Prefusion-stabilized SARS-COV-2 spike s2 subunit as antigen for broad pan-coronavirus vaccines

Abstract

Provided herein is a stable mutant coronavirus spike protein stem domain in a prefusion conformation comprising: an S2 subunit only, that has been modified to comprise; at least one additional intra-monomeric disulfide bond that stabilizes the S2 subunit; and 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus pre-fusion, S2-only spike protein maintains the prefusion conformation. Coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS-CoV-2, existing variants, or an emerging variant thereof.

Description

PREFUSION-STABILIZED SARS-COV-2 SPIKE S2 SUBUNIT AS ANTIGEN FOR BROAD PAN-CORONAVIRUS VACCINESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 599,201, filed November 15, 2023, the entire contents of which are incorporated herein by reference..TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates in general to the field of a coronavirus spike glycoprotein with improved expression and stability, and more particularly, to a prefusion-stabilized SARS-CoV-2 spike S2 subunit as antigen for broad pan-coronavirus vaccines with improved expression and stability.STATEMENT OF FEDERALLY FUNDED RESEARCH

[0003] This invention was made with government support under AI165072 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0004] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on November 14, 2024, is named “LJII2030WO.xml” and is 13,762 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTION

[0005] Without limiting the scope of the invention, its background is described in connection with SARS- CoV-2.

[0006] The 800 million human infections with SARS-CoV-2 and the likely emergence of new variants and additional coronaviruses necessitate a better understanding of the essential spike glycoprotein and the development of immunogens that foster broader and more durable immunity. The S2 fusion subunit is more conserved in sequence, is essential to function, and would be a desirable immunogen to boost broadly reactive antibodies. It is, however, unstable in structure and in its wild-type form, cannot be expressed alone without irreversible collapse into a six-helix bundle. In addition to the irreversible conformational changes of fusion, biophysical measurements indicate that spike also undergoes a reversible breathing action. However, spike in an open, “breathing” conformation has not yet been visualized at high resolution.

[0007] Most of the current SARS-CoV vaccines are based on different formulations of the Spike (S) glycoprotein, which is the major target for neutralizing and protective antibodies, and an important contributor to the overall T cell response. CoV S proteins are composed of an SI subunit that mediates interactions with host cells, and an S2 subunit, which contains the machinery that drives virus-cell membrane fusion. The SI subunit comprises an N-terminal domain (NTD), a receptor-binding domain(RBD), and two subdomains (CTDs). The S2 subunit includes a helix-loop region spanning from the fusion peptide (FP) to heptad repeat 1 (HR1) constituting a metastable structure that during fusion of the virus with host cells undergoes a transition to the post-fusion conformation that has a long, stable a-helix. The stalk region of S can be further divided into “hip”, “knee”, and “ankle” regions in which the knee functions as a hinge that provides considerable flexibility and allows movement of S on the viral surface.

[0008] Given the role of SI in direct interactions with the host receptor, the majority of vaccine -induced antibodies and antibodies elicited upon natural infection target this subunit, which contains the immunodominant RBD. However, vaccine pressures could increase the frequency of mutation events that cause amino acid changes that are concentrated in the S 1 subunit, and the RBD in particular, of most SARS- CoV-2 variants described thus far. The presence of these mutations allows the variants to escape neutralizing mAbs and dampen immunity after either vaccination of natural infections.

[0009] What is needed are novel antigens and vaccines that will not be susceptible to the mutations found in the SI subunit. These antigens must be stable and easy to manufacture.SUMMARY OF THE INVENTION

[0010] As embodied and broadly described herein, an aspect of the present disclosure relates to a mutant coronavirus pre-fusion, S2-subunit only spike protein comprising: the S2 subunit only, that has been modified to comprise; at least one additional intra-monomeric disulfide bond that stabilizes the S2 subunit; and 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus prefusion, S2-only spike protein maintains the prefusion conformation. In one aspect, a furin cleavage site loop has been deleted. In another aspect, the intra-monomeric disulfide bond is formed between N717C- A1070C; I788C-A876C; Y789C-A879C; K790C-A879C; orN801C-N928C. In another aspect, the 1, 2, 3, 4, or 5 proline mutations are selected from F817P, A892P, A899P, A942P, P986K, K986P, V987P, and P987V. In another aspect, the proline mutations are not K986P and V987P mutations. In another aspect, the higher stability is selected from: increased temperature stability, increased freeze / thaw stability, or increased lyophilization / resuspension stability. In another aspect, the mutant coronavirus spike protein further comprises a purification peptide at an amino-terminus, a carboxy-terminus, or both. In another aspect, the mutant coronavirus spike protein has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence to SEQ ID NO: 1. In another aspect, the coronavirus is wherein the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS- CoV-2, Alpha (B.l.1.7 and Q lineages), Beta (B.1.351 and descendent lineages), Gamma (P. l and descendent lineages), Epsilon (B.1.427 and B. 1.429), Eta (B.1.525), Iota (B. 1.526), Kappa (B.1.617.1), Mu (B.1.621, B. 1.621.1), Zeta (P.2), Delta (B.1.617.2 and AY lineages), and Omicron (B. 1.1.529) or its variants BA.l, BA.2, rBA.3, BA.4, BA.5, BA.2.75 (including BH and CH), BA.2.86, XBB, XBB 1.5, XBB.1.16, XBB 1.5 like (including F456L, EG.5, FL. 1.5.1, XBB 1.16.6, and FE. l), XBB.1.9.1, XBB. 1.9.2, XBB.2.3, CH.1. 1, BA.2.74, KP.2, KP.2.3, KP.3, KP.3. 1. 1, and / or LB. 1, or an emerging variant thereof. In another aspect, the coronavirus is SARS-CoV-2.

[0011] As embodied and broadly described herein, an aspect of the present disclosure relates to a nucleic acid encoding a mutant coronavirus pre-fusion, S2-subunit only spike protein comprising: the S2 subunit only, that has been modified to comprise; at least one additional intra-monomeric disulfide bond that stabilizes the S2 subunit; and 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus pre-fusion, S2-only spike protein maintains the profusion conformation. In one aspect, the nucleic acid further comprises a vector.

[0012] As embodied and broadly described herein, an aspect of the present disclosure relates to a cell comprising a nucleic acid encoding a mutant coronavirus pre-fusion, S2-subunit only spike protein comprising: the S2 subunit only, that has been modified to comprise; at least one additional intra- monomeric disulfide bond that stabilizes the S2 subunit; and 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability. In one aspect, the cell is a human cell.

[0013] As embodied and broadly described herein, an aspect of the present disclosure relates to a vaccine composition comprising a mutant coronavirus pre-fusion, S2-subunit only spike protein comprising: the S2 subunit only, that has been modified to comprise; at least one additional intra-monomeric disulfide bond that stabilizes the S2 subunit; and 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, and a pharmaceutically acceptable excipient. In one aspect, the vaccine further comprises an adjuvant.

[0014] As embodied and broadly described herein, an aspect of the present disclosure relates to a nanoparticle comprising a vaccine composition comprising a mutant coronavirus pre-fusion, S2-subunit only spike protein comprising: the S2 subunit only, that has been modified to comprise; at least one additional intra-monomeric disulfide bond that stabilizes the S2 subunit; and 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability. In one aspect, the nanoparticle comprises at least two mutant coronavirus spike proteins. In another aspect, the mutant coronavirus spike proteins are formed into dimers, trimers, or multimers. In another aspect, the nanoparticles comprise ferritin nanoparticles, polymeric nanoparticles, or both.

[0015] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of making a mutant coronavirus pre-fusion, S2-subunit only spike protein comprising: obtaining a nucleic acid sequence encoding a coronavirus spike protein; and modifying the nucleic acid sequence of the coronavirus spike protein such that an amino acid sequence expressed by the nucleic acid sequence comprises: an S2 subunit only, that has been modified to comprise; at least one additional intra-monomeric disulfide bond that stabilizes the S2 subunit; and 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus pre-fusion, S2-subunit only spike protein maintains a profusion conformation. In another aspect, the method further comprises the step of expressing the mutant coronavirus spike protein in a bacteria, fungi, mammalian cell, avian cell, insect cell, or plant cell. In another aspect, a furin cleavage site loop has been deleted. In another aspect, the intra-monomeric disulfide bond is formed between N717C-A1070C; I788C-A876C; Y789C-A879C; K790C-A879C; or N801C- N928C. In another aspect, the 1, 2, 3, 4, or 5 proline mutations are selected from F817P, A892P, A899P, A942P, P986K, K986P, V987P, and P987V. In another aspect, the proline mutations are not K986P andV987P mutations. In another aspect, the higher stability is selected from: increased temperature stability, increased freeze / thaw stability, or increased lyophilization / re suspension stability. In another aspect, further comprises a purification peptide at an amino-terminus, a carboxy-terminus, or both. In another aspect, the mutant coronavirus spike protein has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence to SEQ ID NO: 1. In another aspect, the coronavirus is wherein the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS- CoV-2, Alpha (B.l.1.7 and Q lineages), Beta (B.1.351 and descendent lineages), Gamma (P. l and descendent lineages), Epsilon (B.1.427 and B. 1.429), Eta (B.1.525), Iota (B. 1.526), Kappa (B.1.617.1), Mu (B.1.621, B. 1.621.1), Zeta (P.2), Delta (B.1.617.2 and AY lineages), and Omicron (B. 1.1.529) or its variants BA.l, BA.2, rBA.3, BA.4, BA.5, BA.2.75 (including BH and CH), BA.2.86, XBB, XBB 1.5, XBB.1.16, XBB 1.5 like (including F456L, EG.5, FL. 1.5.1, XBB 1.16.6, and FE. l), XBB.1.9.1, XBB. 1.9.2, XBB.2.3, CH.1. 1, BA.2.74, KP.2, KP.2.3, KP.3, KP.3. 1. 1, and / or LB. 1, or an emerging variant thereof.

[0016] As embodied and broadly described herein, an aspect of the present disclosure relates to a nucleic acid sequence encoding a mutant coronavirus pre-fusion, S2-subunit only spike protein comprising: one or more mutations and deletions that change an amino acid sequence of a coronavirus spike protein S2 subunit only, comprising; at least one additional intra-monomeric disulfide bond that stabilizes the S2 subunit; and 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus pre-fusion, S2-subunit only spike protein maintains a prefusion conformation. In one aspect, the coronavirus is wherein the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS-CoV-2, Alpha (B. l.1.7 and Q lineages), Beta (B.1.351 and descendent lineages), Gamma (P. l and descendent lineages), Epsilon (B.1.427 and B.1.429), Eta (B. 1.525), Iota (B.1.526), Kappa (B.1.617.1), Mu (B.1.621, B.1.621.1), Zeta (P.2), Delta (B.1.617.2 and AY lineages), and Omicron (B. 1.1.529) or its variants BA.l, BA.2, rBA.3, BA.4, BA.5, BA.2.75 (including BH and CH), BA.2.86, XBB, XBB 1.5, XBB.1.16, XBB 1.5 like (including F456L, EG.5, FL. 1.5.1, XBB 1.16.6, and FE. l), XBB.1.9.1, XBB.1.9.2, XBB.2.3, CH.1.1, BA.2.74, KP.2, KP.2.3, KP.3, KP.3. 1.1, and / or LB. l, or an emerging variant thereof. In another aspect, the mutant coronavirus spike protein has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence to express a protein of SEQ ID NO: 1.

[0017] As embodied and broadly described herein, an aspect of the present disclosure relates to a vector comprising a nucleic acid sequence encoding a mutant coronavirus spike protein comprising: one or more mutations and deletions that change an amino acid sequence of a coronavirus spike protein S2 subunit only, comprising; at least one additional intra-monomeric disulfide bond that stabilizes the S2 subunit; and 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus pre-fusion, S2- only spike protein maintains the prefusion conformation. In one aspect, the vector is selected for expression in a bacteria, fungi, mammalian cell, avian cell, insect cell, or plant cell. In another aspect, the vector is in a bacteria, fungi, mammalian cell, avian cell, insect cell, or plant cell. In another aspect, the coronavirus is wherein the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta),SARS-CoV-2, Alpha (B. 1.1.7 and Q lineages), Beta (B.1.351 and descendent lineages), Gamma (P. l and descendent lineages), Epsilon (B.1.427 and B. 1.429), Eta (B.1.525), Iota (B. 1.526), Kappa (B.1.617.1), Mu (B.1.621, B. 1.621.1), Zeta (P.2), Delta (B.1.617.2 and AY lineages), and Omicron (B. 1.1.529) or its variants BA.l, BA.2, rBA.3, BA.4, BA.5, BA.2.75 (including BH and CH), BA.2.86, XBB, XBB 1.5, XBB.1.16, XBB 1.5 like (including F456L, EG.5, FL. 1.5.1, XBB 1.16.6, and FE. l), XBB.1.9.1, XBB. 1.9.2, XBB.2.3, CH.1. 1, BA.2.74, KP.2, KP.2.3, KP.3, KP.3. 1. 1, and / or LB. 1, or an emerging variant thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

[0019] FIGS. 1A-1F. Structure of the prefusion SARS-CoV-2 S2 subunit. (FIG. 1A) Side view of the cryo-EM density map of the trimeric SARS-CoV-2 S2 subdomain. Each protomer is shaded differently, and glycan densities are black. FIG. IB) Side (left) and top (right) views of the S2-DS5 molecular model, gray scale as in (FIG. 1A). (FIG. 1C) and (FIG. ID) Alignment of S2-DS5 with the S2 from the full-length stabilized Hexapro (PDB:6XKL). (FIG. 1C) shows a side view of both trimers with one protomer left side (S2-DS5) and middle (Hexapro). Zoomed view of the ‘open state’ of S2-DS5 by an opening and twisting motion. (FIG. ID) shows the top views with the differences in distances between the P987 of two neighboring protomers. (FIG. IE) S2-DS5 molecular model zooming the not previously solved loop 833- 856, and (FIG. IF) the novel inter-protomeric interaction between the fusion peptide loop 882-898 (right) and the loop 1033-1037 (left), in comparison with S2 in the close state from HXP (structure showed in grey). Contacts are shown in black dash lines for S2-DS5 and grey for HP.

[0020] FIGS. 2A - 2E. Cryo-EM analysis of the 6C10Fab / S2-DS5 interaction and relative conservation of epitope contact residues. (FIG. 2A) Side view of the cryo-EM density map of S2-DS5 ectodomain in complex with Fab 6C10. S2-DS5 are as in FIGS. 1A to IF, and the 6C10 Fab density is top right and top left (heavy chain) and area adjacent and below the top left and top right (light chain). (FIG. 2B) Side (left) and top (right) views of the 6C10Fab / S2-DS5 molecular model complex, shaded as in (FIG. 2A). (FIG. 2C) Zoom of the footprint of one 6C10 Fab to one S2-DS5 protomer. The 6C10 Fab is shown as ribbon representation and the S2-DS5 as the cryo-EM density. The footprint is colored darker than the rest of the S2. (FIG. 2D) S2-DS5 interactions with 6C10. The left panel shows a close-up view of interactions made by 6C10 CDRH1, H2, and framework region 2. Right panel shows the 6C10 CDRH3 interactions and the interaction of the 6C10 light chain (N92 and N93) with the residue R765 of S2-DS5. (FIG. 2E) Sequence alignment of the 6C10 conformational epitope (spike region 733-765 and loop 833-856) from the five human-infective beta-coronaviruses (The sequences used in the alignment are: SARS-CoV-2 (NCBI GenBank: QII57161.1, SEQ ID NO: 2), SARS-CoV (NCBI GenBank: ABF65836.1, SEQ ID NO: 3), MERS-CoV (NCBI GenBank: AKN11072.1, SEQ ID NO: 4), HCoV-OC43 (NCBI GenBank:YP 009555241.1, SEQ ID NO: 5), and HCoV-HKUl (NCBI GenBank: ABD75513.1, SEQ ID NO: 6). Similarity in conservation with SARS-CoV-2 is shown shaded , and the interaction positions with the Fab are marked with an asterisk.DETAILED DESCRIPTION OF THE INVENTION

[0021] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0022] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

[0023] The present inventors recognized that difficulties in making fusion subunits alone in their prefusion conformation is a loss for immunogen development, as the fusion subunit is more conserved than the receptor-binding subunit. Between SARS-CoV-2 and SARS-CoV, for example, the S2 fusion subunits are 90% conserved while the SI N-terminal domain and receptor binding domains are 50% and 76% conserved, respectively (13). Targeting S2-reactive immune responses offers an opportunity to build cross- reactive protection bridging from previous coronavirus infections, and extending to coronaviruses yet to emerge. As an example, convalescent sera from patients infected with SARS-CoV or SARS-CoV-2, who were unlikely to have ever been exposed to MERS, nonetheless include antibodies that react with or neutralize MERS (14, 15),' and immunization with S2-based constructs elicited a broadly cross-reactive IgG antibody response that recognized the spike proteins of not only SARS-CoV-2 variants, but also SARS- CoV- 1 and the four endemic human coronaviruses (16). Meanwhile, other individuals, naive to SARS- CoV -2, had immune responses that reacted with regions of its S2, presumably from prior common cold CoV infections (17). Several known S2-reactive antibodies are neutralizing and protective against infection and pathology in vivo (18-20). “Boosting” with the conserved fusion subunit after a whole -envelope spike would be an attractive strategy for building broader immunity (21), but unfortunately, the wild-type fusion subunit for these viruses can not be expressed alone in the correct pre-fusion conformation.

[0024] In addition to the dramatic and irreversible pre-fusion to post-fusion refolding event, the viral surface glycoproteins are also thought to exhibit reversible, conformational “breathing” motions (22, 23). A low-resolution structure of the respiratory syncytial virus (RSV) F trimer, for example, suggests that F protein exists in both open and closed conformations (24). Breathing motions have been described for hantaviruses (25), flaviviruses (25, 26) and retroviruses. Motion in the envelope (Env) protein of HIV-1has been observed in both soluble and virion-surface forms (27), (28), and is thought to be required for binding of the HIV-1 receptor and coreceptor. Antibodies that cause Env trimer dissociation have been described and may depend on the same breathing motions required for receptor binding. For SARS-CoV- 2, molecular simulations of spike protein also suggest a dynamic prefusion state, including opening of the monomers from the trimeric stem interface (29). This motion could enhance accessibility of receptorbinding domains and expose the conserved trimer interface for recognition by antibody (30). Although supported by molecular dynamics and biochemical and biophysical measurements, the structure of an open state of SARS-CoV-2 has not yet been determined.

[0025] As used herein, the term “mutant coronavirus spike protein” refers to an engineered fusionmediating S2 subunit of SARS-CoV-2, that stably remains in its prefusion conformation alone, that is, in the absence of the receptor-binding S 1 clamp . Electron microscopy was used to confirm that the engineered S2 indeed stably remains in its prefusion conformation. Also described herein is competition mapping of novel human mAbs against S2 from both convalescent and vaccinated individuals, studies which were facilitated by the new existence of a pre-fusion S2-only antigen. One of these mAbs allowed the determination of a cryo-EM structure at 2.9 A of the prefusion S2. Notably, this structure illustrates a high- resolution view of S2 in an open conformation that reflects prior biophysical predictions. It was found that human mAbs from both convalescent (not yet vaccinated) and vaccinated (but not yet infected) individuals react with an upper, inner surface of the open S2, an epitope that is accessible in a “breathing” open S2, but masked in a closed S2, suggesting that the breathing motion of the full-length spike required to expose this epitope happens in both natural infection and in the context of vaccination. Notably, the open conformation of S2 reveals a greater number of intramonomer stabilizing interactions in the trimer base as well as an alternate position and conformation of the fusion peptide.

[0026] The present invention thus provides a novel pre-fusion S2-only immunogen and research tool but also a high-resolution view of a conformational state of spike relevant for antibody recognition and the immune response. The over 800 million human SARS-CoV-2 infections, coupled with the likely emergence of new variants and new coronaviruses, necessitates development of additional vaccine strategies featuring conserved sites such as S2 that foster broad and durable immunity.

[0027] A central goal for SARS-CoV-2 vaccines is to reduce incidence of symptomatic disease through generation of enduring protective immunity. However, the recent emergence of SARS-CoV-2 variants of concern (VOC) poses a risk to first-generation vaccine efficacy and durability of both infection- and vaccine-induced humoral immunity. Lineage B.1.351 (informally known as the South African variant) is particularly concerning due to substitutions that confer increased transmissibility and reduced sensitivity to neutralization by heterotypic convalescent and vaccine-induced sera. Development of structurally designed vaccine candidates with improved immunogenicity and breadth of coverage is critical for controlling emergent VOC.

[0028] To address these issues associated with current spike constructs and emergence of VOC, the present inventors developed spike proteins containing different proline substitutions, cleavage site linkers,and interprotomer disulfide bonds. The present disclosure describes the production of “VFLIP” (five (V) prolines, Flexibly-Linked, Inter-Protomer disulfide) spikes that remain trimeric without exogenous trimerization motifs, and which have enhanced thermostability relative to earlier spike constructs. Surface plasmon resonance (SPR) and cryo-EM analysis confirm the native-like antigenicity of VFLIP and its improved utility for structural biology applications. Moreover, mice immunized with the VFLIP spike elicited significantly more potent neutralizing antibody responses against live SARS-CoV-2 D614G and B.1.351 compared to those immunized with S-2P. Taken together, the data demonstrate that VFLIP is a thermostable, covalently-linked, native-like spike trimerthat represents a next-generation research reagent, diagnostic tool, immunogen, and vaccine.

[0029] Multiple families of viruses that cause severe human disease have an essential surface fusion protein. This protein, whether termed env in retroviruses, GP in filoviruses, GPC in arenaviruses, hemagglutinin in influenza viruses, or spike in coronaviruses, is the essential piece of viral machinery that drives the virus into the cell. These critical proteins are targeted by neutralizing antibodies (nAbs) and are a major component of vaccines. Both antibodies and vaccines are challenged, however, by mutagenic substitutions that escape antibody recognition. Developing vaccines that target conserved, unchanging parts of the fusion protein is key for long-lasting, broadly active immune protection.

[0030] The present invention includes the design and study of a set of SARS-CoV-2 prefusion stabilized S2 constructs bearing VFLIP features (i.e., inter-monomeric disulfide bond at positions Y707 and T883, and relocation of prolines at positions 986 and 987). The invention includes stabilized S2 constructs that can be used as protective vaccine immunogens against emerging hCoVs. In one example, the stabilized S2 constructs can be displayed on ketol-acid reductoisomerase (KARI) nanoparticles that exhibit efficacy against divergent hCoV species. In addition, antibodies that are cross-reactive with the stem domain of different S proteins serve as valuable tools for prospective serological surveillance of future CoV spillover into humans.

[0031] As used herein, the terms “profusion” and “profusion conformation” refer to a coronavirus spike protein S2-only subunit that has been engineered the fusion-mediating S2 subunit of SARS-CoV-2, that stably remains in its profusion conformation alone, in the absence of the receptor-binding S 1 clamp. Viral fusion proteins contain two subunits: one which mediates membrane fusion and the other which mediates initial receptor binding. The receptor-binding subunit (SI) is more subject to selective pressure and mutation, while the fusion subunit (S2) remains more conserved in sequence. The two subunits are formed by enzymatic cleavage of a polyprotein precursor in the producer cell, and although cleaved apart, remain associated on the viral surface. As long as they are associated, one (the receptor-binding subunit) serves as a clamp on the conformation of the other (the fusion subunit). On the surface of the virus, the fusion subunit exists in a metastable, initial, pre-fusion conformation, interacting with or intertwined with the receptor-binding subunit. Upon attachment to the receptor and often, exposure to low pH, the receptorbinding subunit releases its “clamp” and the fusion subunit irreversibly rearranges into a more stable, six- helix bundle structure called the “post-fusion conformation”. This energetically favorable conformationalrearrangement can thwart the neutralizing antibody response. For example, multiple neutralizing antibodies have been discovered that target only the metastable prefusion conformation and are unable to recognize the post-fusion, six-helix bundle conformation (1-12). The greater inherent stability of the postfusion six-helix bundle means that without its clamping receptor-binding subunit, the fusion protein, when expressed alone, adopts only the post-fusion six-helix bundle conformation. The metastability of the fusion subunit has challenged development of immunogens or research reagents that constitute the prefusion S2 (or gp41) alone.

[0032] As used herein, the term “antigen” refers to a mutant SARS-CoV-2 spike protein containing one or more epitopes (either linear, conformational or both) that will stimulate a host’s immune-system to make a humoral and / or cellular antigen-specific response. The term is used interchangeably with the term “immunogen.” Normally, a B-cell epitope will include at least about 5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope, such as a CTL epitope, will include at least about 7-9 amino acids, and a helper T-cell epitope at least about 12-20 amino acids. Normally, an epitope will include between about 7 and 15 amino acids, such as, 9, 10, 12 or 15 amino acids. The term includes polypeptides, which include modifications, such as deletions, additions and substitutions (generally conservative in nature) as compared to a native sequence, so long as the protein maintains the ability to elicit an immunological response, as defined herein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts, which produce the antigens.

[0033] As used herein, the term “adjuvant” refers to a substance that non-specifically changes or enhances an antigen-specific immune response of an organism to the antigen. Generally, adjuvants are non-toxic, have high-purity, are degradable, and are stable. With respect to the present disclosure, an adjuvant may be selected from aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as Bordatella pertussis or Mycobacterium tuberculosis-derived proteins. Suitable adjuvants are commercially available as, for example, Freund’s Incomplete Adjuvant and Complete Adjuvant (Pifco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; and Quil A. Suitable adjuvants also include, but are not limited to, toll-like receptor (TLR) agonists, particularly toll-like receptor type 4 (TLR-4) agonists (e.g., monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetics or analogs), aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, liposomes, oil-in-water emulsions, MF59, and squalene. In some embodiments, the adjuvants are not bacterially-derived exotoxins. In an embodiment, adjuvants may include adjuvants which stimulate a Th 1 type response such as 3DMPL or QS21. Adjuvants may also include certain synthetic polymers such as poly amino acids and co-polymers of amino acids, saponin, paraffin oil, and muramyl dipeptide. Adjuvants also encompass genetic adjuvants such asimmunomodulatory molecules encoded in a co-inoculated DNA, or as CpG oligonucleotides. The coinoculated DNA can be in the same plasmid construct as the plasmid immunogen or in a separate DNA vector. The reader can refer to Vaccines (Basel). 2015 Jun; 3(2): 320-343 for further examples of suitable adjuvants.

[0034] As used herein, the term “immunological response” refers to an immune response to an antigen or composition that triggers in a subject a humoral and / or a cellular immune response to a mutant SARS- CoV-2 spike protein of the present disclosure. For purposes of the present disclosure, a “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and / or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells (CTLs). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A “cellular immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and / or other white blood cells, including those derived from CD4+ and CD8+ T-cells. Hence, an immunological response may include one or more of the following effects: the production of antibodies by B-cells; and / or the activation of suppressor T-cells and / or gamma-delta T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest. These responses may serve to neutralize infectivity, and / or mediate antibody-complement, or antibody-dependent cell cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art. In many instances, it will be desirable to have multiple administrations of the vaccine, usually not exceeding six to ten immunizations, more usually not exceeding four immunizations, e.g., one or more, usually at least about three immunizations. The immunizations will normally be at from two to twelve-week intervals, more usually from three-to-five-week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain protective levels of the antibodies. The course of the immunization may be followed by assays for antibodies for the supernatant antigens. The assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescent agents, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays.

[0035] The present disclosure can be used to generate one or more diagnostic and / or therapeutic antibodies against the novel antigens of the present disclosure. The antibodies can include polyclonal antibodies, such as those from immunized animals, but also include monoclonal antibodies made in vitro or in vivo. Both the polyclonal and monoclonal antibodies can be used in, e.g., radioimmunoassays, enzyme-linkedimmunosorbent assays, immunocytopathology, and flow cytometry for in vitro diagnosis, and in vivo for diagnosis and immunotherapy of human disease. Both the pan-specific and / or monoclonal antibodies of the present disclosure can be used for diagnosis and / or therapy of C0VID19. Monoclonal antibodies may be generated by immunizing an animal, such as a mouse, isolating B cells from the immunized animal and fusing them with immortalized cells, as described by, e.g., Kohler and Milstein (1975, Nature 256:495- 497), or as described by Kozbor et al. (1983, Immunology Today 4:72), or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96), relevant portions incorporated herein by reference. Alternatively, a clone encoding at least the Fab portion of the antibody is optionally obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246: 1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937), relevant portions incorporated herein by reference. For human use, the complementarity determining regions (CDRs) of the light and heavy chains of the monoclonal antibody can be engineered into a human antibody backbone or framework to make humanized antibodies.

[0036] In an aspect of the present disclosure, a method of diagnosing a coronavirus infection in a subject. In certain aspects, the method includes: (a) contacting a biological sample obtained from the subject with the mutant coronavirus spike protein provided herein including embodiments thereof, and (b) detecting binding of one or more antibodies to said mutant coronavirus spike protein, thereby diagnosing the coronavirus infection in said subject. In certain embodiments, the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS-CoV-2, or an emerging variant thereof. SARS- CoV-2 variants include the Wuhan parental sequence with or without the D614G mutation, Alpha (B.l.1.7 and Q lineages), Beta (B. 1.351 and descendent lineages), Gamma (P. l and descendent lineages), Epsilon (B.1.427 and B. 1.429), Eta (B.1.525), Iota (B.1.526), Kappa (B.1.617.1), Mu (B.1.621, B. 1.621. 1), Zeta (P.2), Delta (B.1.617.2 and AY lineages), and Omicron (B.1.1.529) or its variants BA.l, BA.2, rBA.3, BA.4, BA.5, BA.2.75 (including BH and CH), BA.2.86, XBB, XBB 1.5, XBB.1.16, XBB 1.5 like (including F456L, EG.5, FL. 1.5.1, XBB 1.16.6, and FE. l), XBB.1.9.1, XBB.1.9.2, XBB.2.3, CH.1.1, BA.2.74, KP.2, KP.2.3, KP.3, KP.3.1.1, and / or LB. l.

[0037] In an aspect of the present disclosure, a method of diagnosing a SARS-CoV-2 infection in a subject. In certain aspects, the method includes: (a) contacting a biological sample obtained from the subject with the mutant coronavirus spike protein provided herein including embodiments thereof, and (b) detecting binding of one or more antibodies to said mutant coronavirus spike protein, thereby diagnosing the SARS- CoV-2 infection in said subject.

[0038] In an aspect of the present disclosure, a method for evaluating effectiveness of a coronavirus vaccine in a subject. In certain aspects, the method comprises (a) contacting a biological sample from a subject who has been administered with a vaccine for a coronavirus with the mutant coronavirus spike protein described herein, (b) detecting antibodies in the biological sample that specifically bind to the mutant coronavirus spike protein, and (c) performing quantitative and qualitative analysis of the antibodiesdetected in the biological sample, thereby evaluating effectiveness of the coronavirus vaccine in the subject. In certain embodiments, the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU 1 (beta), SARS-CoV-2, or an emerging variant thereof. SARS-CoV-2 variants include the Wuhan parental sequence with or without the D614G mutation, Alpha (B.l.1.7 and Q lineages), Beta (B.1.351 and descendent lineages), Gamma (P. 1 and descendent lineages), Epsilon (B. 1.427 and B.1.429), Eta (B.1.525), Iota (B. 1.526), Kappa (B. 1.617.1), Mu (B.1.621, B. 1.621.1), Zeta (P.2), Delta (B.1.617.2 and AY lineages), and Omicron (B. 1.1.529) or its variants BA.l, BA.2, rBA.3, BA.4, BA.5, BA.2.75 (including BH and CH), BA.2.86, XBB, XBB 1.5, XBB.1.16, XBB 1.5 like (including F456L, EG.5, FL. 1.5.1, XBB 1.16.6, and FE. l), XBB. 1.9.1, XBB.1.9.2, XBB.2.3, CH. 1.1, BA.2.74, KP.2, KP.2.3, KP.3, KPA 1.1, and / or LB.l.

[0039] In an aspect of the present disclosure, a method for evaluating effectiveness of a SARS-CoV-2 vaccine in a subject. In certain aspects, the method comprises (a) contacting a biological sample from a subject who has been administered with a vaccine for a coronavirus with the mutant coronavirus spike protein described herein, (b) detecting antibodies in the biological sample that specifically bind to the mutant coronavirus spike protein, and (c) performing quantitative and qualitative analysis of the antibodies detected in the biological sample, thereby evaluating effectiveness of the SARS-CoV-2 vaccine in the subject.

[0040] As used herein, the terms an “immunogenic composition” and “vaccine” refer to a composition that comprises a mutant SARS-CoV-2 spike protein, or a nucleic acid that expresses the mutant SARS- CoV-2 spike protein, where administration of the immunogenic composition or vaccine to a subject results in the development in the subject of a humoral and / or a cellular immune response to the antigenic molecule of interest, and by extension, to the virus.

[0041] As used herein, the term “substantially purified” refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically, in a sample a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0042] As used herein, the term a “coding sequence” or a sequence which “encodes” a mutant SARS- CoV-2 spike polypeptide, refers to a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory sequences (or “control elements”) and in vitro or in vivo. The boundaries of the coding sequence are determined by a start codon at the 5 ’ (amino) terminus and a translation stop codon at the 3 ’ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3’ to the coding sequence.

[0043] As used herein, the term “control elements”, includes, but is not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3 ’to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences, and / or sequence elements controlling an open chromatin structure.

[0044] As used herein, “nanoparticles” refer to any particles, which are between 1 and 100 nanometers in size. The present disclosure includes formulations comprising the mutant coronavirus spike proteins of the present disclosure formed into nanoparticles or microparticles. In one example, nanoparticles or microparticles are formed with a protein and / or into a polymer matrix. The polymer matrix can be made with, e.g., poly (L-glycolic acid) (PLGA), polyglycolic acid (PGA), polylactic acid (PLA), poly(L-lactic acid) (PLLA), poly(epsilon-Caprolactone) PCL, Poly(methyl vinyl ether-co-maleic anhydride), polyglycolide, poly-L-lactide, poly-D-lactide, poly(amino acids), polyethyleneglycol PEG), polydioxanone, polycaprolactone, polygluconate, polylactic acid-polyethylene oxide copolymers, polyorthoesters, polyhydroxybutyrate, polyanhydride, polyphosphoester, poly(alpha-hydroxy acid), ferritin, chitosan, alginate, collagen, dextran, polyester, cellulose, carboxymethyl cellulose, modified cellulose, collagen, or combinations thereof. In some examples, the nanoparticles are partially or fully biodegradable.

[0045] As used herein, the term “nucleic acid” includes, but is not limited to, DNA or RNA that encodes the mutant SARS-CoV-2 spike proteins of the present disclosure, whether expressed or optimized for prokaryotic or eukaryotic expression. The term also captures sequences that include any of the known base analogs of DNA and RNA.

[0046] As used herein, 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 promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when active. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, 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.

[0047] As used herein, the term “recombinant” refers to a polynucleotide that encodes the mutant SARS- CoV-2 spike protein whether from the viral genome, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and / or (2) is linked to a polynucleotide other than that to which it is linked in nature. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. “Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cell cultures,” and other such terms denoting prokaryotic microorganisms or eukaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the originalparent, due to accidental or deliberate mutation. Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.

[0048] Techniques for determining amino acid sequence “similarity” are well known in the art. In general, “similarity” means the exact amino acid to amino acid comparison of two or more polypeptides at the appropriate place, where amino acids are identical or possess similar chemical and / or physical properties such as charge or hydrophobicity. A so-termed “percent similarity” then can be determined between the compared polypeptide sequences. Techniques for determining nucleic acid and amino acid sequence identity also are well known in the art and include determining the nucleotide sequence of the mRNA for that gene (usually via a cDNA intermediate) and determining the amino acid sequence encoded thereby and comparing this to a second amino acid sequence. In general, “identity” refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.

[0049] “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0050] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0051] As used herein, a “comparison window” includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full-length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. NatT. Acad. Sci. USA 85:2444, by computerized implementations ofthese algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995)).

[0052] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative -scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0053] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0054] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identicalis that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0055] The term “mutant coronavirus spike protein” or “VFLIP” as provided herein includes any of the recombinant or naturally-occurring forms of a coronavirus spike protein, or variants or homologs thereof that maintain coronavirus Spike protein activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to coronavirus S2 subunit Spike Protein). In aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring coronavirus Spike protein polypeptide. In embodiments, coronavirus Spike protein is the protein as identified by the UniProt reference number P0DTC2, or a variant, homolog or functional fragment thereof.

[0056] Two or more polynucleotide sequences can be compared by determining their “percent identity.” Two or more amino acid sequences likewise can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or peptide sequences, is generally described as the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be extended to use with peptide sequences using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986), relevant portion incorporated herein by reference. Suitable programs for calculating the percent identity or similarity between sequences are generally known in the art.

[0057] As used herein, the term a “vector” refers to a nucleic acid capable of transferring gene sequences to target cells (e.g., bacterial plasmid vectors, viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, “vector construct,” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of one or more sequences of interest in a host cell. Thus, the term includes cloning and expression vehicles, as well as viral vectors. The term is used interchangeable with the terms “nucleic acid expression vector” and “expression cassette.”

[0058] Many suitable expression systems are commercially available, including, for example, the following: baculovirus expression (Reilly, P. R., et al., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992); Beames, et al., Biotechniques 11:378 (1991); Pharmingen; Clontech, Palo Alto, Calif.)), vaccinia expression systems (Earl, P. L., et al., “Expression of proteins in mammalian cells using vaccinia” In Current Protocols in Molecular Biology (F. M. Ausubel, et al. Eds.), Greene Publishing Associates & Wiley Interscience, New York (1991); Moss, B., et al., U.S. Pat. No. 5,135,855, issued Aug. 4, 1992), expression in bacteria (Ausubel, F. M., et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, Inc., Media Pa.; Clontech), expression in yeast(Rosenberg, S. and Tekamp-Olson, P., U.S. Pat. No. RE35,749, issued, Mar. 17, 1998, herein incorporated by reference; Shuster, J. R., U.S. Pat. No. 5,629,203, issued May 13, 1997, herein incorporated by reference; Gellissen, G., et al., Antonie Van Ueeuwenhoek, 62(l-2):79-93 (1992); Romanos, M. A., et al., Yeast 8(6):423-488 (1992); Goeddel, D. V., Methods in Enzymology 185 (1990); Guthrie, C., and G. R. Fink, Methods in Enzymology 194 (1991)), expression in mammalian cells (Clontech; Gibco-BRL, Ground Island, N.Y.; e.g., Chinese hamster ovary (CHO) cell lines (Haynes, J., et al., Nuc. Acid. Res. 11:687-706 (1983); 1983, Lau, Y. F„ et al., Mol. Cell. Biol. 4: 1469-1475 (1984); Kaufinan, R. J., “Selection and coamplification of heterologous genes in mammalian cells,” in Methods in Enzymology, vol. 185, pp 537- 566. Academic Press, Inc., San Diego Calif. (1991)), and expression in plant cells (plant cloning vectors, Clontech Laboratories, Inc., Palo-Alto, Calif., and Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.; Hood, E„ et al., J. Bacteriol. 168: 1291-1301 (1986); Nagel, R„ et al., FEMS Microbiol. Lett. 67:325 (1990); An, et al., “Binary Vectors”, and others in Plant Molecular Biology Manual A3: 1-19 (1988); Miki, B. L. A., et al., pp. 249-265, and others in Plant DNA Infectious Agents (Hohn, T., et al., eds.) Springer-Verlag, Wien, Austria, (1987); Plant Molecular Biology: Essential Techniques, P. G. Jones and J. M. Sutton, New York, J. Wiley, 1997; Miglani, Gurbachan Dictionary of Plant Genetics and Molecular Biology, New York, Food Products Press, 1998; Henry, R. J., Practical Applications of Plant Molecular Biology, New York, Chapman & Hall, 1997), relevant portions of any of the above are incorporated herein by reference.

[0059] As used herein, the term “subject” refers to any member of the subphylum chordata, including, but not limited to, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered. The system described above is intended for use in any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly.

[0060] As used herein, the terms “pharmaceutically acceptable” or “pharmacologically acceptable” refer to a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual in a formulation or composition without causing any unacceptable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0061] As used herein, the term “administering” refers to oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, ortransdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “co-administer” it is meant that a composition describedherein is administered at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy. The compounds of the invention can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation). The compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0062] As used herein, the term “co-administer” refers to a compound or composition described herein that is administered at the same time, just prior to, or just after the administration of one or more additional therapies. The compounds provided herein can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation). The compositions of the present disclosure can be delivered transdermally, by a topical route, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. The preparations may also be combined with inhaled mucolytics (e.g., rhDNase, as known in the art) or with inhaled bronchodilators (short- or long-acting beta agonists, short- or long-acting anticholinergics), inhaled corticosteroids, or inhaled antibiotics to improve the efficacy of these drugs by providing additive or synergistic effects. The compositions of the present invention can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, nanoparticles, pastes, jellies, paints, powders, and aerosols. Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions. The compositions of the present invention may additionally include components to provide sustained release and / or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). In another embodiment, the formulations of the compositions of the present invention can be delivered by the use ofliposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46: 1576-1587, 1989).

[0063] The compositions of the present invention may additionally include components to provide sustained release and / or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug -containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). In embodiments, the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46: 1576-1587, 1989). The compositions of the present invention can also be delivered as nanoparticles, such as protein nanoparticles.

[0064] As used herein, the term “pharmaceutically acceptable” is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.

[0065] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer’s solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinylpyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0066] The term “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

[0067] The term “preparation” is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0068] The pharmaceutical preparation is optionally in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The unit dosage form can be of a frozen dispersion.

[0069] The term “vaccine” refers to a composition that can provide active acquired immunity to and / or therapeutic effect (e.g., treatment) of a particular disease or a pathogen. A vaccine typically contains one or more agents that can induce an immune response in a subject against a pathogen or disease, i.e., a target pathogen or disease. The immunogenic agent stimulates the body’s immune system to recognize the agent as a threat or indication of the presence of the target pathogen or disease, thereby inducing immunological memory so that the immune system can more easily recognize and destroy any of the pathogen on subsequent exposure. Vaccines can be prophylactic (e.g., preventing or ameliorating the effects of a future infection by any natural or pathogen, or of an anticipated occurrence of cancer in a predisposed subject) or therapeutic (e.g., treating cancer or infection in a subject who has been diagnosed with the cancer or infection). The administration of vaccines is referred to vaccination. In embodiments, a vaccine composition can provide nucleic acid, e.g., mRNA that encodes antigenic molecules (e.g., peptides) to a subject. The nucleic acid that is delivered via the vaccine composition in the subject can be expressed into antigenic molecules and allow the subject to acquire immunity against the antigenic molecules. In the context of the vaccination against infectious disease, the vaccine composition can provide mRNA encoding antigenic molecules that are associated with a certain pathogen, e.g., one or more peptides that are known to be expressed in the pathogen (e.g., pathogenic bacterium or virus).

[0070] Pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized sepharose (TM), agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants).

[0071] As used herein, the term “treatment” refers to any of (i) the prevention of infection or reinfection with SARS-CoV-2, as in a traditional vaccine, (ii) the reduction or elimination of symptoms, and (iii) the substantial or complete elimination of the pathogen in question. Treatment may be effected prophylactically (prior to infection) or therapeutically (following infection).

[0072] As used herein, the term “effective dose” refers to that amount of one or more mutant SARS-CoV- 2 spike proteins of the disclosure sufficient to induce immunity, to prevent and / or ameliorate an infection or to reduce at least one symptom of an infection and / or to enhance the efficacy of another dose of a SARS- CoV-2. An effective dose may refer to the amount of a mutant SARS-CoV-2 spike protein sufficient to delay or minimize the onset of an infection. An effective dose may also refer to the amount of a mutant SARS-CoV-2 spike protein that provides a therapeutic benefit in the treatment or management of an infection. Further, an effective dose is the amount with respect to a mutant SARS-CoV-2 spike protein of the disclosure alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of an infection. An effective dose may also be the amount sufficient to enhance a subject’s (e.g., a human’s) own immune response against a subsequent exposure to an infectious agent. Levels of immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and / or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay. In the case of a vaccine, an “effective dose” is one that prevents disease and / or reduces the severity of symptoms.

[0073] As used herein, the term “immune stimulator” refers to a compound that enhances an immune response via the body’s own chemical messengers (cytokines). These molecules comprise various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro- inflammatory activities, such as interferons, interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc. The immune stimulator molecules can be administered in the same formulation as the mutant SARS-CoV- 2 spike proteins of the disclosure or can be administered separately. Either the protein or an expression vector encoding the protein can be administered to produce an immunostimulatory effect.

[0074] As used herein, the term “protective immune response” or “protective response” refers to an immune response mediated by antibodies against an infectious agent, which is exhibited by a vertebrate (e.g., a human), which prevents or ameliorates an infection or reduces at least one symptom thereof. Mutant SARS-CoV-2 spike proteins of the disclosure can stimulate the production of antibodies that, for example, neutralize infectious agents, blocks infectious agents from entering cells, blocks replication of saidinfectious agents, and / or protect host cells from infection and destruction. The term can also refer to an immune response that is mediated by T-lymphocytes and / or other white blood cells against an infectious agent, exhibited by a vertebrate (e.g., a human), that prevents or ameliorates flavivirus infection or reduces at least one symptom thereof.

[0075] As used herein, the term “antigenic formulation” or “antigenic composition” refers to a preparation which, when administered to a vertebrate, e.g., a mammal, will induce an immune response.

[0076] As used herein, the terms “immunization” or “vaccine” are used interchangeably to refer to a formulation which contains one or more of the mutant SARS-CoV-2 spike proteins of the present disclosure, which is in a form that is capable of being administered to a vertebrate and which induces a protective immune response sufficient to induce immunity to prevent and / or ameliorate an infection and / or to reduce at least one symptom of an infection and / or to enhance the efficacy of another dose of the mutant SARS-CoV-2 spike proteins. Typically, the vaccine comprises a conventional saline or buffered aqueous solution medium in which the composition of the present disclosure is suspended or dissolved. In this form, the composition of the present disclosure can be used conveniently to prevent, ameliorate, or otherwise treat an infection. Upon introduction into a host, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies and / or cytokines and / or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and / or other cellular responses.

[0077] The practice of the present disclosure employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Short Protocols in Molecular Biology, 4th ed. (Ausubel et al. eds., 1999, John Wiley & Sons); Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag); Fundamental Virology, Second Edition (Fields & Knipe eds., 1991, Raven Press, New York), relevant portion incorporated herein by reference.

[0078] Difficulties in making fusion subunits alone in their pre-fusion conformation is a loss for immunogen development, as the fusion subunit is more conserved than the receptor-binding subunit. Between SARS-CoV-2 and SARS-CoV, for example, the S2 fusion subunits are 90% conserved while the SI N-terminal domain and receptor binding domains are 50% and 76% conserved, respectively (13). Targeting S2-reactive immune responses offers an opportunity to build cross-reactive protection bridging from previous coronavirus infections, and extending to coronaviruses yet to emerge. As an example, convalescent sera from patients infected with SARS-CoV or SARS-CoV-2, who were unlikely to have ever been exposed to MERS, nonetheless include antibodies that react with or neutralize MERS (14, 15); and immunization with S2-based constructs elicited a broadly cross-reactive IgG antibody response thatrecognized the spike proteins of not only SARS-CoV-2 variants, but also SARS-CoV-1 and the four endemic human coronaviruses (16). Meanwhile, other individuals, naive to SARS-CoV-2, had immune responses that reacted with regions of its S2, presumably from prior common cold CoV infections (17). Several known S2-reactive antibodies are neutralizing and protective against infection and pathology in vivo (18-20). “Boosting” with the conserved fusion subunit after a whole -envelope spike would be an attractive strategy for building broader immunity (21), but unfortunately, the wild-type fusion subunit for these viruses can not be expressed alone in the correct pre-fusion conformation.

[0079] In addition to the dramatic and irreversible pre-fusion to post-fusion refolding event, the viral surface glycoproteins are also thought to exhibit reversible, conformational “breathing” motions (22, 23). A low-resolution structure of the respiratory syncytial virus (RSV) F trimer, for example, suggests that F protein exists in both open and closed conformations (24). Breathing motions have been described for hantaviruses (25), flaviviruses (25, 26) and retroviruses. Motion in the envelope (Env) protein of HIV-1 has been observed in both soluble and virion-surface forms (27), (28), and is thought to be required for binding of the HIV-1 receptor and coreceptor. Antibodies that cause Env trimer dissociation have been described and may depend on the same breathing motions required for receptor binding. For SARS-CoV- 2, molecular simulations of spike protein also suggest a dynamic prefusion state, including opening of the monomers from the trimeric stem interface (29). This motion could enhance accessibility of receptorbinding domains and expose the conserved trimer interface for recognition by antibody (30). Although supported by molecular dynamics and biochemical and biophysical measurements, the structure of an open state of SARS-CoV-2 has not yet been determined.

[0080] The inventors have engineered the fusion-mediating S2 subunit of SARS-CoV-2, to stably remain in its prefusion conformation alone, in the absence of the receptor-binding SI clamp. Evidence from electron microscopy shows that the engineered S2 indeed stably remains in its prefusion conformation. Also described herein is competition mapping of novel human mAbs against S2 from both convalescent and vaccinated individuals, studies that were facilitated by the new existence of a pre-fusion S2-only antigen. One o f these mAbs allowed the determination of a cryo-EM structure at 2.9 A of the prefusion S2. Notably, this structure illustrates a high-resolution view of S2 in an open conformation that reflects prior biophysical predictions. It was found that human mAbs from both convalescent (not yet vaccinated) and vaccinated (but not yet infected) individuals react with an upper, inner surface of the open S2, an epitope that is accessible in a “breathing” open S2, but masked in a closed S2, showing that the breathing motion of the full-length spike required to expose this epitope happens in both natural infection and in the context of vaccination. Notably, the open conformation of S2 reveals a greater number of intramonomer stabilizing interactions in the trimer base as well as an alternate position and conformation of the fusion peptide.

[0081] The following sequences can be used with the present invention, SARS-CoV-2 (NCBI GenBank: MT 121215.1 ), SARS-CoV (NCBI GenBank: ABF65836.1), MERS-CoV (NCBI GenBank: AKN 11072. 1 ),HCoV-OC43 (NCBI GenBank: YP 009555241.1), and HCoV-HKUl (NCBI GenBank: ABD75513.1), relevant sequences incorporated herein by reference.

[0082] This work provides a novel pre-fusion S2-only immunogen and research tool but also a high- resolution view of a conformational state of spike relevant for antibody recognition and the immune response. The over 800 million human SARS-CoV-2 infections, coupled with the likely emergence of new variants and new coronaviruses, necessitates the development of additional vaccine strategies featuring conserved sites, such as S2, that foster broad and durable immunity.

[0083] Example 1. Design of the SARS-CoV-2 stem domain in the prefusion conformation.

[0084] The introduction of disulfide bonds can substantially increase the stability of viral glycoproteins, as has been demonstrated for HIV, LASV and RSV (4, 5, 7, 21, 31). The inventors have previously described the development of an engineered SARS-Cov-2 spike protein, termed SVFLIP, that contains five (V) proline substitutions (one subtracted from Hexapro to restore a native, stabilizing salt bridge between K986 and D748 (32)), a flexible linker (FL) in place of the native furin cleavage site, as well as a novel inter-protomer (IP) disulfide bond between residues Y707C-T883C, located in the interior of the S2 stem which maintains spike in its trimeric organization without an exogenous trimerization domain (33, 34). The S 1 region of this construct, and the FL from VFLIP, were genetically removed to create a SVFLIP S2- only construct (residues 691-1208) to serve as the basis for further engineering.

[0085] To stabilize the prefusion conformation of S2 in the absence of SI, the inventors used the Disulphide by Design server (35) to computationally predict optimal disulfide bonds in the VFLIP-S2 ectodomain. Of the 31 pairs identified, it was found that ten were likely to form the stabilizing disulfide bond in the pre-fusion (PDB: 6XKL), but not the post-fusion conformation (PDB: 7E9T) (36-38).

[0086] Each of these ten intra-monomeric disulfide bonds was engineered into the S2 subunit bearing VFLIP substitutions. To assess the viability of these constructs as potential vaccine antigens, the inventors comprehensively examined the feasibility of high-yield expression for each of the ten constructs in ExpiCHO cells, as well as conformation, protein aggregation and stability. The inventors also expressed the wild-type S2 ectodomain (residues 691-1208), which naturally refolds into the post-fusion conformation, as a control. Two constructs, DS2 and DS3, had minimal-to-no expression and were not characterized further. The remaining eight constructs, however, yielded a sufficient amount of protein to evaluate by size-exclusion chromatography (SEC). Seven of the eight constructs eluted as a major peak at 14 mL, with only minor shoulder and aggregation peaks. The eighth, DS1, eluted earlier, at 13 mL. Of note, four of the engineered constructs (DS1, DS5, DS6, and DS9) yielded more than more than 5 mg purified, monodisperse protein from a 50 mL culture of transiently transfected ExpiCHO cells (extrapolated yield: >100 mg / L). Non-reducing SDS-PAGE showed that all four proteins retain a covalently-linked trimeric state with efficient disulfide bond formation, as evidenced by a band at the expected molecular weight of 200 kDa and a lack of detectable low molecular weight species. The inventors did not observe higher molecular weight species that might indicate aberrant disulfide bond formation. Meanwhile, underreducing conditions, all constructs exhibited the monomeric molecular weight of the wild-type S2 (~80 kDa), showing that trimerization was anchored as expected by the Y707C-T883C disulfide bond.

[0087] FIGS. 1A-1F. Structure of the prefusion SARS-CoV-2 S2 subunit. (FIG. 1A) Side view of the cryo-EM density map of the trimeric SARS-CoV-2 S2 subdomain. Each protomer is shaded differently, and glycan densities are black. FIG. IB) Side (left) and top (right) views of the S2-DS5 molecular model, gray scale as in (FIG. 1A). (FIG. 1C) and (FIG. ID) Alignment of S2-DS5 with the S2 from the full-length stabilized Hexapro (PDB:6XKL). (FIG. 1C) shows a side view of both trimers with one protomer left side (S2-DS5) and middle (Hexapro). Zoomed view of the ‘open state’ of S2-DS5 by an opening and twisting motion. (FIG. ID) shows the top views with the differences in distances between the P987 of two neighboring protomers. (FIG. IE) S2-DS5 molecular model zooming the not previously solved loop 833- 856, and (FIG. IF) the novel inter-protomeric interaction between the fusion peptide loop 882-898 (right) and the loop 1033-1037 (left), in comparison with S2 in the close state from HXP (structure showed in grey) Contacts are shown in black dash lines for S2-DS5 and grey for HP.

[0088] Negative stain electron microscopy (ns-EM) showed that constructs DS4, DS5, DS6, DS7 and DS 10 adopt the expected ‘pear’ shape of S2 in its prefusion state. In contrast, wild-type S2 adopted only the characteristic stick shape of the post-fusion conformation. The remaining constructs were highly heterogeneous and did not yield any clear 2D classes. Next, the thermostability of DS4, DS5, DS6, DS7, and DS10 was evaluated by Differential Scanning Calorimetry (DSC). Of these, DS5 exhibited the highest thermostability with a Tm of 58.1±0.1 °C, compared to the lowest Tm, 54.8±0.1 °C, of DS6.

[0089] Engineered S2 construct, S2-DS5, was used because it stably presents only the pre-fusion conformation, exhibits one of the highest expression yields (-150 mg / L; similar to full-length SVFLIP ectodomain) and the greatest thermostability, showing its utility as both a potential immunogen and a research tool to elicit and map pre-fusion S2-specific antibodies.

[0090] Example 2. Binding kinetics and antigenic communities of anti-S2 antibodies isolated from human subjects.

[0091] Most antibody epitope mapping efforts have focused on the SI subunit and its receptor-binding domain. Comparatively fewer antibodies are known, and fewer epitopes mapped, in the S2 subunit. The inventors previously isolated and characterized a panel of antibodies that target the spike protein from convalescent (unpublished) and vaccinated (39) subjects. Approximately one-fourth react to regions outside the N-terminal and receptor-binding domains of SI, showing recognition of the S2 subunit. High- throughput SPR (Carterra LSA) was used to map the conformational specificity using SVFLIP-D614G, S2-DS5, and post-fusion S2 and evaluated cross-reactivity of these anti-S2 antibodies to S2-DS5-XBB 1.5, SVFLIP-XBB1.1, and SVFLIP-SARS-CoV-1. Of the 48 mAbs tested, two recognized full-length D614G Spike (S1+S2 domains) only, with no recognition of pre-fusion S2-DS5 or post-fusion S2 alone, showing that their epitopes involve S 1 in some way. Two mAbs were specific for post-fusion S2, with very weak or non-existent binding to full spike or pre-fusion S2-DS5. The remaining 44 mAbs, however, recognized both full-length Spike and the pre-fusion stabilized S2. One mAb (3B 11) recognized S2-DS5 but not post-fusion S2, showing that this mAh may bind to an epitope presented only in the prefusion conformation of S2. Aside from this single mAh, other Spike / DS5-S2-reactive mAbs also bound to post-fusion S2, and curiously, often did so with higher affinity. It was also found that 40 mAbs bound to XBB1.1 and SARS- CoV-1 spikes.

[0092] FIGS. 2A - 2E. Cryo-EM analysis of the 6C10Fab / S2-DS5 interaction and relative conservation of epitope contact residues. (FIG. 2A) Side view of the cryo-EM density map of S2-DS5 ectodomain in complex with Fab 6C10. S2-DS5 are as in FIGS. 1A to IF, and the 6C10 Fab density is top right and top left (heavy chain) and area adjacent and below the top left and top right (light chain). (FIG. 2B) Side (left) and top (right) views of the 6C10Fab / S2-DS5 molecular model complex, shaded as in (FIG. 2A). (FIG. 2C) Zoom of the footprint of one 6C10 Fab to one S2-DS5 protomer. The 6C10 Fab is shown as ribbon representation and the S2-DS5 as the cryo-EM density. The footprint is colored darker than the rest of the S2. (FIG. 2D) S2-DS5 interactions with 6C10. The left panel shows a close-up view of interactions made by 6C10 CDRH1, H2, and framework region 2. Right panel shows the 6C10 CDRH3 interactions and the interaction of the 6C10 light chain (N92 and N93) with the residue R765 of S2-DS5. (FIG. 2E) Sequence alignment of the 6C10 conformational epitope (spike region 733-765 and loop 833-856) from the five human-infective beta-coronaviruses (The sequences used in the alignment are: SARS-CoV-2 (NCBI GenBank: QII57161.1, SEQ ID NO: 2), SARS-CoV (NCBI GenBank: ABF65836.1, SEQ ID NO: 3), MERS-CoV (NCBI GenBank: AKN11072.1, SEQ ID NO: 4), HCoV-OC43 (NCBI GenBank: YP 009555241.1, SEQ ID NO: 5), and HCoV-HKUl (NCBI GenBank: ABD75513.1, SEQ ID NO: 6). Similarity in conservation with SARS-CoV-2 is shown shaded , and the interaction positions with the Fab are marked with an asterisk.

[0093] Next, 30 of the S2-DS5 reactive mAbs were sorted into epitope communities. S2-DS5 was preincubated with >10x the KD of each mAb and complexes injected over the same array used for kinetic studies. This panel of mAbs were sorted into four communities (S2-A to S2-D). Interestingly, the S2-A community, which contains mAbs from both convalescent and vaccinated subjects, represents a convergent solution to this particular antigenic region, as all but two members originate from the 3-30 VH germline.

[0094] Example 3. Structural characterization of an engineered S2 domain.

[0095] Representative nsEM 2D classes of the antigen-only S2-DS5 structure demonstrate that it adopts the pre-fusion conformation. To add molecular mass and a fiducial to improve cryoEM resolution, complexes of S2-DS5 were made with an antibody from each of four representative communities, and evaluated them by nsEM. One complex, of Fab 6C10 bound to S2-DS5, yielded a good distribution of particles and orientations, including top-down and side views, which was suitable for high-resolution 3D structure determination. Antibody 6C10 was identified in a convalescent individual, from PBMCs donated early in the pandemic prior to the availability of vaccines. Two datasets from two batches of the complex were independently collected. A final set of 407,314 particles of the complex led to a 2.9 A reconstruction, with Cl symmetry and no masks applied (FIG. 1A). Three copies of the 6C10 Fab fragment are bound to the profusion S2 trimer.

[0096] In the map, residues 706-1149 of S2 can be modeled (FIG. IB). As in pre-fusion structures of the complete spike, the C-terminal peptide (residues 1150-1208) is not visible. A density for each of 6 N- linked glycans attached to each S2 monomer (18 per trimer) was observed located in positions N709, N717, N801, N1074, N1098, and N1134. The newly engineered I788C-A876C intra-monomer disulfide, which holds S2 in the pre-fusion state, and the SVFLIP -derived Y707C-T883C inter-monomer disulfide, which maintains the trim er in the absence of an exogenous domain, are also both clearly ordered in the cryo-EM maps. The inventors further observe clear density for a loop comprising residues 833-856 located in the equatorial part of the full-length spike, under the CD 1 domain (FIG. IE). This loop has been poorly ordered in all pre-fusion structures of spike thus far (40-45).

[0097] Example 4. The breathing conformation.

[0098] Previous biophysical experiments demonstrated that S2 adopts closed and open states in the context of complete spike, whether wild-type or moderately stabilized by two prolines (30). However, all previous structures are of a similar closed conformation, in which a tightly trimerized S2 is capped by S 1. The high- resolution structure of the open conformation remains unknown.

[0099] Comparison of the S2 in the S2-DS5 / 6C10_Fab complex presented here to the S2 in previous spike structures, shows that this complex has captured a more open conformation. The difference in structure between this open and previous closed states is essentially a rigid-body, screw-opening motion from the bottom membrane-proximal region (MPER) toward the upper reaches of each S2 protomer. This motion opens the monomers from the vertical axis by 10° degrees with a concomitant rotation of each protomer apex counter-anticlockwise 26° degrees (FIG. 1C). In the open state, the apex of each protomer (defined by the position of Pro 987) is 50 A; in the closed state, the distance between each protomer’s Pro 987 is 23 A (FIG. ID). Analysis of S2 trimers using the PDBePISA server reported a total solvent-accessible surface of 22,300 A2 in the closed state and 23,700 A2 in the open state. In the closed conformation, the S2 trimer buries an average of 2,230 A2 in each interprotomeric interface with a predicted AG = -20.5 kcal mol-1, whereas the open S2 trimer buries 1,260 A2, with a predicted AG = -20. 1 kcal mol-1.

[0100] The protomers themselves only differ between closed and open states by 0.76 A r.m.s.d.; hence the motion for the most part is rigid body with each protomer top rotating open relative to the others. At the base, where the three protomers meet, the [3-sheet-containing membrane-proximal region (MPER) (residues 1070-1138) from each monomer mediates interactions with its neighbor. These interactions, together with a short a-helix before the N-termini of HR2 (residues 686-705), confer most of the quaternary interactions visualized in the cryo-EM S2-DS5 map.

[0101] Interestingly, the open conformation of S2 appears to stabilize interprotomer interactions formed by the wild-type residues in the S2 base. This is perhaps paradoxical that an open conformation induces more stable interactions, so restated, the opening action of the upper part of S2 acts to stabilize and anchor the base where the protomers connect. In full, closed spike, the upper parts of S2 interact with S 1. Relaxing the top into an open conformation may favor stabilization in the base; as the rigid body expansion of the top occurs with a concomitant compression of the base, satisfied by formation of chemically favorableinteractions. The resulting greater number of stabilizing interactions in the open conformation is most visible in loop 885-897 in the base, which corresponds to the recently described fusion peptide of the spike (38). In closed structures, whether formed by S-2P or Hexapro spikes, residues 886-891 form a short a- helix, residues Phe888 and Gly880 of the same protomer are in close contact with each other, and Gly889 and Ala890 interact with Lysl045, Glyl046, and Tyrl047 from the 1037-1047 loop of the adjacent protomer, with a predicted AG = -3.3 kcal mol-1 for the closed-state interactions (FIG. IF). In the open structure, there is an elongation of the 885-897 fusion peptide loop towards the 1037-1047 loop of the neighboring protomer. Residues Phe888, Gly889 and Ala890 are instead in closer contact to 1038-1041 (Lys-Arg-Val-Asp) (FIG. IF), with a predicted AG = -4.0 kcal mol-1 for the open-state interactions. Note that the structural differences in the base are related to the open conformation and not engineering or antibody binding, as all of these contacts are made by wild-type residues that are distant from the DS5 disulfide and distant from the Fab 6C10 footprint. Thus, this newly visualized interaction at the S2 trimeric base connection point may be important to maintaining the integrity of the trimer in the splayed-open or breathing conformation.

[0102] Example 5. Structural definition of the 6C10 epitope.

[0103] Cryo-EM analysis of 6C10 Fab bound to S2-DS5 shows stoichiometric symmetry, in which one copy of the 6C10 Fab is stably bound to each protomer of S2-DS5 (FIGS. 2A-2B). The heavy- and lightchain complementarity determining regions (CDRs) bind ~ 1,290 A2 of the molecular surface on each S2 protomer, with the majority of the interface area, ~87%, contributed by the heavy chain. 6C10 Fab approaches the S2-DS5 protomer from the side and bridges a novel quaternary epitope formed between the regions 733-772 and 844-862 of the apex of the S2 domain. Those two regions have no defined function yet, other than flanking the S2’ cleavage site (808-820).

[0104] The majority of the 6C10 paratope is contributed by CDRH3. The 20 amino acids of CDRH3 line the lateral side of the S2 apex (FIG. 2D) form four main stabilizing H-bonds. There, CDRH3 residues R102, Y105, S108, and Yl l l form hydrogen bonds with N848, K733, N737, and R765 of the spike, respectively. CDRH3 R102 also forms a salt bridge with N848 in S2. The CDRH2 forms additional hydrogen bonds between N52 of the antibody and Q853 of spike, N54 with L849, S55 N745 and Q853, and Q58 of the antibody with E748 and Q853 of S2. Y60 in antibody FR2 forms an additional hydrogen bond with residue S750 of the spike. Light chain N92 and N93 interact with N737 and R765 of S2.

[0105] Access to the 6C10 epitope requires a dynamic opening of the spike. There are two polypeptide sections that contribute to the epitope: residues 733-772 and 844-862. Residues 733-772 are fully occluded in the closed, complete spike. Further, in the closed conformation of spike, the heavy chain of 6C10 would clash with the position of the S 1 N-terminal domain of the neighboring closed protomer, and the light chain of 6C10 would clash with the SI C-terminal portion adjacent to the furin cleavage site. Based on the structure, those clashes could not be resolved by mere positioning of the RBDs “up” or “down”, or by the natural motion of the NTD necessary for lifting of the RBD - the spike protomers must breathe open relative to each other. The second section of the epitope, residues 844-862 have become disordered. This regionis accessible in the full-length spike and might be a starting point for, or sufficient for antibody binding. That hypothesis is supported by the fact that the two regions that form the 6C10 epitope are separated from each other in the postfusion conformation, although 6C10 can still recognize S2 in the postfusion conformation in addition to complete spike.

[0106] Despite the apparent complexity of the conformational epitope recognized by 6C10, the antibody community S2-C (competition group) to which 6C10 belongs is the most abundant among the panel of anti-S2 antibodies. The panel of mAbs was initially sorted using an S-2P full-length spike, which is prefusion, but has a demonstrated tendency to “breathe” (30). Moreover, residues contained in the footprint of 6C10 are highly conserved: 7 out of the 10 residues on the S2 that form H-bonds to 6C10 are conserved among the five human infective [3-CoVs (FIG. 2E).

[0107] Since the outbreak of COVID- 19, numerous vaccines have been developed and several have been authorized for use in humans. However, the emergence of variants of concern bearing numerous mutations has evaded antibody responses, particularly against SI -containing epitopes. Reactivity against S2 is broader and more durable against these variants, and may have played important roles in mitigating and preventing SARS-CoV-2 disease. Further, it has also been reported that pre-existing immunity against S2 epitopes conserved with common cold coronaviruses may have also mitigated SARS-CoV-2 severity (17, 40-42). Notably, the B cells of COVID-19 patients have increased somatic hypermutation and higher affinity against spike proteins of common cold coronaviruses (HCoV) than do pre-pandemic individuals, indicating that SARS-CoV-2 exposure might train the HCoV-induced B cells to produce more effective SARS-CoV-2 NAbs (43-45).

[0108] The present invention includes a structure-based design of a prefusion stabilized S2 ectodomain, with high thermostability, yield, and homogenous maintenance of a pre-fusion conformation. Five versions of S2 were engineered, which each yield a stable, prefusion molecule in the absence of an antibody or an SI subunit. These stabilized S2 ectodomains in isolation did not yield a high-resolution map, but did yield low-resolution (~7 A) cryoEM maps. These maps and nsEM reconstructions confirm the prefusion shape of stabilized S2 in the absence of a bound antibody. Complexing the engineered S2-DS5 with an antibody isolated from a SARS-CoV-2 convalescent patient allowed a higher resolution, 2.9 A cryo-EM structure to be obtained. The conformation of S2 is identical between the 7 A unbound and the 2.9 A antibody-bound structures.

[0109] Conformational changes on the S2 and repositioning of the fusion peptide. It has been suggested that full spike undergoes a sequential motion where lifting of RBDs to the “up” position reduces the contacts of the SI to S2 and allows S2 to “breathe” in a reversible motion from closed, to open and back (30). This “breathing” of the SARS-CoV-2 spike has been observed biophysically, and postulated by many (46) (47), but not yet visualized. All structures thus far for SARS-CoV-2 spike are of a closed conformation. For respiratory syncytial virus (RSV) and HIV, an open conformation has been visualized at very low resolution (24, 27, 28). Molecular simulations of ‘breathing and tilting’ have been also described for the flu HA, suggesting that these motions could potentially affect the attachment to the hostcell and the release of progeny viruses, and the open conformation is accessible to a broadly protective human antibody, FluA-20, suggesting that keeping it in that position could be a way to develop more effective drugs against the flu virus (48). In this work, the inventors present the first open conformation of a fusion subunit of a class 1 fusion protein at high resolution. The inventors also reveal that adopting the open conformation is essential for full recognition by an antibody identified in convalescent individual, and likely others in its competition group.

[0110] By way of explanation, but not a limitation of the present invention, the opening motion of S2 might be driven by residues in the recently described fusion peptide (FP, residues 876-909) (38). Of these residues, Phe888 in the center of the fusion peptide might be particularly important. Phe888 is fully hidden from solvent in the closed conformation, but in the open conformation is the main residue responsible for the new interactions described here between the fusion peptide loop 885-897. Further, in the open conformation, Phe888 is fully solvent accessible. Note that Phe 888 is completely conserved among all human infective CoVs, and that in the subsequent, triggered, postfusion, six-helix bundle conformation, Phe888 is located in the center of the fusion peptide inserted into the target membrane (38). Short peptides designed from the internal fusion peptide region of SARS-CoV-2 S2 have shown high conformationdependent inhibitory activity against the virus (49). Hence, taken together, this structure, of an open, breathing conformation of the FP illuminates steps in the fusion cascade and provides a new template for fusion inhibition.

[0111] Use of the open, breathing state in spike function and spike vaccination. Although it has not been directly demonstrated whether reversible breathing occurs in wild-type spikes on virions, it is possible that the ‘open’ S2 may also reflect an intermediate along the pathway to S 1 shedding, during the transition from the prefusion conformation to the postfusion conformation. If the ‘open’ stage is an on-pathway intermediate, antibodies or ligands that trap the protein in this state may block the protein along the pathway to fusion.

[0112] 6C10 recognizes two polypeptidic portions at the apex of S2: residues 733-772 and 844-862, with at least ten specific hydrogen bonds to 733-772 and at least six hydrogen bonds and a salt bridge to 844- 862. Of these sections, 733-772 is fully masked in the closed spike, while 844-862 is accessible. One of these sections alone may be sufficient for binding, as 6C10 also recognizes post-fusion S2 in which 733- 772 and 844-862 are too far apart to be simultaneously recognized by the same Fab fragment. Based on solvent accessibility, one might expect region 844-862 to be the initial attachment site for 6C10. Further, the initial attachment to 844-862 can best occur when S2 is in the pre-fusion conformation as 844- 862 is structurally different between pre- and post-fusion conformations, and antibody access to residues 844-862 is sterically blocked in the post-fusion conformation Similarly, mice immunized with an S-2P version of MERS S induced antibodies with epitopes present only in the profusion core of [3-coronaviruses (980-1006) (2, 21), showing that these antibodies may have also been selected only by a spike in the open conformation.

[0113] Although the 6C10 epitope is visualized here at high-resolution in the context of an S2-only structure, this epitope exists and is available in a conformation also available to the complete spike in the prefusion conformation. Antibodies in the same competition group as 6C10 were also identified in individuals vaccinated with S-2P, a form of spike stabilized to favor the pre-fusion conformation (50-52), but not yet infected. 6C10 also recognizes VFLIP formats of SARS-CoV-2 and SARS-CoV-1 spike with additional proline substitutions to more completely eliminate pre- to post-fusion conformational transitions of spike (33, 34, 53), further demonstrating that the epitope must be accessible in the presence of SI and in the prefusion S1 / S2 complex. The fact that 6C10 was identified in a person early in the pandemic, who was convalescent from COVID-19, but had not yet been vaccinated, suggests that these epitopes are exposed in natural infection. Further, these studies on the array of S2-reactive antibodies, from both infected (but not yet vaccinated) and vaccinated (but not previously infected) individuals, demonstrate that such epitopes are present in the vaccine-elicited repertoire as well as the natural infection-elicited repertoire. Breathing of the SARS-CoV-2 spike occurs in natural infection and in two-proline stabilized S-2P spike vaccination.

[0114] The alternative conformation presents new druggable sites. Antibodies against conserved epitopes in S2 can be used for the recognition of variants and coronaviruses yet to emerge. In general, antibodies against S2 do not neutralize in cell culture as effectively as those against SI, although the capacity to neutralize may depend on how the assay is organized (presence or absence of TMPRSS2), different cell types, for example. Further, antibodies may be protective in the absence of neutralization by tagging infected cells for destruction, or through a mechanism not recapitulated in monotypic neutralization assays. Fewer antibodies against S2 may also be described thus far in the literature, because S2 is less immunogenic. Boosting with an S2-only subunit would improve elicitation of such antibodies, but was previously thwarted by the lack of a prefusion S2-only antigen in the pre-fusion conformation with which to achieve boosts. Antibodies against S2 may also be less prevalent due to the previous technical challenges with identifying and characterizing such antibodies, even if they had been present in vaccinees or convalescent individuals. The provision here of a prefusion S2-only protein will facilitate the identification and characterization of more such S2-reactive antibodies.

[0115] Bacterial strains. E. coli strain Rosetta DE3 (Novagen) was grown in lysogeny broth. The genotype is: F-ompT hsdSB (rB-mB) gal dem (DE3) pRARE (CamR). Selection markers were used at the indicated concentrations: kanamycin (100 pg / mL); chloramphenicol (28.3 pg / mL).

[0116] Cell lines. Expi-CHO cells were obtained from Thermo Fisher Scientific and maintained in ExpiCHO-Expression Medium (Thermo Fisher Scientific). Vero CCL-81 cells were purchased from ATCC and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Coming) supplemented with 2% FBS, 1% penicillin-streptomycin, 1% HEPES buffer, and 1% non-essential amino acids.

[0117] Design of SARS-CoV-2 S2 variants. SARS-CoV-2 S2 variants (residues 691-1211) were initially designed using the VFLIP construct Wuhan strain (Genbank: MN908947), which contains 5 proline substitutions (F817P, A892P, A899P, A942P, and V967P), and an inter-protomer disulfide bond formedby Y707C-T883C. All variants were cloned into a PhCMV mammalian expression vector containing a C- terminal foldon trimerization domain followed by an HRV-3C cleavage site and a Twin-Strep-Tag.

[0118] Transient transfection and protein purification. SARS-CoV-2 S2 variants were transiently transfected in ExpiCHO-S cells (Thermo Fisher). CHO cells were maintained and transfected according to the manufacturer’s protocols. For all ExpiCHO cultures, the manufacturer’s “High Titer” protocol was used with a 7-day culture incubation to assess relative expression. Briefly, plasmid DNA and Expifectamine were mixed in Opti-PRO SFM (Gibco) according to the manufacturer’s instructions and added to the cells. On day 1, cells were fed with manufacturer-supplied feed and enhancer as specified in the manufacturer’s protocol, and cultures were moved to a shaker incubator set to 32 °C, 5% CO2 and 115 RPM. On day 7, cultures were clarified by centrifugation, followed by addition of BioLock (IBA Life Sciences), passage through a 0.22 pM sterile filter, and purification on an AKTA go system (Cytiva) using a 5mL Strep-Tactin XT column equilibrated with TBS buffer (25mM Tris pH 7.6, 200mM NaCl, 0.02% NaN3), and eluted in TBS buffer supplemented with lOmM d-desthiobiotin (Sigma Aldrich). Proteins were then purified by size-exclusion-chromatography (SEC) on a Superdex 6 Increase 10 / 300 column (Cytiva) in the same TBS buffer.

[0119] Differential Scanning Calorimetry. Thermal stability of the SARS-CoV-2 S2 constructs was analyzed by Nano DSC (TA instruments). The corresponding protein (500 pg) was buffer-exchanged to phosphate-buffered saline (PBS) and loaded into the sample wells. The temperature ramped from 20 °C to 100°C at 1 °C per minute. The resulting thermogram was corrected by subtracting a buffer blank data set and baseline-correcting before fitting to a thermodynamic model to extract the exact melting temperature (Tm).

[0120] Negative stain electron microscopy (NS-EM). NS-EM was utilized to visualize the SARS-CoV-2 S2 designs, as well as the complexes formed with the S2-DS5 and the Fabs fragments of the human mAbs 6C10, lAl l, 6El, and 4B3. To prepare the complexes, S2-DS5 and Fab in a 1:2 molar ratio, were incubated overnight at 4°C. The samples were then injected over a gel filtration column (Superose 6 10 / 30, GE Life Sciences) equilibrated with 20mM Tris pH 8.0 and 150mM NaCl. The complex peak was diluted to 0.02mg / ml, and 3 pL of the peak was applied to a carbon film 400 mesh grid for 1 minute. The grid was then washed three times with 20 pL Milli-Q water and stained three times with 20 pL drops of 1% uranyl formate, with the first two drops briefly, and the third time for 1 minute. The grid was blotted with Whatman filter paper after each application of liquid and air dried. Micrographs were collected on a Titan Halo, operating with an accelerating voltage of 300kV and fitted with a Gatan K3 camera. A magnification corresponding to a pixel size of 1.7A / pixel was used.

[0121] Beacon-based antibody discovery. Activated memory B cells were loaded onto OptoSelect UK chips (Bruker) and isolated as single cells in nanoliter pens using OEP light cages. Cells were screened in a 30 minute time course assay for secretion of antibodies that bound to streptavidin beads (Spherotech) coated with lOpg / mL biotinylated SARS-CoV-2 S-2P, as reported previously (39). Secreted antibodieswere detected with Ipg / mL goat anti-human IgG (H+L)-Alexa Fluor 594 (Invitrogen), which was added to the cell culture media used to resuspend the antigen-coated beads.

[0122] Synthesis of cDNA from antigen-positive cells was carried out on-chip using the OptoSeq BCR kit (Bruker), according to the manufacturer’s directions. First-strand reaction products were exported on mRNA capture beads and deposited into individual wells of a 96-well plate. Total cDNA was amplified using Platinum SuperFi II polymerase (Invitrogen). After enzymatic cleanup, antibody heavy and light chain variable domains were amplified with two rounds of nested PCR using Platinum II Hot-Start polymerase (Invitrogen). The resulting PCR products were assessed using 96w E-gels (Thermo Fisher) and paired wells were Sanger sequenced. Sequences were annotated using the bioinformatics platform PipeBio (PipeBio ApS, Horsens, DK).

[0123] Unique VH and VL domains were cloned into linearized human antibody expression vectors (human IgG I and kappa light chain) using Gibson assembly (NEB) according to the manufacturer’s directions. Ligation reactions were transformed into 5-alpha F’lq competent E. coli cells (NEB). QIAprep 96 Turbo Miniprep kits (Qiagen) were used to isolate plasmids according to the manufacturer’s instructions. Briefly, S block wells containing 1.1 mb LB media spiked with antibiotic were inoculated with single colonies and incubated overnight at 37 °C with agitation. DNA extraction was carried out with Qiagen buffer solutions and protocols. Plasmids were sequenced to ensure that the genes were in-frame, and that the cloned heavy and light chain variable domains matched PCR sequences.

[0124] High-throughput SPR binding kinetics. Binding kinetics measurements were performed on the Carterra LSA platform using a CDMP sensor chip (Carterra). The chip was activated with a freshly prepared solution of 130 mM l-ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC) (Pierce PG82079) and 33 mM N-hydroxysulfosuccinimide (Sulfo-NHS) (ThermoFisher Scientific 24510) in 0.1 M MES pH 5.5 using the SFC. A surface capture lawn was prepared with 50 ug / mL of goat antiHuman IgG Fc secondary antibody (VWR, 103255-066) in 10 mM sodium acetate (pH 4.5) / 0.01 % Tween. Unreactive esters were quenched with a 7-minute injection of 1 M ethanolamine-HCl (pH 8.5). Purified mAbs were captured in quadruplicate using the 96PH with IX HBSTE buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.01% Tween-20) as running buffer and antibody diluent.

[0125] A two-fold dilution series of the spike construct was prepared in IxHBSTE-BSA buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20, supplemented with 0.5 mg / ml BSA). Protein was injected onto the chip surface (at 25 °C) using the SFC, from the lowest to the highest concentration, without regeneration in between. Five buffer injections of the buffer before the lowest nonzero concentration were used for signal stabilization. For each concentration, baseline data were collected for 120 seconds, association data for 300 seconds and dissociation data for 900 seconds. After the titration of each analyte, the huFc capture lawn surface was regenerated with two pulses (17 seconds per pulse) of lOmM Glycine, pH 2.0 and mAbs re-captured in between each analyte. The running buffer for all kinetic steps was IxHBSTE-BSA.

[0126] Titration data were processed with the Kinetics software package (Carterra), including reference subtraction, buffer subtraction and data smoothing. Analyte binding time courses for each antibody were fitted to a 1 : 1 Langmuir model to derive ka, kd and KD values.

[0127] High-throughput SPR epitope binning. Epitope binning was performed on the Carterra LSA® HT- SPR using an HC30M sensor chip (Carterra). The chip was activated with a freshly prepared solution of 130 mM l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Pierce PG82079) and 33 mM N-hydroxysulfosuccinimide (Sulfo-NHS) (ThermoFisher Scientific 24510) in 0.1 M MES pH 5.5 using the SFC. Antibodies were diluted to 5 pg / mL in 10 mM sodium acetate (pH 4.5) / 0.01 % Tween and immobilized in quadruplicate using the 96PH for 15 minutes. Unreactive esters were quenched with a 7- minute injection of 1 M ethanolamine -HC1 (pH 8.5).

[0128] Epitope binning for S2-DS5 was performed with a premix assay format at 25 °C. A mixture of 50 nM of S2-DS5 was preincubated with 200 nM of each analyte antibody for at least 30 minutes before injecting over the array for 4 minutes. The sample injection for every sixth cycle was S2-DS5 only instead of antibody-S2-DS5 mixture. The surface was regenerated each cycle with double pulses (30 seconds per pulse) of 10 mM Glycine pH 2.0. Data was processed and analyzed with Epitope® software (Carterra). Data was referenced using unprinted locations (nearest reference spots) on the array for both experimental set ups. The response of each injection cycle was normalized to the response level in S2-DS5 only cycles. Competition results were visualized as a heat map that depicts blocking relationships of analyte / ligand pairs. Clones that suffered from severe loss of activity or lack of complete dissociation from S2-DS5 when used as ligands were excluded from analysis. Antibodies with similar patterns of competition are clustered together in a dendrogram and are assigned to shared communities.

[0129] Antibody expression. 25ml culture of ExpiCHO cells were maintained as described above. On the day of transfection, plasmids encoding heavy and light chains (10 pg each) were mixed with 80 pl Expifectamine, allowed to sit for 1-2 minutes, and added to cells dropwise over 5 minutes. The flask containing the transfected cells was allowed to shake at 120 rpm in an incubator maintained at 37 °C, 8% CO2. The cells were fed 18-22 hours after transfection with a mixture of 6 ml cold feed and 150 pl cold enhancer and moved to a 32 °C incubator with 5% CO2 and incubated with agitation at 120 rpm.

[0130] Antibody purification. The cells were harvested 7-8 days post transfection by centrifugation at 4,000 g for 30 minutes. The supernatant was filtered using a 0.22 pm filter and the pH was adjusted to 7.5. Packed protein A beads (2 ml; #Praesto AP Purolite Life Sciences, PR00300-310) were washed with TBS and incubated with the supernatant for between 30 minutes and 2 hours at RT. The mixture was passed through a gravity column and the beads were washed with 20 CV TBS to remove unbound proteins; IgG was eluted using 6 ml of elution buffer (100 mM glycine pH 2.2). Neutralization buffer (900 pl, IM Tris pH 8) was added to bring the pH to neutral. IgG was dialyzed into TBS pH 7.5 overnight at 4 °C. Aliquots (Img / ml) were made and stored at -20 °C.

[0131] Digestion of IgG to Fabs. Each antibody IgG (2mg) was incubated with 4% papain w / w for 2 hours at 37 °C. L-cysteine (Sigma) was added to a final concentration of 15 mM and incubated for 1 hourat 37 °C. Digestion was quenched with 50 mM iodoacetamide. The Fc portion was removed by passing the mixture over a column containing protein A beads. Fab was recovered from the flowthrough and buffer-exchanged to PBS using a Vivaspin 10k concentrator.

[0132] Cryo-EM data collection and processing. The S2-DS5 / 6C10_Fab complex was concentrated to 1 mg / ml and electron microscopy grids were prepared by placing a 3 pL aliquot of the sample on a plasma- cleaned C-flat grid (2 / 1C-3T, Protochips Inc) that was then immersed in liquid ethane for vitrification (VitroBot). Grids were loaded into a Titan Krios G3 electron microscope (Thermo Fisher Scientific) equipped with a K3 direct electron detector (Gatan, Inc.) at the end of a BioQuantum energy filter, using an energy slit of 20eV. The microscope was operated with an accelerating voltage of 300kV. Grids were imaged with a pixel size of 0.66 A in counting mode. Data was acquired using the software EPU. Two independent batches of the complex S2-DS5 / 6C10_Fab were frozen. A total of 5022 and 7422 movie stacks were motion-corrected using the patch motion correction, and the CTF parameters were determined using the patch CTF estimation in Warp (54). Warp picker was used to select a total of 1,137,885 particles. After two-dimensional classification and several rounds of hetero-refinement and non-uniform refinement in cryoSPARC-v2 (55) (Structure Biotechnology Inc.), a set of 407,314 refinements yielded a 2.9 A Cryo- EM density map with Cl symmetry.

[0133] Cryo-EM model building and structure analysis. Structure prediction of the 6C10 Fab was performed with AlphaFold server (56). Model building was performed in Coot 0.9.8.7 ((57, 58) and guided by the PDB model 6XKL. Model refinement and validation was performed in Phenix 1.20 (59). For visualization purposes, the map was processed by DeepEMhancer (60) and ChimeraX 1.6 (61, 62) was used to prepare representations of the structure. The surface areas and interactions S2-DS5 / 6C10_Fav were analyzed using the PDBePISA server (63).

[0134] Destabilization of the post-fusion conformation by removal of portions of S2 that are buried by S 1 in full-length spike.

[0135] The high sequence conservation of the S2 subunit in beta-CoV S proteins is likely related to the complex and precisely timed refolding events that essential for membrane fusion. During transition of S2 from a pre-to-post conformation, HR1 plays a central role by rearranging the 3 short a-helix in the prefusion state to a long a-helix that exposes and positions the fusion peptide towards the cell host membrane, and in the process acquiring a more energetically favored state. Destabilization of the post fusion conformation through deletion of particular regions of the HR1 may produce a more stable pre-fusion S2. Moreover, when the S2 is expressed alone without the SI subunit, two loops, termed Loop A (aa 738-760) and Loop B (aa 966-1000) are solvent-exposed. In full-length S protein, these loops, which are highly enriched in hydrophobic amino acids, are protected by the SI domain and when S2 is expressed alone, may promote aggregation. In addition, these loops could have immunodominant epitopes that can elicit a strong antibody response that is neither neutralizing nor protective, and they may preclude selection of other less immunogenic epitopes that are important in neutralizing or protective responses.

[0136] SARS-CoV-2 VFLIP Wuhan variant Delta SI drives high yield expression of heterogenous pre- and post-fusion S2 subunits.

[0137] SIIAYTMSLGAENSVACSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLL LQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQCYKTPPIKDFGGFNFSQILPDPSKPSKRSPIE DLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSCLLAGTICS GWTFGAGPALQIPFPMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQ DVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKPEAEVQIDRLITGRLQSLQTYVTQQLIRA AEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTA PAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQP ELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ (SEQ ID NO: 1)

[0138] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0139] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0140] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0141] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” 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, although the disclosure supports a definition that refers to only alternatives and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0142] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods providedherein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. As used herein, the phrase “consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method / process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method / process steps or limitation(s)) only.

[0143] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0144] As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0145] Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the invention(s) set out in any claims that may issue from this disclosure. Specifically, and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any invention(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the invention(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure, but should not be constrained by the headings set forth herein.

[0146] All of the compositions and / or 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 skillin the art that variations may be applied to the compositions and / or 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. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0147] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.

[0148] For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A mutant coronavirus pre-fusion, S2-subunit only spike protein comprising: the S2 subunit only, that has been modified to comprise;(1) at least one additional intra-monomeric disulfide bond that stabilizes the S2 subunit; and(2) 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus pre-fusion, S2-only spike protein maintains the profusion conformation.

2. The mutant coronavirus spike protein of claim 1, wherein a furin cleavage site loop has been deleted.

3. The mutant coronavirus spike protein of claim 1, wherein the intra-monomeric disulfide bond is formed between N717C-A1070C; I788C-A876C; Y789C-A879C; K790C-A879C; or N801C-N928C.

4. The mutant coronavirus spike protein of claim 1, wherein the 1, 2, 3, 4, or 5 proline mutations are selected from F817P, A892P, A899P, A942P, P986K, K986P, V987P, and P987V.

5. The mutant coronavirus spike protein of claim 1, wherein the proline mutations are not K986P and V987P mutations.

6. The mutant coronavirus spike protein of claim 1, wherein the higher stability is selected from: increased temperature stability, increased freeze / thaw stability, or increased lyophilization / resuspension stability.

7. The mutant coronavirus spike protein of claim 1, further comprising a purification peptide at an amino-terminus, a carboxy-terminus, or both.

8. The mutant coronavirus spike protein of claim 1, wherein the mutant coronavirus spike protein has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence to SEQ ID NO: 1.

9. The mutant coronavirus spike protein of any one of claims 1-8, wherein the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS-CoV-2, Alpha (B.1.1.7 and Q lineages), Beta (B.1.351 and descendent lineages), Gamma (P.l and descendent lineages), Epsilon(B.1.427 and B. 1.429), Eta (B. 1.525), Iota (B. 1.526), Kappa (B. 1.617.1), Mu (B.1.621, B.1.621.1), Zeta (P.2), Delta (B.1.617.2 and AY lineages), and Omicron (B.1.1.529) or its variants BA. l, BA.2, rBA.3, BA.4, BA.5, BA.2.75 (including BH and CH), BA.2.86, XBB, XBB 1.5, XBB.1.16, XBB 1.5 like (including F456L, EG.5, FL. 1.5.1, XBB 1.16.6, and FE.l), XBB.1.9.1, XBB.1.9.2, XBB.2.3, CH.1.1, BA.2.74, KP.2, KP.2.3, KP.3, KP.3.1.1, and / or LB. l, or an emerging variant thereof.

10. The mutant coronavirus spike protein of any one of claims 1-9, wherein the coronavirus is SARS-CoV-2.

11. A nucleic acid encoding the mutant coronavirus spike protein of any one of claims 1-10.

12. The nucleic acid of claim 11, further comprising a vector.

13. A cell comprising the mutant coronavirus spike protein of any one of claims 1-10 or the nucleic acid of claim 11.

14. The cell of claim 13, wherein the cell is a human cell.

15. A vaccine composition comprising the mutant coronavirus spike protein of any one of claims 1- 10 or nucleic acid of either claim 11 or claim 12, and a pharmaceutically acceptable excipient.

16. The vaccine composition of claim 15, further comprising an adjuvant.

17. A nanoparticle comprising the mutant coronavirus spike protein of any one of claims 1-10.

18. The nanoparticle of claim 17, wherein the nanoparticle comprises at least two of the mutant coronavirus spike protein of any one of claims 1-10.

19. The nanoparticle of claim 17, wherein the mutant coronavirus spike proteins are formed into dimers, trimers, or multimers.

20. The nanoparticle of any one of claims 17-19, wherein the nanoparticles comprise ferritin nanoparticles, polymeric nanoparticles, or both.

21. A method of making a mutant coronavirus pre-fusion, S2-subunit only spike protein comprising: obtaining a nucleic acid sequence encoding a coronavirus spike protein; and modifying the nucleic acid sequence of the coronavirus spike protein such that an amino acid sequence expressed by the nucleic acid sequence comprises: an S2 subunit only, that has been modified to comprise; at least one additional intra-monomeric disulfide bond that stabilizes the S2 subunit; and1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus prefusion, S2-subunit only spike protein maintains a prefusion conformation.

22. The method of claim 21, further comprising the step of expressing the mutant coronavirus spike protein in a bacteria, fungi, mammalian cell, avian cell, insect cell, or plant cell.

23. The method of claim 21, wherein a furin cleavage site loop has been deleted.

24. The method of claim 21, wherein the intra-monomeric disulfide bond is formed between N717C- A1070C; I788C-A876C; Y789C-A879C; K790C-A879C; or N801C-N928C.

25. The method of claim 21, wherein the 1, 2, 3, 4, or 5 proline mutations are selected from F817P, A892P, A899P, A942P, P986K, K986P, V987P, and P987V.

26. The method of claim 21, wherein the proline mutations are not K986P and V987P mutations.

27. The method of claim 21, wherein the higher stability is selected from: increased temperature stability, increased freeze / thaw stability, or increased lyophilization / re suspension stability.

28. The method of claim 21, further comprising a purification peptide at an amino-terminus, a carboxy-terminus, or both.

29. The method of claim 21, wherein the mutant coronavirus spike protein has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole sequence to SEQ ID NO: 1.

30. The method of any one of claims 21-29, wherein the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS-CoV-2, Alpha (B. l.1.7 and Q lineages), Beta (B.1.351 and descendent lineages), Gamma (P. l and descendent lineages), Epsilon (B.1.427 and B.1.429), Eta (B.1.525), Iota (B.1.526), Kappa (B.1.617.1), Mu (B.1.621, B. l.621.1), Zeta (P.2), Delta (B. l.617.2 and AY lineages), and Omicron (B. 1.1.529) or its variants BA. l, BA.2, rBA.3, BA.4, BA.5, BA.2.75 (including BH and CH), BA.2.86, XBB, XBB 1.5, XBB.1.16, XBB 1.5 like (including F456L, EG.5, FL. 1.5.1, XBB 1.16.6, and FE.l), XBB.1.9.1, XBB. 1.9.2, XBB.2.3, CH.1.1, BA.2.74, KP.2, KP.2.3, KP.3, KP.3.1.1, and / or LB.l, or an emerging variant thereof.

31. A nucleic acid sequence encoding a mutant coronavirus pre-fusion, S2-subunit only spike protein comprising: one or more mutations and deletions that change an amino acid sequence of a coronavirus spike protein S2 subunit only, comprising; at least one additional intra-monomeric disulfide bond that stabilizes the S2 subunit; and1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus prefusion, S2-subunit only spike protein maintains a prefusion conformation.

32. The nucleic acid of claim 31, wherein the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS-CoV-2, Alpha (B. l.1.7 and Q lineages), Beta (B.1.351 and descendent lineages), Gamma (P.l and descendent lineages), Epsilon (B. 1.427 and B. 1.429), Eta(B.1.525), Iota (B.1.526), Kappa (B.1.617.1), Mu (B.1.621, B. l.621.1), Zeta (P.2), Delta (B. l.617.2 and AY lineages), and Omicron (B. 1.1.529) or its variants BA. l, BA.2, rBA.3, BA.4, BA.5, BA.2.75 (including BH and CH), BA.2.86, XBB, XBB 1.5, XBB.1.16, XBB 1.5 like (including F456L, EG.5, FL. 1.5.1, XBB 1.16.6, and FE.l), XBB.1.9.1, XBB. 1.9.2, XBB.2.3, CH.1.1, BA.2.74, KP.2, KP.2.3, KP.3, KP.3.1.1, LB. l, or an emerging variant thereof.

33. The nucleic acid of claim 31, wherein the mutant coronavirus spike protein has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity across the whole sequence to express a protein of SEQ ID NO: 1.

34. A vector comprising a nucleic acid sequence encoding a mutant coronavirus spike protein comprising: one or more mutations and deletions that change an amino acid sequence of a coronavirus spike protein S2 subunit only, comprising;at least one additional intra-monomeric disulfide bond that stabilizes the S2 subunit; and1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus prefusion, S2-only spike protein maintains the profusion conformation.

35. The vector of claim 34, where the vector is selected for expression in a bacteria, fungi, mammalian cell, avian cell, insect cell, or plant cell.

36. The vector of claim 34, where the vector is in a bacteria, fungi, mammalian cell, avian cell, insect cell, or plant cell.

37. The vector of claim 34, wherein the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS-CoV-2, Alpha (B. l.1.7 and Q lineages), Beta (B.1.351 and descendent lineages), Gamma (P. l and descendent lineages), Epsilon (B.1.427 and B. 1.429), Eta (B. 1.525), Iota (B.1.526), Kappa (B.1.617.1), Mu (B. 1.621, B.1.621.1), Zeta (P.2), Delta (B. 1.617.2 and AY lineages), and Omicron (B. 1.1.529) or its variants BA. l, BA.2, rBA.3, BA.4, BA.5, BA.2.75 (including BH and CH), BA.2.86, XBB, XBB 1.5, XBB.1.16, XBB 1.5 like (including F456L, EG.5, FL. 1.5.1, XBB 1.16.6, and FE. l), XBB.1.9.1, XBB.1.9.2, XBB.2.3, CH.1.1, BA.2.74, KP.2, KP.2.3, KP.3, KP.3.1.1, LB.l, or an emerging variant thereof.

Images