FIG. 20). The partially open conformations are characterized by CHChain-B forming an extensive interaction interface with the polar residues along UHChain-C (FIG. 21a-c, FIG. 18, FIG. 19, and FIG. 20). Interestingly, residues V991 and T998 in the CHs did not contribute to stabilizing interprotomer contacts in either the closed or partially open conformations (FIG. 21a-c). On the contrary, residues D994, R995, and Q1002, which are one or two helical turns away from V991 and T998, form stabilizing interprotomer interactions in both the closed and partially open conformations (FIG.21a-c).

[0285] The opening mechanism of HexaPro-SS-Δstalk underscored two residues located in the proximity of the CH apex, V991 and T998, as best candidates for cavity-filling substitutions aimed at improving the stability of the closed conformation. 87 4893-1038-0739, v.1Cavity-filling is a common strategy utilized to stabilize proteins by introducing hydrophobic side chains to occupy hollow regions within the protein (Eriksson, A. E. et al 1992; McLellan, J. S. et al 2013). It was hypothesized that introducing a bulky hydrophobic sidechain at positions 991 and 998, such as in tryptophan, may contribute to forming novel, interprotomer, stabilizing interactions, which, in turn, could slow down opening or fully stabilize S2 trimer in the closed prefusion conformation.

[0286] Tryptophan cavity-filling substitutions slow down prefusion-stabilized S2 trimer opening. Aiming to stabilize the S2 trimer in the closed prefusion conformation, three HexaPro-SS-Δstalk tryptophan variants were designed. Three engineered models in the closed conformation were generated from the base construct: i) HexaPro-SS-V991W, incorporating V991W substitution; ii) HexaPro-SS-T998W, including T998W substitution; iii) a double substitution variant, named HexaPro-SS-2W, containing both V991W and T998W substitutions. In a similar way to the base construct, WE simulations were performed for the three HexaPro-SS-Δstalk tryptophan variants to interrogate their stability and, possibly, opening mechanism. Interestingly, each engineered S2 trimer swung away from the initial closed conformation to sample partially open and open conformations. There were 149, 187 and 195 opening pathways across 35, 47, and 56 µs of aggregate sampling for HexaPro- SS-V991W, HexaPro-SS-T998W, and HexaPro-SS-2W, respectively (FIG. 10, FIG. 12, FIG. 15, FIG. 22, and FIG. 23, and Tables 3,5). The closed-to-open transition is consistent across all four simulated S2 constructs, with chain-chain separation proceeding stepwise. Although opening occurred, it is important to note that WE is a path-sampling approach used to enhance the sampling efficiency of conformational transitions that are rarely observed by means of conventional MD (Zuckerman, D. M. et al 2017). That is, sampling an open conformation does not itself imply that the closed conformation is unstable; rather, the WE method allowed for the observation of a slow dynamic behavior that is physically accessible to the protein. The WE simulations indicate that trimer opening is somehow affected by the introduction of the V991W and T998W substitutions. There are differences in the aggregate sampling time to observe the first opening event during WE simulations (i.e., including the time spent in the closed state) (FIG. 24a), with HexaPro-SS-T998W and HexaPro-SS-2W requiring an additional ~6.3 µs and ~4.9 µs of sampling, respectively, and a corresponding shift toward overall longer molecular times to opening (FIG. 24b). This may also suggest more time spent in the closed state. In contrast, HexaPro-SS-V991W necessitates less sampling to open for the first time, approximately ~1.5 µs less than the base construct. 88 4893-1038-0739, v.1Analysis of opening event durations as a measure of the molecular time required by the system to transition from the closed state to the open state (i.e., excluding the time spent within the closed state), shows a distribution slightly shifted toward longer durations for the three tryptophan variants (FIG. 25), with HexaPro-SS-T998W exhibiting the longer mean duration followed by HexaPro-SS-2W (Table 5). In this regard, no significant differences are observed for HexaPro-SS-V991W with respect to the base construct, suggesting that V991W might not affect the opening kinetics. One advantage of WE simulations is the possibility of estimating kinetics quantities such as rate constants underlying the investigated transition (Russo, J. D. et al 2022; Suarez, E. et al 2014; Saglam, A. S. et al 2019; Bhatt, D. et al 2010). In this case, by iteratively reweighting the WE trajectories using the WE Equilibrium Dynamics (WEED) (Suarez, E. et al 2014; Bhatt, D. et al 2010) protocol within WESTPA 2.049 to facilitate the convergence of WE simulations toward near equilibrium steady-state conditions (Suarez, E. et al 2014; Saglam, A. S. et al 2019), it was possible to estimate the rate constant of the opening transition (k-opening) for each simulated system (FIG. 26 and Table 5). Similar to the HexaPro-SS-∆stalk base construct, WE MD simulations were performed for HexaPro-SS-V991W, HexaPro-SS-T998W, and HexaPro-SS-2W systems on the TSCC’s RTX 3090 GPU nodes using the WESTPA 2.0 software Pallesen, J. et al 2017) in combination with Amber20 PMEMD.cuda MD engine (Zhou, P. et al 2022; Claireaux, M. et al 2022) to propagate dynamics, CHARMM36m force fields (Wrapp, D. et al 2020; Walls, A. C. et al 2020; Casalino, L. et al 2020) for protein and glycan, and TIP3P (Dacon, C. et al 2022) for water molecules. WE simulations for the three mutants were started from 50 basis states in the closed conformation, each carrying an initial statistical weight of 0.02. WE simulations were carried out using the same progress coordinates and settings detailed for the HexaPro-SS-∆stalk base construct in the WE simulations of HexaPro-SS-∆stalk construct. WE simulations of the HexaPro-SS-V991W mutant were initially carried out for 200 iterations resulting in ~23.5 µs aggregate sampling and 100 successful pathways. To provide a more accurate estimate of kinetic rates, further extended WE simulations were conducted by 101 iterations, achieving a total of 301 iterations, ~35.7 µs aggregate sampling, and 149 successful opening pathways (FIG. 23 and Table 5). At the end of iterations 201 and 261 the trajectories were reweighted using WEED plugin of WESTPA 2.0 to improve convergence toward equilibrium (Bangaru, S. et al 2019; Ozorowski, G. et al 2017). For this purpose, the same settings detailed for the HexaPro-SS-∆stalk base construct were used. WE simulation of the HexaPro-SS-T998W mutant was performed for a first set of 254 iterations resulting in 89 4893-1038-0739, v.1~32.5 µs aggregate sampling and 155 successful opening pathways. Then, WE simulations were continued for additional 101 iterations, reaching a total of 355 iterations, ~47.7 µs aggregate sampling, and 187 successful opening pathways (FIG. 23 and Table 5). Trajectories were reweighted at the end of iterations 255 and 315 using WEED plugin of WESTPA 2.0 (Bangaru, S. et al 2019; Ozorowski, G. et al 2017). For this purpose, the same settings detailed above for the HexaPro-SS-∆stalk base construct were used. WE simulation of the double HexaPro-SS-2W mutant was initially executed for 269 iterations resulting in ~35.4 µs aggregate sampling and 136 successful opening pathways. WE simulation was further extended for additional 132 iterations, reaching a total of 401 iterations, ~56.6 µs aggregate sampling, and 195 successful opening pathways (FIG. 23 and Table 5). Reweighted of trajectories was conducted twice, namely at the end of iterations 269 and 330 using WEED plugin of WESTPA 2.0 (Bangaru, S. et al 2019; Ozorowski, G. et al 2017). For this purpose, the same settings detailed for the HexaPro-SS-∆stalk base construct were used.

[0287] The opening of the HexaPro-SS-Δstalk S2 trimer was estimated to occur’;;; on the order of fractions of milliseconds [kopening = (4.5 ± 1.5) × 104 s-1]. Although the rate constant does not significantly change for HexaPro-SS-V991W [kopening = (1.1 ± 0.5) × 105 s-1], it becomes smaller for HexaPro-SS-2W [kopening = (7.0 ± 2.3) × 103 s-1] and HexaPro-SS-T998W [kopening = (3.4 ± 0.1) × 103 s-1], indicating that T998W induces a roughly 10-fold slower trimer opening. V991W alone does not appear to affect opening kinetics. The calculated values align well with each other and single-molecule fluorescence resonance energy transfer (smFRET) studies reporting similar domain movements of class I fusion glycoproteins occurring in the millisecond-to-second time scales (Lu et al., 2020; Das et al., 2018; Lu et al., 2019).

[0288] As previously performed for the base construct (FIG. 21d), the interprotomer residue-residue interactions for the tryptophan variants were examined to gain a mechanistic understanding of the impact of V991W and T998W on the opening pathways. As highlighted from the contact heatmaps in FIG. 21e and FIGS. 18-20, both V991W and T998W substitutions boost interprotomer interactions in the closed and partially open conformations with respect to the base construct. These interactions promote engagement at the respective substituted positions and the nearby residues located a few helical turns away (FIG. 21e). The increased interhelix interplay observed in the partially open conformations 90 4893-1038-0739, v.1may underlie the longer opening durations observed in HexaPro-SS-T998W and HexaPro- SS-2W.

[0289] V991W and T998W substitutions stabilize S2 in a closed prefusion conformation. Beyond characterizing the trimer opening kinetics for the tryptophan variants, whether V991W and T998W substitutions energetically stabilize HexaPro-SS-Δstalk in the closed prefusion conformation was investigated. Computationally, this can be probed through “alchemical” non-equilibrium free energy calculations where an amino acid (i.e., V or T) is gradually morphed into another one (i.e., W) in a set of non-equilibrium MD simulations, both in the context of the folded and unfolded protein (Seeliger, D. et al 2010).

[0290] Alchemical non-equilibrium free energy calculations - System setup. To build the thermodynamic cycle for free energy calculations, two independent systems representing the folded and unfolded states of the protein were generated. For the unfolded state, a capped tripeptide was used (GXG), where X is the amino acid of interest (either V, T, or W). The wild-type and the two mutated tripeptides were built with Chimera (Sztain, T. et al 2021). For the wild-type folded state system, the same equilibrated HexaPro-SS-∆stalk construct extracted from conventional MD was used. For the constructs used in these calculations glycans were not considered; however, it was noted that the region of the S2 trimer surrounding the central helices does not bear glycans as the surface is buried beneath S1. Mutant systems for the folded state (HexaPro-SS-V991W, HexaPro-SS-T998W, HexaPro-SS-2W) were constructed from the wild-type system using the psfgen 2.0 structure- building tool within VMD via the mutate command (Pormohammad, A. et al 2020). For each variant, mutations were initially incorporated into one protomer only (protomer A), generating the respective A-mutated construct. Next, starting from this construct, a second system was built by introducing the respective mutation also in the second protomer (protomer B). Finally, a third construct incorporating the respective mutation in all three protomers (A, B, and C) was created. At each step, the mutated residues, as well as the residues located within 5 Å of them, were locally minimized for 1000 steps using the AutoIMD tool (Gilman, M. S. A. et al 2019) within VMD (Gilman, M. S. A. et al 2019). The PMX (Huber, G. A. et al 1996) software was used to build hybrid structures and topologies incorporating side chains from both the wild-type and mutant proteins. Generated structures of the solute were then solvated in a cubic TIP3P (Dacon, C. et al 2022) water box using Gromacs 2022.1 software (Zhang, B. W. et al 2010; Russo, J. D. et al 2022), imposing at 91 4893-1038-0739, v.1least 12 Å from the solute edges in each direction. Na+and Cl+ions were added at a concentration of 150 mM to neutralize the systems. Energy minimization of the generated systems was carried out using the steepest descent algorithm in Gromacs 2022.1 software (Zhang, B. W. et al 2010; Russo, J. D. et al 2022).

[0291] Alchemical non-equilibrium free energy calculations – Equilibrium molecular dynamics simulations. Generated systems were initially equilibrated in the isothermal-isobaric ensemble (NPT) for 100 ps with harmonic position restraints applied to all protein heavy atoms. Langevin dynamics (Bogetti, A. T. et al 2022; Salomon-Ferrer, R. et al 2013) was used to couple the temperature at 298.15 K, whereas Berendsen barostat (Scheurer, M. et al 2018) was used to couple the pressure at a target pressure of 1 bar. The particle mesh Ewald algorithm (Starr, T. N. et al 2021) was employed to handle the long- range electrostatic interactions using a real space cut-off of 1 nm, a Fourier spacing of 12 Å, and a spline order of 4. The Verlet cutoff scheme was used in the neighbor list search with a cutoff of 10 Å. The P-LINCS algorithm (Eriksson, A. E. et al 1992) was utilized to constrain all bonds involving hydrogen atoms. All simulations were performed with CHARMM36m force fields (Wrapp, D. et al 2020; Walls, A. C. et al 2020) for protein and TIP3P (Dacon, C. et al 2022) model for water molecules using Gromacs 2022.1 software (Zhang, B. W. et al 2010; Russo, J. D. et al 2022). Next, unrestrained equilibrium simulations were carried out for 20 ns in the NPT ensemble at a target pressure of 1 bar using the Parrinello-Rahman barostat (McLellan, J. S. et al 2013) and a time step of 2 fs. The equilibration procedure was carried out independently for both wild-type and mutant systems in both the folded and unfolded states. In order to aid the convergence of the subsequent non-equilibrium MD simulations and obtain accurate error estimates, the aforementioned procedure was repeated five times for both states (folded and unfolded) of each mutant system. Frames were saved every 100 ps, for a total of 200 frames saved per simulation.

[0292] Alchemical non-equilibrium free energy calculations - Alchemical non- equilibrium molecular dynamics simulations. 200 snapshots were extracted from each preparatory unrestrained, 20 ns-long equilibrium MD simulation (one every 100 ps) for a total of 1000 snapshots for each state (folded and unfolded) considering the 5 replicas. These frames were used to spawn 1000 non-equilibrium MD simulations in the forward direction (wild-type-to-mutant) and 1000 non-equilibrium MD simulations in the reverse direction (mutant-to-wild-type) for both the folded and the unfolded state. A parameter λ was used to 92 4893-1038-0739, v.1control the contribution of the wild-type and mutant side chain to the Hamiltonian such that at λ = 0, the Hamiltonian corresponds to the wild-type protein, whereas at λ = 1 it corresponds to the mutant (Xiang, S. -H. et al 2002). The alchemical transition between wild- type and mutant was achieved in 100,000 steps (200 ps) by switching the λ parameter from 0 to 1 or from 1 to 0, depending on the direction of the transition, using a gradient of 0.00001 / step. During these simulations, the soft-core function and the default parameters for Coulombic and van der Waals interactions were used (Starr, T. N. et al 2020).

[0293] Alchemical non-equilibrium free energy calculations - Free energy calculations. Having multiple mutations at once imposes a strong perturbation to the system. Therefore, more work will be dissipated along the path leading to less accurate free energy estimation. To overcome this caveat, free energy calculations that include multiple mutations can be performed sequentially. Since the HexaPro-SS-∆stalk construct is a homotrimer, three subsequent free energy calculations were necessary (FIG. 27 and FIG. 28). The first one was performed by introducing the mutation in chain A only. Subsequently, another independent calculation was carried out where the mutation was also introduced in chain B, with the mutation already present in chain A. Finally, an independent calculation was performed where the mutation was also induced in chain C, with the mutation already present in both chains A and B. The same workflow was implemented in the case of the double mutant (HexaPro-SS-2W), where six independent calculations were performed sequentially (FIG. 28). For each independent calculation, PMX analyse script was used to extract the work values corresponding to each alchemical transition, and the free energy differences were calculated based on the Crooks fluctuation theorem (Suarez, E. et al 2014) by utilizing the Bennett acceptance ratio (BAR) as a maximum likelihood estimator (Saglam, A. S. et al 2019). The numerical uncertainty of each calculation was estimated by employing a bootstrapping procedure (100 resampling runs for every error estimate). A thermodynamic cycle was then constructed as shown in FIG. 27, and the difference in protein folding free energy upon mutation (ΔΔGfolding) was calculated as follows: ∆∆^^^^^^^^= ∆^^^^^^^^ ^^^^^^− ∆^^^^^^^^ ^^^^^^^^where the total change in free energy for the mutated trimer (ΔGmutated folded) was calculated by combining the ΔG values recovered from each independent calculation (Table 5). The ΔG for 93 4893-1038-0739, v.1the tripeptide was calculated once and multiplied by three to obtain ΔGmutated unfolded. Uncertainty propagation was applied when combining ΔG values.

[0294] Relying on the notion that the free energy of a system is a state variable, it is possible to calculate the difference in the free energy of folding (∆∆Gmutation- folding) between the investigated variant and the base construct (closed prefusion HexaPro- SS-Δstalk), as illustrated in the thermodynamic cycle in FIG. 27. Here, starting from an equilibrated closed conformation of HexaPro-SS-Δstalk, alchemical non-equilibrium free energy calculations were performed to predict the contribution of V991W and / or T998W substitutions to the folding free energy in the three engineered tryptophan variants in the closed conformation (FIG. 28). As a result, in all three cases, the introduced substitutions are stabilizing (FIG. 29). In ascending order (i.e., from the least to the most stabilizing), calculated ∆∆Gmutation-folding for HexaPro-SS-V991W, HexaPro-SS-T998W, and HexaPro-SS-2W, are -2.76 ± 0.11, -8.91 ± 0.26, and -12.18 ± 0.34 kcal / mol (FIG. 29a and Table 5). Hence, these independent simulations predicted the double substitution variant, HexaPro-SS-2W, to be the most stable in the closed prefusion conformation.

[0295] For each system, all the closed conformations that were sampled during the respective WE simulation were extracted, including those that did not result in an opening event. Using this multi-microsecond-long conformational ensemble of closed conformations, interprotomer contacts involving CHs, UHs, and HR1 helices were analyzed to decipher, at the molecular level, the stabilizing effect imparted by the tryptophan substitutions (FIG.31, FIG.32, and FIG.33). Introducing V991W and T998W into HexaPro- SS-Δstalk directly contributes to novel stabilizing contacts, enhancing electrostatic and hydrophobic interaction networks (FIG. 29b-e, FIG. 31, FIG. 32, and FIG. 33). The tryptophan side chains at positions 991 and 998 self-associate to form π–π hydrophobic clusters at the S2 apex surrounding R995 (FIG. 29c-e). Notably, alongside forming a salt bridge with D994 from the adjacent protomer, the side chain of R995 can also adopt an inward-oriented conformation, where it engages in a π–cation–π stack with W991 and W998 from both the same and neighboring protomers (FIG. 29d-e, FIG. 31, and FIG. 32). W998 is sometimes observed to swivel slightly outward, interacting with Y756 and F759 of the same chain and F970 from the adjacent chain. Interestingly, an extensive network of interactions is also preserved in the partially open conformations obtained from the WE simulation, where the presence of intra-protomer π–π and cation–π interactions modulate the orientation of the 94 4893-1038-0739, v.1tryptophans’ sidechain (FIG. 29e). Most importantly, the extensive network of π–π and cation–π interactions at the S2 apex is absent in the HexaPro-SS-Δstalk base construct (FIG. 29c, FIG.31, and FIG.32).

[0296] To experimentally probe the computational predictions, all three tryptophan variants were assessed relative to HexaPro-SS-Δstalk for expression yield in FreeStyle 293F cells (FIG. 34a,b) and thermostability through differential scanning fluorimetry (DSF) (FIG. 34c). In line with the stabilization trend predicted with free energy calculations, expression of HexaPro-SS-2W and, to a lesser extent, HexaPro-SS-T998W, led to higher yields than the parent. In addition, the slightly right-shifted peaks of their size- exclusion chromatography (SEC) profiles hint to more compact structures (FIG. 34b). V991W alone does not significantly improve the expression yield of HexaPro-SS-V991W relative to the base construct. A similar trend is observed from the thermostability profile (FIG. 34c), where HexaPro-SS-2W exhibits the highest melting temperature relative to the control systems (+5 °C shift relative to HexaPro-SS-Δstalk and +16 °C shift relative to HexaPro), followed by HexaPro-SS-T998W, and HexaPro-SS-V991W.

[0297] Based on the SEC data that indicated a ~2-fold increase in purified protein, the 3D structures of both HexaPro-SS-T998W (Δstalk) and HexaPro-SS-2W (Δstalk) were characterized using cryo-EM. For the former, a high-resolution cryo-EM structure was not obtained; indeed, the low-resolution 3D ab-initio map shows twisted S2 single protomers. On the other hand, for HexaPro-SS-2W a high-resolution (2.8 Å) cryo-EM map was generated from 1,622 exposures collected on a 200 kV Talos Glacios. The cryo-EM map of the HexaPro-SS-2W was processed with C3 symmetry and DeepEMhancer64 sharpening.2D classes, especially the top-down views, indicate that the closed prefusion conformation is predominant in HexaPro-SS-2W (FIG. 35). This result is consistent with free energy calculations and DSF experiments. The structure highlights key residue interactions also observed in the WE simulation, showing a densely packed cluster of hydrophobic residues at the S2 trimer’s apex (FIG. 34e-g). In this region, an extended network of π–π interactions encompassing W991, W998, Y756, F759, and F970 surrounds an ordered salt bridge (D994– R995) (FIG. 34e and FIG. 36). Examining the cryo-EM density of each mutation, V991W at the very apex of the S2 trimer shows clear cation–π interactions with R995 (FIG. 34f). T998W shows clear cryo-EM density of the side chain, filling the cavity in between the CHs of the S2 trimer (FIG.34g). By stabilizing the S2 trimer’s apex with V991W and T998W, we 95 4893-1038-0739, v.1were also able to resolve the fusion peptide proximal region (FPPR, residues 835-857). This region had been challenging to characterize in the full-length spike or HexaPro-SS due to its inherent flexibility.

[0298] Sera of mice immunized with closed prefusion-stabilized S2-only antigen neutralize rVSV-SARS-CoV-2 variants. The impact of tryptophan substitutions on S2- only antigen immunogenicity was investigated. Mice with the HexaPro-SS and HexaPro-SS- 2W antigens (including the stalk region) were immunized and the sera was assayed for neutralization against recombinant vesicular stomatitis viruses (rVSVs) bearing the coronavirus spikes of either SARS-CoV-2 Wuhan-1 or Omicron BA.1 variant (FIG. 37 and FIG. 38). Against rVSV-SARS-CoV-2 Wuhan-1, both sera immunized with HexaPro-SS and HexaPro-SS-2W show a significant increase in neutralization (FIG. 37b) as compared to PBS-immunized sera, but do not differ with respect to each other. In contrast, only mice immunized with HexaPro-SS-2W demonstrated a significant increase in the neutralization of rVSV-SARS-CoV-2 Omicron BA.1 compared to PBS-immunized sera.

[0299] To evaluate the impact of the tryptophan substitutions on the preservation of the epitopes on the spike S2 subunit, we performed biolayer interferometry (BLI) experiments. The binding of previously reported neutralizing and non-neutralizing S2- directed antibodies that target the highly conserved stem helix (IgG22,2 S2P623 and CC40.825), fusion peptide (CoV44-79 and CoV91-2724), and apex (B3-1, manuscript under review at the time of writing) were tested against HexaPro-SS (FIG. 37c), HexaPro-SS-2W (FIG. 37c), and HexaPro-SS-T998W (FIG. 39) constructs (including the stalk region). An antibody targeting the RBD of S1 was used as a control (N3-165). BLI shows that the binding affinity of antibodies that target the stem helix or fusion peptide is not affected in HexaPro- SS-2W when compared to HexaPro-SS. However, binding affinity of the non-neutralizing antibody B3-1 targeting the apex of the S2 trimer as in HexaPro-SS-2W was reduced by half with respect to HexaPro-SS (FIG. 37c). This suggests that the opening of the S2 apex is important for B3-1 binding. Interestingly, B3-1 binding is not fully abrogated, suggesting that HexaPro-SS-2W may infrequently undergo opening motions as shown from WE simulations.

[0300] Discussion. The simulation-driven design of SARS-CoV-2 S2-only antigens stabilized in the closed prefusion conformation are described herein. WE path- sampling MD simulations were used to investigate the conformational plasticity of HexaPro- SS-Δstalk, a prefusion-stabilized S2 construct that exhibits flexibility at the apex, leading to 96 4893-1038-0739, v.1splayed open conformations (Hsieh, C. -L. et al 2024). As described herein, the prefusion- stabilized S2 trimer transitions from a closed to open conformation through an asymmetric protomer-protomer separation. Contact map analysis of the interfacial helices (CHs, UHs, and HR1) reveals key residue-residue interactions taking place in the opening pathway. To improve interprotomer stabilization, two residues located in the CH apex, V991 and T998, were identified herein as candidates for cavity-filling substitutions with tryptophan. As described herein, three variants of HexaPro-SS-Δstalk were designed, incorporating V991W, T998W, and V991W+T998W substitutions, namely HexaPro-SS-V991, HexaPro-SS-T998, and HexaPro-SS-2W. Additional WE simulations of the three newly engineered S2 variants showed that T998W and V991W+T998W provide kinetic stabilization by slowing down the S2 trimer opening. Interestingly, when only position 991 is substituted to tryptophan (i.e., in HexaPro-SS-V991W), it does not induce slower opening kinetics despite furthering interprotomer engagement. This could be ascribed to the apical position of W991, which is more solvent-exposed than W998 and, in the absence of a partner hydrophobic residue underneath (i.e., W998 in HexaPro-SS-2W), is not kinetically stabilizing during the opening transition.

[0301] Subsequent alchemical non-equilibrium free energy calculations revealed a negative ∆∆Gmutation-foldingimparted by the tryptophan substitutions, evincing increased stability of the closed prefusion conformation for all three S2-engineered variants, with HexaPro-SS-2W exhibiting superior stability. Strikingly, cellular expression and thermostability characterization of the newly stabilized S2 constructs confirmed the computational predictions, showing higher expression yields and melting temperatures for the three tryptophan variants, with HexaPro-SS-2W surpassing all the other constructs. The improved stability of HexaPro-SS-2W allowed the high-resolution characterization of its closed prefusion structure via cryo-EM. The resolved cryo-EM structure highlights key residue interactions also observed in the WE simulations. As these interactions in the closed state are not protomer-specific, this suggests that the synergistic residue coordination in the S2 interior can dynamically exchange interactions to stabilize a closed conformation. Importantly, immunogenicity assays on sera from mice immunized with HexaPro-SS-2W demonstrated that this stabilized S2-only antigen can neutralize different rVSV-SARS-CoV-2 variants. Furthermore, BLI assays showed that the binding of the non-neutralizing antibody B3-1 to the HexaPro-SS-2W’s apex is reduced by half when compared to the base construct. Reducing the binding affinity of the non-neutralizing antibodies targeting S2 may indicate 97 4893-1038-0739, v.1that closing the S2 trimer with 2W can bias immunization responses towards neutralizing epitopes such as stem helix and fusion peptide as opposed to non-neutralizing epitopes such as the apex.

[0302] The approach described herein underscores the advantage of using dynamic information from molecular simulations to inform immunogen design. Static- structure methods depend on energetic calculations of protein structures in cryogenic conditions or rely on the structural homology of proteins with minor conformational variance. In contrast, the simulations described herein capture dynamic information from an ensemble of conformations at reproduced physiological conditions. The analysis of the representative conformational ensemble may help decipher the residue-specific contributions to conformational stability, shedding light on the impact of mutations in emerging SARS-CoV-2 VOCs. Given the rapid accumulation of sequence modifications in the spike protein, investigating the effects of these mutations may become crucial to design robust vaccines against future variants. The approach described herein, or alternative MD-based approaches such as mesoscale simulations (Casalino, L. et al 2022), algorithms to study allostery (Castelli, M. et al 2024; Ray, D. et al 2022; Sofia, A. et al 2024) and epitope prediction (Sikora, M. et al 2021; Serapian, S. A. et al 2020; Triveri, A. et al 2021) could also be used to characterize short-lived antigenic conformations, dissect the antigenic impact of escape mutations, or reveal novel cryptic epitopes. Considering the challenges to obtain atomic-level details of conformational ensembles from experiments, the mechanistic insights gained from computational methods could play a pivotal role in steering vaccine design efforts. The computational approach used herein enabled the design of novel, highly stable SARS-CoV-2 S2-only immunogens that could serve as the structural basis for future pan-coronavirus vaccine development efforts and may be generally applicable to structure-based vaccine design. Example 2 – Material and Methods

[0303] Modeling of the closed prefusion HexaPro-SS-∆stalk S2 trimer. The all-atom model of the closed prefusion HexaPro-SS-∆stalk S2 trimer, spanning residues 696 to 1141, was constructed from a cryo-EM structure of the SARS-CoV-2 HexaPro spike (PDB ID: 6XKL32). The fusion peptide region (residues 823 to 857) was grafted from a cryo-EM structure of the SARS-CoV-2 3Q-2P spike (PDB ID: 7JJI) (Bangaru et al., 2020). The HexaPro-SS-∆stalk stabilizing mutations (S704C, K790C, and Q957E) were incorporated 98 4893-1038-0739, v.1with VMD’s psfgen 2.0 structure-building tool (Humphrey et al., 1996). The model was glycosylated using the same glycoprofile from Casalino et. al. and parameterized using the all-atom additive CHARMM36m force field for protein and glycans (Huang, J. et al 2017; Huang, J. et al 2013; Guvench, O. et al 2009). The system was solvated with TIP3P76 explicit water molecules in a box with at least 15 Å between the protein and box edges (box size: 134 Å x 140 Å x 153 Å), and the charge was neutralized with Na+ and Cl- ions up to a concentration of 150 mM. The final system accounted for 274,082 atoms.

[0304] All-atom MD simulations of HexaPro-SS-∆stalk. Conventional all- atom MD simulations of the closed prefusion HexaPro-SS-∆stalk system were performed using NAMD3 (Phillips, J. C. et al 2020). An energy minimization was initially performed for 10,800 steps utilizing the conjugate gradient method. During this phase, protein and glycan atoms were subjected to positional harmonic restraints, applying a force constant of 1.0 (kcal / mol) / Å2. Following this, a 0.5 ns NPT (isothermal-isobaric) equilibration was conducted. The harmonic restraints were retained throughout this stage. Next, restraints were released, and an additional 5 ns of NPT equilibration was performed. Subsequently, four replicates of NPT production MD simulations were run (R1 = 601.5 ns, R2 = 655.4 ns, R3 = 744.6 ns, R4 = 627.6 ns) after re-initializing velocities. Control of the temperature and pressure was achieved with a Langevin thermostat (Ermak, D. L. et al 1978; Loncharich, R. J. et al 1992) (310 K) and a Nosé-Hoover Langevin barostat80 (1.01325 bar). All simulations (including equilibration) were performed using an integration time step of 2 fs and the SHAKE81 algorithm to keep covalent bonds involving all hydrogen atoms fixed. Periodic boundary conditions were active, with the particle-mesh Ewald82 method used for long-range electrostatics calculation. Non-bonded interactions, such as van der Waals interactions and short-range electrostatics, were accounted for with a cutoff of 12 Å.

[0305] Gaussian accelerated molecular dynamics (GaMD) simulations. GaMD simulations were performed on the Triton Shared Computing Cluster (TSCC) at the San Diego Supercomputer Center (SDCS) using GaMD (Miao et al., 2015; Wang et al., 2021) with NAMD2.14 (Phillips et al., 2020). The initial state for GaMD simulations was selected from the last frame of the first replica of all-atom conventional MD simulations, which represents a tightly closed conformation that is more compact relative to the initial state used in all-atom MD simulations. To determine the magnitude of the dual boost potential ΔV (Total potential boost + Dihedral potential boost) required to enhance 99 4893-1038-0739, v.1conformational sampling, the reference E threshold for application of the dual harmonic boost potential was assessed at both low acceleration (E=1) and high acceleration (E=2). To obtain the optimal boost potential parameters for GaMD production simulations, an NPT equilibration was performed at 310 K and 1.01325 bar with the following protocol. The system was simulated without a boost applied for 0.4 ns without collecting system potential statistics (Vmin, Vmax, Vavg, σv),then the system was simulated for 1.6 ns where system potential statistics were collected. A boost potential is then applied for the remainder of GaMD equilibration and production simulations. Using the previously calculated system potential statistics, the system was simulated with boost potential parameters fixed for 0.4 ns, then the boost parameters were updated as the system was further equilibrated for 24.6 ns. At the end of system equilibration, the final calculated boost parameters were fixed and maintained for GaMD production simulations. Velocities were randomly initialized, and 3 replicates of NPT GaMD production simulations were run for each acceleration level at 310 K and 1.01325 bar.

[0306] WE MD simulation of HexaPro-SS-∆stalk. The initial HexaPro-SS- ∆stalk construct for WE simulations was extracted from a frame parsed from conventional MD simulations. The dry construct was re-solvated with TIP3P83 water molecules in an orthorhombic box of 138 Å × 150 Å × 168 Å per side, allowing at least 24 Å between the solute and the box edges in all directions, and neutralized with a 150 mM concentration of NaCl. As a result, we obtained a system tallying 328,200 atoms. In order to use the Amber software (Salomon-Ferrer, R. et al 2013) as the engine for the subsequent WE MD simulations while maintaining the CHARMM36m force fields, we used the CHAMBER (Crowley, M. F. et al 2009) program to convert the generated coordinate (.pdb) and topology (.psf) files, and the necessary CHARMM36m parameters files into an Amber readable format (i.e., .prmtop file for topology and parameters and .rst7 file for coordinates). To ensure a more extensive sampling of the initial state in a WE simulation, it is recommended to provide a set of initial, equally weighted conformations (called “basis states”) rather than a single conformation. To accomplish this task, we performed 10 preparatory MD simulations of 50 ns each using Amber20 software (Salomon-Ferrer, R. et al 2013) and CHARMM36m force fields (Huang, J. et al 2017; Huang, J. et al 2013; Guvench, O. et al 2009). The full protocol is described in the SI. From the generated ensemble of closed conformations, we extracted 50 initial basis states, to which we assigned an initial weight of 0.02 each. Criteria used for the selection of basis states are fully detailed in the SI. 100 4893-1038-0739, v.1

[0307] All WE MD simulations were performed using WESTPA 2.0 software (Russo, J. D. et al 2022), exploiting Amber20 PMEMD.cuda (Salomon-Ferrer, R. et al 2013) as MD engine to propagate dynamics. CHARMM36m force fields (Huang, J. et al 2017; Huang, J. et al 2013; Guvench, O. et al 2009) were adopted for protein and glycan, and TIP3P (Jorgensen, W. L. 1983) for water molecules. To enhance sampling of closed-to-open transitions of HexaPro-SS-∆stalk S2 trimer we defined two progress coordinates. The first progress coordinate was set as the area of the triangle having the Cα atom of residues P987A, P987B and P987C as vertices. The second progress coordinate was set as the RMSD of the Cα atoms of the CHs (residues 987-1033) in the simulation construct to the corresponding Cα atoms of the CHs (residues 987-1033) in the HexaPro-SS-∆stalk open crystal structure (PDB ID: 8U1G33). The minimal adaptive binning (MAB) scheme was applied to bin the conformational space defined by the two progress coordinates (Torrillo, P. A. et al 2021). The resampling time interval ^ was set to 100 ps, whereas the target number of independent trajectories run in each bin, M, was set to 8. MD simulations were performed in NPT conditions using a Langevin thermostat (Ermak, D. L. et al 1978; Loncharich, R. J. et al 1992) to maintain the temperature at 300 K and the Monte Carlo barostat (Aqvist, J. et al 2004) to keep the pressure at 1 bar. SHAKE algorithm was used to constrain bonds involving hydrogen atoms (Ryckaert, J. -P. et al 1977). Particle Mesh Ewald method was used to handle long-range electrostatic interactions (Darden, T. et al 1993), whereas non-bonded interactions were calculated with a cutoff of 10 Å. WE simulation of HexaPro-SS-∆stalk was carried out for a total of 315 iterations, ~46 µs aggregate sampling, and 180 successful opening pathways (FIG. 23 and Tables 3,5). In order to be able to provide an estimate of kinetic rates for the opening transition, we facilitated the convergence of WE simulations toward near equilibrium steady-state conditions by applying the WE Equilibrium Dynamics (WEED) protocol twice to reweight the trajectories (Suarez, E. et al 2014; Bhatt, D. et al 2010). Convergence of WE simulations was assessed by monitoring the evolution of the 1D probability distributions relative to each progress coordinate (FIGS. 14b,12b,15b,10b) as well as by tracking the evolution of the kinetic rates’ rolling averages calculated for the opening transition (FIG.26).

[0308] Modeling and WE MD simulations of HexaPro-SS-V991W, HexaPro- SS-T998W, and HexaPro-SS-2W variants. The three HexaPro-SS-∆stalk tryptophan- stabilized systems (HexaPro-SS-V991W, HexaPro-SS-T998W, and HexaPro-SS-2W) were built from the same base construct used in the HexaPro-SS-∆stalk WE simulation. V991W, T998W, or V991W+T998W substitutions were incorporated in each protomer of the 101 4893-1038-0739, v.1respective HexaPro-SS-∆stalk variant using the psfgen 2.0 structure-building tool within VMD (Humphrey, W. et al 1996). A similar system setup and WE simulation settings, as described for HexaPro-SS-∆stalk, were used for each variant. WE simulation of the HexaPro- SS-V991W mutant was carried out for a total of 301 iterations, ~36 µs aggregate sampling, and 149 successful opening pathways (FIG. 23 and Tables 3,5). WE simulation of the HexaPro-SS-T998W mutant was performed for a total of 355 iterations, ~48 µs aggregate sampling, and 187 successful opening pathways (FIG. 23 and Tables 3,5). WE simulation of the double HexaPro-SS-2W mutant was carried out for a total of 401 iterations, ~57 µs aggregate sampling, and 195 successful opening pathways (FIG. 23 and Tables 3,5). For all the mutants, trajectory reweighting was conducted twice using the WEED plugin of WESTPA 2.0 (Suarez, E. et al 2014; Bhatt, D. et al 2010).

[0309] Simulation analysis. WE simulations were analyzed using analysis tools provided by WESTPA 2.0 (Russo, J. D. et al 2022) software, including estimation of kinetic rates. Contact map analysis of the opening pathways was performed with PyContact (Scheurer, M. et al 2018). Contact analysis for the closed conformations was performed with MDAnalysis (Michaud-Agrawal, N. et al 2011). Images relative to the simulations were generated with VMD (Humphrey, W. et al 1996).

[0310] Alchemical non-equilibrium free energy calculations. To build the thermodynamic cycle (FIG. 27) for alchemical non-equilibrium free energy calculations, two independent systems representing the unfolded and folded states of the protein were generated. To approximate the unfolded state, considering the limitations of empirical force fields in simulating unfolded proteins (Piana, S. et al 2011; Freddolino, P. L. et al 2009; Fischer, A. -L. M. et al 2024) and given the unavailability of the S2 trimer unfolded structure, we used a capped tripeptide (G-X-G) built with Chimera (Petterson, E. F. et al 2004), where X is the amino acid of interest (either V, T, or W). It is noted herein that this approach has been commonly adopted in alchemical free energy calculations and has proven to align well with experimental data (Seeliger, D. et al 2010; Gapsys, V. et al 2015; Jones, K. F. M. et al 2023). For the folded state, wild-type system, we used the same equilibrated closed HexaPro- SS-∆stalk construct in the closed conformation extracted from conventional MD. Mutant systems for the folded state (HexaPro-SS-V991W, HexaPro-SS-T998W, HexaPro-SS-2W in the closed conformation) were constructed from the HexaPro-SS-∆stalk base construct using the psfgen 2.0 structure-building tool within VMD (Humphrey, W. et al 1996). For each 102 4893-1038-0739, v.1variant, mutations were initially incorporated into one protomer only (protomer A), generating the respective A-mutated construct. Next, starting from this construct, a second system was built by introducing the respective mutation also in the second protomer (protomer B). Finally, a third construct incorporating the respective mutation in all three protomers (A, B, and C) was created. Free energy calculations were then performed sequentially, and the total change in free energy for the mutated trimer was recovered by combining the ΔG values for each independent calculation. The PMX (Gapsys, V. et al 2015) software was used to build hybrid structures and topologies incorporating side chains from both the wild-type and mutant amino acids. Generated structures of the solute were then solvated in a cubic TIP3P (Jorgensen, W. L. et al 1983) water box using Gromacs 2022.1 software (Abraham, M. J. et al 2015), imposing at least 12 Å from the solute edges in each direction. Na+ and Cl+ ions were added at a concentration of 150 mM to neutralize the systems. For both wild-type and mutant systems in both the folded and unfolded states, equilibrium MD simulations were carried out, independently, for 20 ns in the NPT ensemble at a target pressure of 1 bar using the Parrinello-Rahman barostat (Parrinello, M. et al 1981) and a time step of 2 fs. Equilibrium simulations were performed using Gromacs 2022.1 software (Abraham, M. J. et al 2015) and CHARMM36m force fields (Huang, J. et al 2017; Huang, J. et al 2013) and TIP3P (Jorgensen, W. L. et al 1983) model for water molecules. In order to aid the convergence of the subsequent non-equilibrium MD simulations, the aforementioned procedure was repeated five times for both states (folded and unfolded) of each mutant system. Frames were saved every 100 ps, corresponding to 200 frames saved per simulation. These frames were used to spawn a total of 1000 non-equilibrium MD simulations in the forward direction (wild-type-to-mutant) and 1000 non-equilibrium MD simulations in the reverse direction (mutant-to-wild-type) for both the folded and the unfolded state. The alchemical transition between wild-type and mutant was achieved in 100,000 steps (200 ps) by switching the λ parameter from 0 to 1 or from 1 to 0, depending on the direction of the transition, using a gradient of 0.00001 / step (Seeliger, D. et al 2010). Convergence was assessed by evaluating the intersection between the work values calculated from the forward and reverse non-equilibrium simulations (FIG. 28). During these simulations, the soft-core function and the default parameters for Coulombic and van der Waals interactions were used (Gapsys, V. et al 2012). PMX (Gapsys, V. et al 2015) analysis script was used to extract the work values corresponding to each alchemical transition, and the free energy differences were calculated based on the Crooks fluctuation theorem (Crooks, G. E. et al 1999) by utilizing the Bennett acceptance ratio (BAR) as a maximum likelihood 103 4893-1038-0739, v.1estimator (Shirts, M. R. et al 2003). A thermodynamic cycle was then constructed as shown in FIG.27, and the difference in protein folding free energy upon mutation (ΔΔGfolding) was calculated.

[0311] Protein expression. V991W, T998W, and V991W / T998W substitutions were introduced into HexaPro-SS (SARS-CoV-2 HexaPro S, encompassing amino acid residues 697-1208, with S704C, K790C, and Q957E substitutions) and HexaPro- SS-Δstalk (encompassing amino acid residues 697-1141, with S704C, K790C, and Q957E substitutions) constructs containing a C-terminal fold on trimerization motif, an HRV 3C cleavage site, 8x His tag, and Twin-Strep-tag. Plasmids (0.125 mg) encoding HexaPro-SS, HexaPro-SS-Δstalk, and the tryptophan variants were transiently transfected into 250 mL FreeStyleTM 293F cells (ThermoFisher, cat #R79007) with 25 kDa linear polyethylenimine (PEI) (Polysciences cat# 3966-2), and sterile-filtered kifunensine was added 3 hours post- transfection to a final concentration of 5 uM. The medium was harvested and filtered 6 days post-transfection. StrepTactin Sepharose resin (IBA, cat #2-1201-002) was used for affinity purification, and the elution fraction was applied onto a Superose™ 6 Increase 10 / 300 GL (Cytiva, cat #29091596) size-exclusion chromatography (SEC) column in a running buffer of 2 mM Tris pH 8.0, 200 mM NaCl, and 0.03% NaN3. Peak fractions were eluted, combined, and concentrated using 30K Amicon Ultra centrifugal filters. Endotoxin-free protein samples for mouse immunizations were prepared using Pierce™ High Capacity Endotoxin Removal Spin Columns (Thermo Scientific, cat # 88274).

[0312] Cryo-EM sample preparation. The SARS-CoV-2 HexaPro-SS-2W (Δstalk) construct was diluted to 0.8 mg / mL in 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% NaN3. A 4 uL aliquot of the sample was placed onto a glow discharged UltrAuFoil R 1.2 / 1.3 300 mesh gold grid (Electron Microscopy Sciences, Cat. # Q350AR13A). The grid was then subjected to a 5-second wait time in a 22 ºC, 100% humidity chamber, blotted for 4 seconds with -3 force, and plunge frozen into liquid ethane using a Vitrobot Mark IV (ThermoFisher). A total of 1,764 micrographs were collected on a 200 kV Talos Glacios (ThermoFisher) equipped with a Falcon 4 detector. Data were collected at 150,000x magnification, corresponding to 0.94 Å / pix. A full description of the cryo-EM data collection can be found in Table 6. 104 4893-1038-0739, v.1Table 6: EM data collection for HexaPro-SS-2W-∆stalk EM data collection Microscope FEI Glacios Voltage (kV) 200 Detector Falcon 4 Magnification (nominal) 150,000 Pixel size (Å / pix) 0.94 Exposure rate (e- / pix / sec) 3.79 Exposure (e- / Å2)50Defocus range (µm) 1.5-2.5 Tilt angle ( ̊ ) 0 Micrographs collected 1,764 Micrographs used 1,622 Particles extracted (total) 744,038 Automation software SerialEM Sample HexaPro-SS-2W-Δstalk Particles 249,447 Symmetry C3 Map sharpening B-factor -107.3 Unmasked resolution at 0.5 FSC (Å) 3.2 Masked resolution at 0.5 FSC (Å)3.0Unmasked resolution at 0.143 FSC (Å) 2.8 Masked resolution at 0.143 FSC (Å) 2.8 Model refinement and validation statistics Refinement package ChimeraX ISOLDE, PhenixRefinement toolPhenix real-space refinementmin global, local_grid_search, adp, occupancy, Refinement strategies reference model restraints, ss restraints, rotamer restraints, Ramachandran restraints, NCS constraints Composition Amino acids 1311 RMSD bonds (Å) 0.003 RMSD angles (º) 0.49 Average B-factors Amino acids 53.3 Ramachandran Favored (%) 97.7 Allowed (%) 2.3 Outliers (%) 0 Rotamer outliers (%) 0 Clash score2.86C-beta outliers (%) 0 CaBLAM outliers (%) 2.08 CC (mask) 0.77 MolProbity score 1.14 EMRinger score4.19105 4893-1038-0739, v.1

[0313] Cryo-EM data processing and model building. CryoSPARC (Punjani, A. et al 2017) Live v4.0.2 was used for motion correction, CTF estimation, micrograph curation, and particle picking. Particles were picked using template picker with an extraction box size of 240 (pix). The selected particles and accepted micrographs were exported into CryoSPARC v4.0.2 for ab-initio 3D reconstruction, heterogenous refinement, and homogeneous refinement. C3 symmetry was applied in the final reconstruction. The C3 map was further sharpened using DeepEMhancer (Sanches-Garcia, R. et al 2021) via the Cosmic2 (Cianfrocco, M. A. et al 2017) science gateway. ChimeraX (Petterson, E. F. et al 2004), Isolde (Croll, T. I. et al 2018), Phenix (real-space refinement) (Liebschner, D. et al 2019; Afonine, P. V. et al 2018) and Coot (Emsley, P. et al 2010) were used interactively to build the model. A full description of cryo-EM data processing can be found in FIG.35.

[0314] Differential scanning fluorimetry. Solutions containing a final protein concentration of 1 µM and 6.25X SYPRO Orange Protein Gel Stain (Thermo Fisher, cat #S5692) were added to the wells of a 96-well qPCR plate. Continuous fluorescence measurements (λex=465 nm, λem=580 nm) were performed using a Roche LightCycler 480 II at a temperature ramp rate of 4.4 °C / minute increasing from 22 °C to 95 °C. Data were plotted as the negative of the derivative of fluorescence as a function of temperature.

[0315] Biolayer interferometry. Anti-hIgG Fc Capture (AHC) Biosensors (Sartorius, cat # 18-5064) were pre-wet in 1X HBS-EP+ buffer (Cytiva, Cat # BR100669) for 10 minutes. IgGs (with IgG G4 (Paellesen, J. et al 2017) as an isotype control) were diluted in the same buffer to 25 nM at a total volume of 200 uL per well. HexaPro, HexaPro-SS, and tryptophan-stabilized constructs were diluted in the 1X HBS-EP+ buffer to a final concentration of 100 nM in 200 uL per well. The AHC biosensors first underwent 60 seconds of baseline in buffer and were then dipped into IgG wells for capture to a level of 0.6 nm. The captured biosensors were then transferred back to buffer wells for another 60 seconds of baseline and then dipped into wells containing spike protein constructs for a 300-second association followed by transfer into wells containing buffer for another 300-second dissociation. Data were then reference subtracted (isotype control) and fit globally for both association and dissociation to a 1:1 binding model.

[0316] Cell culture. Vero cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, high glucose; Gibco) supplemented with 2% heat-inactivated fetal bovine serum (FBS; Bio-Techne), 1% penicillin-streptomycin (P / S; Thermo Fisher 106 4893-1038-0739, v.1Scientific), and 1% GlutaMAX (Thermo Fisher Scientific). 293FT cells (Thermo Fisher Scientific) were cultured in DMEM supplemented with 10% FBS, 1% P / S, and 1% GlutaMAX.

[0317] Generation of rVSV-SARS-CoV-2 viruses. A plasmid encoding the VSV antigenome was engineered to replace its native glycoprotein G with the CoV spike of interest. The spike sequence of rVSV-SARS-CoV-2 Wuhan-1 was obtained from GenBank (MN908947.3) while that of rVSV-SARS-CoV-2 Omicron was generated by incorporating the defining mutations of B.1.1.529 (as reported by CoVariants (Hodcroft, E. et al)) in the Wuhan sequence. DNA fragments for both sequences were produced by Twist Bioscience. As previously reported, a 19 amino acid C-terminal deletion was made in each CoV spike. An enhanced green fluorescent protein (eGFP) reporter gene was included in the VSV antigenome as a separate transcriptional unit. Plasmid-based rescue of the rVSVs was carried out (Dieterle, M. E. et al 2020; Kleinfelter, L. M. et al 2015). 293FT cells were transfected using polyethyleneimine with the VSV antigenome plasmid along with helper plasmids expressing T7 polymerase and VSV proteins: N, P, M, G, and L. Supernatants from transfected cells were transferred to Vero cells 48 hours post-transfection. The appearance of eGFP-positive cells confirmed the presence of virus. Viral clones were plaque-purified on Vero cells and propagated by cell subculture. RNA was isolated from viral supernatants, and Sanger sequencing was performed to confirm the spike gene sequence. Viral stocks were concentrated via ultracentrifugation in an SW28 rotor at 28,000 rpm for 4 hours. Virus was aliquoted and stored at -80° C. The generation of all rVSV-CoVs and their use in tissue culture was done at biosafety level 2 and was approved by the Environmental Health and Safety Department and Institutional Biosafety Committee at Albert Einstein College of Medicine.

[0318] In vivo immunization. 6–8-week-old female C57BL / 6J mice were purchased from The Jackson Laboratory (JAX: 000664). Mice (n = 6) were immunized with 10 µg of each S2 antigen construct or PBS adjuvanted with 10 µg of Sigma Adjuvant System (Sigma) and boosted three weeks after in the same fashion. Constructs and adjuvant were each diluted in endotoxin-free PBS (Millipore) to a total volume of 100 µL before they were mixed to obtain a total of 200 µL per mouse. 200 µL of immunogen / adjuvant were delivered by intraperitoneal injection for each mouse. Mice were bled one week prior to prime (week - 1), boost (week 2), and four weeks post-boost (week 7). Serum was collected from each 107 4893-1038-0739, v.1mouse, heat-inactivated for 30 min at 56 ˚C, aliquoted, and stored at -80 ºC for subsequent use in rVSV-CoV neutralization assays.

[0319] rVSV-CoV neutralization assays. Serial 3-fold dilutions were made for each mouse serum sample starting at a 1:10 dilution in DMEM supplemented with 2% FBS, 1% P / S, and 1% GlutaMAX. Mouse sera was incubated with rVSV-SARS-CoV-2 (Wuhan or Omicron) for 1 hour at room temperature. Media was removed from Vero cells in a 96- well cell culture plate (Corning 3585) and 40 µl virus / sera mixture was added to well in singlicate. The cells were incubated with the virus / sera mixture at 37oC and 5% CO2 for 10 hours. The cells were then fixed with 16% paraformaldehyde (PFA; Thermo Fisher Scientific) to a final PFA concentration of 4%, washed with 1x PBS and stained for cell nuclei in 1x PBS with Hoechst 33342 (Invitrogen) at a dilution of 1:2000. Viral infection was quantified by automatic enumeration of GFP-positive cells from captured images using Cytation 5 automated fluorescence microscope (BioTek) with analysis done by Gen5 (version 3.12) data analysis software (BioTek). The area under the curve (AUC) of sera sample was calculated using GraphPad Prism software (Version 9.3.1) with the following parameters: baseline Y=0; minimum peak height less than 0 Y units high. Each sera group was evaluated for normality and homoscedasticity by QQplot and by Bartlett’s test, respectively. Ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed for each neutralization study. All statistical testing was performed using GraphPad Prism software (Version 9.3.1). Example 3

[0320] To form ferritin nanoparticles with the stabilized S2 subunit trimers, a ferritin molecule derived from the insect Trichoplusia ni (iFerr) was used since it self- assembles naturally as a 24-mer with twelve copies each of a heavy and light chain (termed iFerr HC (SEQ ID NO: 7) and iFerr LC (SEQ ID NO: 8), respectively) (see U.S. Pat. 10,961,283, which is incorporated herein by reference in its entirety). The S2 protomer sequence (SEQ ID NO: 9 or 10) was linked to the N-terminus of the heavy or light chain ferritin sequence by a 5 amino acid peptide linker (positions 481-485 of SEQ ID NOs: 3-6). SARS-CoV-2 SSS-np comprises substitutions of S704C, K790C, F817P, A892P, A899P, A942P, Q957E, K986P, and V987P (with positions relative to SEQ ID NO: 1), with the iFerr HC fusion corresponding to SEQ ID NO: 5 and the iFerr LC fusion corresponding to SEQ ID NO: 6. SARS-CoV-2 SSS-WW-np comprises substitutions of S704C, K790C, F817P, 108 4893-1038-0739, v.1A892P, A899P, A942P, Q957E, K986P, V987P, V991W, and T998W (with positions relative to SEQ ID NO: 1), with the iFerr HC fusion corresponding to SEQ ID NO: 3 and the iFerr LC fusion corresponding to SEQ ID NO: 4.

[0321] Antigenicity of SARS-CoV-2 stabilized spike stem-nanoparticles (SSS- nps). The antigenicity of SARS-CoV-2 SSS-np and SARS-CoV-2 SSS-WW-np was tested using three anti-fusion peptide antibodies (CoV44-62, CoV77-39, VN01H1) and one anti- stem helix antibody (S2P6) by biolayer interferometry (BLI) using the Octet HTX instrument (fortéBio). Recombinant antibodies were immobilized on the pre-hydrated anti-human Fc (AHC) biosensors. After a brief equilibration in assay buffer (1% BSA in PBS), the biosensors were dipped in solutions of SARS-CoV-2 SSS-np or SARS-CoV-2 SSS-WW-np for 5  min to record binding events. Biosensors were then dipped in assay buffer to allow the SSS-nps to dissociate from the antibodies for 5 min. All assay steps were performed at 30°C with agitation set at 1,000 r.p.m. Data are shown in response unit (nm shift) at the maximum binding (at the end of incubation with SSS-nps) (FIG. 1). Of note, the fusion peptide epitope that is recognized by the three antibodies is partly occluded in the closed state spike trimer, and the observed weaker bindings of these antibodies to SARS-CoV-2 SSS-WW-np indicates more complete trimer closure.

[0322] Size exclusion chromatography (SEC) profiles of the SARS-CoV-2 SSS- nps. SSS-nps were purified from culture supernatants of Expi293 cells transfected with expression vector DNA plasmids encoding either SARS-CoV-2 SSS-np or SARS-CoV-2 SSS-WW-np with a GNA lectin. Eluates from the GNA lectin column were further purified by SEC using a Superose 6 Increase 10 / 300 GL column. The peak around the retention volume of 10.5-11 ml corresponds to fully assembled nanoparticles (FIG. 2). The yields are 2-4 mg / L and 4-6 mg / L for SARS-CoV-2 SSS-np and SARS-CoV-2 SSS-WW-np, respectively. As such, the V991W / T998W substitutions (WW) enhance protein expression in the context of SARS-CoV-2 SSS-np.

[0323] Negative stain electron microscopy images of SARS-CoV-2 SSS-nps. Purified SSS-nps were diluted to 0.02 mg ml-1with buffer containing 10 mM HEPES, pH 7.0, and 150 mM NaCl, and adsorbed onto glow-discharged carbon-coated copper mesh grids for 15 s. The grids were washed with the buffer and stained with 0.7% uranyl formate. Images were obtained at a magnification of 50,000 semi-automatically on an FEI Tecnai T20 transmission electron microscope (TEM) operated at 200 kV, equipped with a 2,048- by 109 4893-1038-0739, v.12,048-pixel Eagle charge-coupled-device (CCD) camera using SerialEM (Mastronarde, 2005). The pixel size was 0.44 nm and particles were picked automatically using software developed in house. Reference-free 2D classification was performed using SPIDER (Shaikh et al., 2008) and Relion 1.4 (Scheres, 2012). 2D class averages were shown in FIG. 3. SSS trimers are projected outward and clearly visible. SSS trimers on SARS-CoV-2 SSS-np showed a mixture of open- and closed states (FIG. 3, left). SSS trimers on SARS-CoV-2 SSS-WW-np showed closed state exclusively (FIG.3, right).

[0324] Serum antibody titers in mice immunized with SARS-CoV-2 SSS-nps. BALB / cJ mice (n = 10) were immunized intramuscularly with 10 µg of immunogens with AddaVax adjuvant (50% v / v final) three times with 4 weeks intervals. Serum samples taken at 2 weeks after the final immunization were analyzed by ELISA. ELISA plates were coated with 2 µg well-1of recombinant SARS-CoV-2 WA-1 S6P (Hexapro) or SARS-CoV-2 XBB S2P protein for overnight at 4°C. The plates were then blocked and incubated with serially diluted serum samples starting at 1 / 100. Plates were washed with PBST and incubated with goat anti-mouse IgG-HRP secondary antibody (GE Healthcare) at 1 / 5000 in PBS. After washing plates were developed using TMB substrate and the reactions were stopped with 1N sulfuric acid. The absorbance at 450 nm was measured and endpoint titers were calculated using GraphPad Prism (v9) software. Serum antibody titers to spike proteins of SARS-CoV-2 WA-1 S6P (hexapro) and SARS-CoV-2 XBB S2P were shown as endpoint dilutions, respectively (FIG. 4). Each symbol represents an individual mouse and the horizontal bar indicates the geometric mean of the group. Horizontal dashed lines denote the lower limit of detection. N.T., not tested.

[0325] Serum antibody titers in Syrian hamsters immunized with SARS-CoV-2 SSS-np and SSS-WW-np. Male Syrian hamsters (n = 15) were immunized with adjuvant alone, SARS-CoV-2 S2P, SARS-CoV-2 SSS-np, or SARS-CoV-2 SSS-WW-np intramuscularly thrice at weeks 0, 4, and 8. All protein and nanoparticle vaccines were given at 10 µg with AddaVax adjuvant. Serum samples were collected at week 10 and ELISA binding antibodies were measured against SARS-CoV-2 S6P (ancestral strain) and SARS- CoV-2 S2P of EG.5.1 subvariant (FIG. 40). Lower and upper limit of detection are indicated as dash lines in FIG.40.

[0326] Protection of Syrian hamsters immunized with SARS-CoV-2 SSS-np and SSS-WW-np. Male Syrian hamsters (n = 15) were immunized with adjuvant alone, 110 4893-1038-0739, v.1SARS-CoV-2 S2P, SARS-CoV-2 SSS-np, or SARS-CoV-2 SSS-WW-np intramuscularly thrice at weeks 0, 4, and 8. All protein and nanoparticle vaccines were given at 10 µg with AddaVax adjuvant. All animals were intranasally challenged at week 12 with SARS-CoV-2 EG.5.1 subvariant (10,000 PFU). Five animals from each group were euthanized on days 2, 4, and 7 for virological readout (FIG.41).

[0327] Serum neutralizing antibody titers in mice immunized with SARS-CoV- 2 SSS-nps. Neutralization assays were performed with pseudovirus expressing the spike protein of the ancestral SARS-CoV-2 WA-1 (Washington 1) strain using the 293 Flpin- TMPRSS2-ACE2 cells. The day before performing the neutralization assay, cells were plated into 96-well white / black isoplates (Perkin Elmer) at 10,000 cells well-1. Serially diluted serum samples were incubated with pseudovirus for 45 min at 37°C before added to cells. Following 2 hours of incubation, wells were replenished with 150 µl of fresh media and incubated for 3 days. Cells were lysed at 72 h post-infection, and luciferase activity was measured. Serum dilutions to reach 50% neutralization (IC50) were calculated using GraphPad Prism (v9) and plotted (FIG. 5). Each symbol represents an individual mouse, and the horizontal bar indicates the geometric mean of the group. Horizontal dashed lines denote the lower limit of detection.

[0328] Cross-reactive binding of SARS-CoV-2 NPs to various betacoronaviruses. BALB / cJ mice (n = 10) were immunized intramuscularly with 10 µg of immunogens with AddaVax adjuvant (50% v / v final) three times with 4 weeks intervals. Serum samples taken at 2 weeks after the final immunization were analyzed by ELISA. ELISA plates were coated with 2 µg well-1of recombinant antigen for overnight at 4°C. The plates were then blocked and incubated with serially diluted serum samples starting at 1 / 100. Plates were washed with PBST and incubated with goat anti-mouse IgG-HRP secondary antibody (GE Healthcare) at 1 / 5000 in PBS. After washing plates were developed using TMB substrate and the reactions were stopped with 1N sulfuric acid. The absorbance at 450 nm were measured and endpoint titers were calculated using GraphPad Prism (v9) software. Serum antibody titers to spike proteins of SARS-CoV, OC43, MERS-CoV and HKU1 are shown as endpoint dilutions, respectively, in FIG. 6. Each symbol represents an individual mouse and the horizontal bar indicates the geometric mean of the group. Horizontal dashed lines denote the lower limit of detection. 111 4893-1038-0739, v.1

[0329] FPLC profiles of the SARS-CoV and combo NPs. SSS-nps were purified from culture supernatants of Expi293 cells transfected with expression vector DNA plasmids encoding either SARS-CoV SSS-WW-np, SARS-CoV-2(HC) / SARS-CoV(LC) SSS-WW-np or SARS-CoV(HC) / SARS-CoV-2(LC) SSS-WW-np with a GNA lectin. Eluates from the GNA lectin column were further purified by SEC using a Superose 6 Increase 10 / 300 GL column. Size exclusion chromatography (SEC) profiles of the SARS- CoV SSS-WW-np and SARS-CoV-2 / SARS-CoV combination nanoparticles are shown in FIG. 7. The peak around the retention volume of 10.5 ml corresponds to fully assembled nanoparticles.

[0330] Negative stain EM of SARS-CoV and SARS-CoV-2 / SARS-CoV combo SSS-WW-nps. Purified SSS-WW-nps were diluted to 0.02 mg ml-1with buffer containing 10 mM HEPES, pH 7.0, and 150 mM NaCl, and adsorbed onto glow-discharged carbon-coated copper mesh grids for 15 s. The grids were washed with the buffer and stained with 0.7% uranyl formate. Images were obtained at a magnification of 50,000 semi-automatically on an FEI Tecnai T20 transmission electron microscope (TEM) operated at 200 kV, equipped with a 2,048- by 2,048-pixel Eagle charge-coupled-device (CCD) camera using SerialEM (Mastronarde, 2005). The pixel size was 0.44 nm and particles were picked automatically using software developed in house. Reference-free 2D classification was performed using SPIDER (Shaikh et al., 2008) and Relion 1.4 (Scheres, 2012).2D class averages are shown in FIG. 8. SSS trimers are projected outward and clearly visible. SSS trimers on SARS-CoV SSS-WW-np (left), SARS-CoV-2(HC) / SARS-CoV(LC) SSS-WW-np (upper right) and SARS-CoV(HC) / SARS-CoV-2(LC) SSS-WW-np (bottom right) showed closed states. * * *

[0331] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. 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Claims

CLAIMS 1. An engineered protein comprising an engineered coronavirus S2 protein ectodomain that comprises a sequence at least 90% identical to the sequence of positions 697-1159 of SEQ ID NO: 1 or 2, wherein the engineered coronavirus S2 protein ectodomain comprises the following substitution(s) relative to the sequence of SEQ ID NO: 1: V991W and / or T998W.

2. The engineered protein of claim 1, wherein the engineered coronavirus S2 protein ectodomain further comprises the following substitutions relative to the sequence of SEQ ID NO: 1: K986P and V987P.

3. The engineered protein of claim 1, wherein the engineered coronavirus S2 protein ectodomain further comprises the following substitutions relative to the sequence of SEQ ID NO: 1: F817P, A892P, A899P, A942P, K986P, and V987P.

4. The engineered protein of any one of claims 1-3, wherein the engineered coronavirus S2 protein ectodomain comprises the following substitutions relative to the sequence of SEQ ID NO: 1: F817P, A892P, A899P, A942P, K986P, V987P, V991W, and T998W.

5. The engineered protein of any one of claims 1-4, wherein the engineered coronavirus S2 protein ectodomain further comprises the following substitutions relative to the sequence of SEQ ID NO: 1: S704C and K790C.

6. The engineered protein of any one of claims 1-5, wherein the engineered coronavirus S2 protein ectodomain further comprises the following substitutions relative to the sequence of SEQ ID NO: 1: Q957E.

7. The engineered protein of any one of claims 1-6, wherein the engineered coronavirus S2 protein ectodomain comprises the following substitutions relative to the sequence of SEQ ID NO: 1: S704C, K790C, F817P, A892P, A899P, A942P, Q957E, K986P, V987P, V991W, and T998W.

8. The engineered protein of any one of claims 1-7, wherein the engineered coronavirus S2 protein ectodomain further comprises an additional engineered disulfide bond, an additional cavity filling substitution, and / or a substitution that provides an electrostatic or polar interaction. 1224893-1038-0739, v.

19. The engineered protein of any one of claims 1-8, wherein the engineered coronavirus S2 protein ectodomain further comprises an engineered disulfide bond comprising paired cysteine substitutions at positions corresponding to: Y707C / P792C, Y707C / T883C, S704C / K790C, A713C / L894C, Q755C / N969C, G757C / S968C, G891C / P1069C, S1030C / D1041C, G1035C / V1040C, S735C / T859C, I770C / A1015C, L727C / S1021C, V911C / N1108C, A672C / A694C, A1080C / I1132C, S659C / S698C, V722C / A930C, A903C / Q913C, S974C / D979C, P728C / V951C, V736C / L858C, S884C / A893C, P807C / S875C, T791C / A879C, G799C / A924C, A570C / V963C, T874C / S1055C, V729C / A1022C, L822C / A1056C, Q965C / S1003C, A972C / Q992C, I980C / Q992C, A1078C / V1133C, H1088C / T1120C, I870C / S1055C, T1117C / D1139C, T1116C / Y1138C, I896C / Q901C, G885C / Q901C, F1103C / P1112C, G889C / L1034C, E819C / S1055C, A972C / I980C, I1081C / N1135C, or E819C / Q1054C.

10. The engineered protein of any one of claims 1-9, wherein the engineered coronavirus S2 protein ectodomain further comprises a cavity filling substitution selected from: T724M, I1013F, H1058W, Q901M, S875F, H1088W, L1141F, V1040F, T778L, L938F, V963L, R1039F, V826L, A899F, Q779M, L894F, H1058F, H1058Y, V1040Y, H1088Y, V1104I, R1000Y, R1000W, A944F, T724I, A944Y, S730L, A890V, D1118F, or S1003V.

11. The engineered protein of any one of claims 1-10, wherein the engineered coronavirus S2 protein ectodomain further comprises a proline substitution selected from: F817P, L865P, T866P, A892P, A899P, T912P, A893P, Q895P, K921P, L922P, N978P, A942P, G946P, or S975P.

12. The engineered protein of any one of claims 1-11, wherein the engineered coronavirus S2 protein ectodomain further comprises an electrostatic interaction substitution selected from: T961D, T961E, L966E, L966D, T859K, T752K, T912R, L828K, L828R, S730R, T961D, A766E, P862E, T859K, Q957E, G769K, G769E, T778Q, A713S, or I1130Y.

13. The engineered protein of any one of claims 1-12, wherein the engineered coronavirus S2 protein ectodomain comprises a sequence that has at least 95% identity to positions 697- 1159 of SEQ ID NO:

1.

14. The engineered protein of any one of claims 1-12, wherein the engineered coronavirus S2 protein ectodomain comprises a sequence that has at least 95% identity to positions 697- 1159 of SEQ ID NO: 2.

15. The engineered protein of any one of claims 1-12, wherein the engineered coronavirus S2 protein ectodomain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

9.

16. The engineered protein of any one of claims 1-15, wherein the engineered coronavirus S2 protein ectodomain comprises the amino acid sequence of SEQ ID NO:

9.

17. The engineered protein of any one of claims 1-16, wherein the C-terminal residue of the engineered coronavirus S2 protein ectodomain is linked to a protein nanoparticle subunit by a peptide linker, or is directly linked to the protein nanoparticle subunit.

18. The engineered protein of claim 17, wherein the protein nanoparticle subunit is a ferritin molecule derived from the insect Trichoplusia ni.

19. The engineered protein of claim 18, wherein the engineered protein comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 3 or 4.

20. The engineered protein of claim 18, wherein the engineered protein comprises the amino acid sequence of SEQ ID NO: 3 or 4.

21. A protein nanoparticle, comprising the engineered protein of any one of the prior claims.

22. The nanoparticle of claim 21, further comprising an additional antigen.

23. The nanoparticle of claim 22, wherein the engineered protein and the additional antigen are both engineered proteins of any one of claims 1-20.

24. The nanoparticle of claim 23, wherein the engineered protein and the additional antigen share a sequence identity of at least 90%.

25. The nanoparticle of claim 22, wherein the additional antigen is an engineered protein comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 27 or 28, of which the C-terminal residue is linked to a protein nanoparticle subunit by a peptide linker, or is directly linked to the protein nanoparticle subunit. 1244893-1038-0739, v.

126. The nanoparticle of claim 22, wherein the additional antigen is an engineered protein comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 23 or 24.

27. The engineered protein of any one of claims 1-16, wherein the engineered coronavirus S2 protein ectodomain is fused or conjugated to a trimerization domain.

28. The engineered protein of claim 27, wherein the engineered coronavirus S2 protein ectodomain is fused to a trimerization domain.

29. The engineered protein of claim 28, wherein the trimerization domain is positioned C- terminally relative to the engineered coronavirus S2 protein ectodomain.

30. The engineered protein of claim 29, wherein the trimerization domain comprises a T4 fibritin trimerization domain.

31. The engineered protein of any one of claims 1-16, wherein the engineered coronavirus S2 protein ectodomain is fused or conjugated to a transmembrane domain.

32. The engineered protein of claim 30, wherein the engineered coronavirus S2 protein ectodomain is fused to a transmembrane domain.

33. The engineered protein of claim 31 or 32, wherein the transmembrane domain comprises a beta-coronavirus spike protein transmembrane domain.

34. The engineered protein of claim 33, wherein the transmembrane domain comprises a homologous beta-coronavirus spike protein transmembrane domain.

35. The engineered protein of claim 33, wherein the transmembrane domain comprises a SARS-CoV-2 transmembrane domain.

36. The engineered protein of any one of the prior claims, further comprising a coronavirus S1 protein ectodomain.

37. The engineered protein of claim 36, wherein the engineered protein comprises an amino acid sequence at least 95% identical to the amino acid sequence of positions 16-1208 of SEQ ID NO: 1 or 2. 1254893-1038-0739, v.

138. The engineered protein of claim 36, wherein the engineered protein comprises an amino acid sequence at least 95% identical to the amino acid sequence of positions 319-1208 of SEQ ID NO: 1 or 2.

39. An engineered coronavirus trimer comprising at least one subunit according to any one of claims 27-38.

40. The engineered trimer of claim 39, wherein the trimer is stabilized in a prefusion conformation relative to a trimer of wildtype S protein subunits.

41. The engineered trimer of claim 39, wherein the trimer comprises at least one engineered disulfide bond between subunits.

42. The engineered trimer of claim 41, wherein the at least one engineered disulfide bond between subunits is selected from: Y707C and P792C; Y707C and T883C; S704C and K790C; A713C and L394C; Q755C and N969C; G757C and S968C; G891C and P1069C; S1030C and D1041C; and G1035C and V1040C.

43. The engineered trimer of claim 41 or 42, wherein the at least one engineered disulfide bond between subunits is selected from: V705C and A983C; T547C and N968C; T961C and S758C; and / or T961C and Q762C.

44. A nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence of an engineered protein of any one of claims 1-38.

45. The nucleic acid of claim 44, wherein the nucleic acid comprises a DNA expression vector.

46. The nucleic acid of claim 44, wherein the nucleic acid comprises an mRNA.

47. An immunogenic composition comprising the engineered protein, protein nanoparticle, engineered trimer, or nucleic acid molecule of any one of the prior claims, and a pharmaceutically acceptable carrier.

48. The composition of claim 47, further comprising an adjuvant.

49. A method of preventing coronavirus infection or a disease associated with coronavirus infection in a subject, comprising administering to the subject an effective amount of the immunogenic composition according to claim 47 or 48. 1264893-1038-0739, v.

150. The method of claim 49, wherein the method elicits a beta-coronavirus cross-reactive immune response.

51. The method of claim 49, wherein the method elicits the production of pan-coronavirus antibodies in the subject.

52. The immunogenic composition of claim 47 or 48 for use in inducing an immune response to a SARS-CoV-2 S ectodomain in a subject.

53. Use of the engineered protein, protein nanoparticle, engineered trimer, or nucleic acid molecule of any one of the prior claims in the manufacture of an immunogenic composition for inducing an immune response to a SARS-CoV-2 S ectodomain. 1274893-1038-0739, v. 1

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