Coronavirus antigen variants

Abstract

The present disclosure provides isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof, wherein the variant coronavirus spike protein has one or more modifications compared to the native coronavirus spike protein that increase recruitment by a receptor binding domain (RBD) of the variant coronavirus spike protein in an up conformation; and / or decrease recruitment by the RBD of the variant coronavirus spike protein in a down conformation; and / or increase expression of the variant coronavirus spike protein compared to the native coronavirus spike protein; and / or increase adoption of a prefusion conformation; and / or decrease shedding of the S1 subunit of the variant coronavirus spike protein; and / or improve localization of the variant coronavirus spike protein to a host cell membrane. The present disclosure also provides compositions comprising the isolated immunogenic polypeptides, as well as methods of making and using such compositions. [Figure 1] TIFF2025504887000062.tif42148

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 304,560, filed January 28, 2022, and U.S. Provisional Patent Application No. 63 / 480,375, filed January 18, 2023, each of which is incorporated by reference in its entirety herein.

[0002] The present disclosure relates to the field of immunology, and in particular to immunogenic compositions. More specifically, the present disclosure relates to methods and compositions involving coronavirus spike protein variants that can be used to elicit an immune response against coronaviruses. [Background technology]

[0003] Coronaviruses are a large family of viruses that usually cause mild to moderate upper respiratory tract illnesses, such as the common cold. However, three new coronaviruses, MERS-CoV, SARS-CoV and SARS-CoV-2, have emerged from reservoir animals over the past two decades and have caused severe and widespread illness and death. SARS coronavirus (SARS-CoV) emerged in November 2002 and caused Severe Acute Respiratory Syndrome (SARS). Middle East Respiratory Syndrome (MERS) is caused by MERS coronavirus (MERS-CoV). Transmitted from reservoir animals in camels, MERS was identified in September 2012 and continues to cause sporadic and localized outbreaks. The third novel coronavirus to emerge this century is called SARS-CoV-2. It causes coronavirus disease 2019 (COVID-19), which emerged from China in December 2019 and was declared a global pandemic by the World Health Organization on March 11, 2020. Summary of the Invention [Problem to be solved by the invention]

[0004] Coronavirus infection is mediated by the receptor binding domain (RBD) of the coronavirus spike glycoprotein, which binds to the ACE2 receptor on the surface of the host cell membrane. Although the coronavirus spike sequence and structure are known and several immunogenic compositions exist to elicit an immune response against the spike, there remains a need for spike protein antigens with increased protein expression, stability and immunogenic conformation compared to currently used immunogenic compositions to more effectively stimulate a protective immune response against coronaviruses. [Means for solving the problem]

[0005] The present disclosure provides methods and compositions involving coronavirus spike protein variants. The variants can elicit an immune response against coronaviruses more effectively than naturally occurring coronavirus spike proteins. Increasing the immunogenicity of viral antigens, for example by improving the expression of viral antigens, improving the stability of viral antigens, and / or increasing the number of neutralization-sensitive epitopes in viral antigens, is a desirable outcome in vaccine safety and efficacy. The present disclosure is based, at least in part, on the discovery that one or more specific amino acid modifications can be made to native coronavirus spike protein sequences to produce variant coronavirus spike proteins with improved in vivo expression, improved stability of the prefusion conformation, and / or increased exposure of neutralization-sensitive epitopes that can result in more immunogenic antigens.

[0006] It is contemplated that any embodiment described herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Additionally, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0007] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description, but it should be understood that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

[0008] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram of various domains of a coronavirus spike protein according to some embodiments disclosed herein. [Diagram 2] FIG. 1 illustrates the conformational changes undergone by the coronavirus spike protein prior to fusion with the host cell membrane. [Diagram 3] FIG. 1 illustrates several configurations of the up and down conformations assumed by the receptor-binding domains of various coronavirus spike proteins. [Figure 4] FIG. 1 illustrates the experimental strategy for designing and screening variant coronavirus spike proteins. [Diagram 5] FIG. 1 illustrates the conformation of the coronavirus spike protein before and after fusion with a host cell membrane, as well as the location of three amino acid residues for the SARS-CoV-2 spike protein sequence that, in some embodiments, has been modified to produce a variant coronavirus spike protein. [Figure 6]FIG. 1 illustrates the location of three amino acid residues in the SARS-CoV-2 spike protein sequence that, in some embodiments, have been modified to produce variant coronavirus spike proteins, and the effect of these modifications on spike protein expression and localization to the surface of the host cell membrane. [Figure 7A] FIG. 1 illustrates the placement of a single amino acid residue with respect to the SARS-CoV-2 spike protein sequence, demonstrating the effect of this modification on the conformation assumed by the receptor-binding domain of the variant coronavirus spike protein. [Figure 7B] FIG. 1 illustrates the placement of residues when modified relative to the SARS-CoV-2 spike protein sequence (B), demonstrating the effect of this modification on the conformation assumed by the receptor-binding domain of the variant coronavirus spike protein. [Figure 8] 1 is a graph showing protein expression, ACE2 binding and 3022 binding levels of variant coronavirus spike proteins according to some embodiments disclosed herein. [Figure 9] FIG. 10 describes some of the amino acid residues that, in some embodiments, have been modified to produce variant coronavirus spike proteins. In some instances, these specific modifications are used in addition to other amino acid residue modifications, such as some of the combinations in FIG. 10. [Figure 10] FIG. 9 describes some of the amino acid residues that, in some embodiments, have been modified to produce variant coronavirus spike proteins, and the effect of these modifications on protein expression, ACE2 binding, and 3022 binding by variant coronavirus spike proteins. In some examples, the modifications are made in addition to the modifications described in FIG. 9 as indicated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] I. Definition Examples Throughout this application, the term "about" is used according to its obvious and ordinary meaning in the field of cell and molecular biology to indicate that a value includes the standard deviation of the inherent variation or error for the measurement or quantification method being used to determine the value. For example, in some embodiments, the term "about" can encompass a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the measurement or quantification.

[0011] The use of the word "a" or "an," when used in conjunction with the term "comprising," can mean "one," but this is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0012] The phrase "and / or" means "and" or "or." To illustrate, A, B and / or C includes A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C. In other words, "and / or" operates as an inclusive or.

[0013] The phrase "essentially all" is defined as "at least 95%; if essentially all members of a group have a particular property, then at least 95% of the members of the group have that property. In some examples, essentially all means that equal to, at least any one of, or between any two of 95, 96, 97, 98, 99, or 100% of the members of the group have that property.

[0014] The compositions and methods for their use may "comprise," "consist essentially of," or "consist" of any of the components or steps disclosed throughout this specification. Throughout this specification, unless the context requires otherwise, 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") will be understood to be inclusive or open-ended and to imply the inclusion of a described step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. It is contemplated that embodiments described herein in the context of the term "comprising" may also be practiced in the context of the terms "consisting of" or "consisting essentially of." Compositions and methods "consisting essentially of" any of the disclosed components or steps limit the claims to the specified materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure. The word "consisting of" (as well as any form of "consisting of," such as "consist of" and "consists of") is meant to include and be limited to whatever precedes the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or essential, and that other elements may not be present.

[0015] Reference throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0016] The terms "inhibiting" or "reducing" or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result. The terms "improving," "promoting" or "increasing" or any variation of these terms includes any measurable increase to achieve a desired result or production of a protein or molecule.

[0017] As used herein, the term "reference", "standard" or "control" refers to the value that is compared to.For example, the agent, subject, population, sample or value of interest is compared to the agent, subject, population, sample or value of reference, standard or control.The reference, standard or control can be tested and / or determined substantially simultaneously with the test or determination of the agent, subject, population, sample or value of interest, and / or can be determined or characterized under conditions or circumstances that are comparable to the agent, subject, population, sample or value of interest that is being evaluated.

[0018] The term "DNA" as used herein means a nucleic acid molecule that comprises deoxyribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and / or thymine (T)). For example, DNA can contain all or most of the deoxyribonucleotide residues. As used herein, the term "deoxyribonucleotide" means a nucleotide that lacks a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. Without any limitation, DNA can encompass double-stranded DNA, antisense DNA, single-stranded DNA, isolated DNA, synthetic DNA, recombinantly produced DNA, and modified DNA.

[0019] As used herein, a "protein", "polypeptide" or "peptide" refers to a molecule that comprises at least two amino acid residues. As used herein, the term "wild type" or "native" refers to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, a wild type version of a protein or polypeptide is used, but in many embodiments of the disclosure, a modified protein or polypeptide is used to generate an immune response. The terms described above can be used interchangeably. A "modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, has been altered relative to a wild type protein or polypeptide. In some embodiments, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered for one activity or function, but still retain wild type activity or function in other respects, such as immunogenicity. A protein, when specifically referred to herein, is generally a reference to a native (wild type) or recombinant (modified) protein. A protein can be directly isolated from the organism in which it is native, can be produced by recombinant DNA / exogenous expression methods, can be produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In certain embodiments, there are isolated nucleic acid segments and recombinant vectors that incorporate a nucleic acid sequence that encodes a polypeptide (e.g., an antigen or fragment thereof). The term "recombinant" can be used in conjunction with the name of a polypeptide or a specific polypeptide, and generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is a product of replication of such a molecule.

[0020] The term "isolated" can refer to a nucleic acid or polypeptide that is substantially free of cellular, bacterial, or viral materials, or the culture medium from which it originated (if produced by recombinant DNA techniques), or chemical precursors or other chemicals (if chemically synthesized). Additionally, an isolated compound refers to a compound that can be administered to a subject as an isolated compound; in other words, a compound may not simply be considered "isolated" when it is attached to a column or embedded in an agarose gel. Additionally, an "isolated nucleic acid fragment" or an "isolated peptide" is a nucleic acid or protein fragment that does not naturally occur as a fragment, and / or is not typically in a functional state, and / or has been altered or removed from its natural state via human intervention. For example, DNA that is naturally present in a living animal is not "isolated," but synthetic DNA, or DNA that is partially or completely separated from the coexisting materials of its natural state, is "isolated." An isolated nucleic acid can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a cell into which the nucleic acid was delivered.

[0021] Immune response refers to humoral response, cellular response, or both humoral and cellular response in an organism.Immune response can be measured by assays including, but not limited to, the presence or amount of antibody that specifically recognizes protein or cell surface protein, the assay that measures T cell activation or proliferation, and / or the modulation in terms of the activity or expression of one or more cytokines.

[0022] II. Viruses As considered herein, without any limitation, the compositions and methods herein can be used as modalities for treating and / or preventing and / or reducing the severity or medical / health risks of several diseases and / or conditions in a mammal, including coronavirus infection in humans. The methods described herein include administration of the compositions described herein to a mammal, such as a human. For example, in one embodiment, such methods of use of the compositions herein include variant coronavirus spike protein or peptide vaccines or variant coronavirus spike protein or peptide coding nucleic acid vaccines, preferably to induce robust neutralizing antibodies and associated / concomitant T cell responses to achieve protective immunization with minimal vaccine doses.

[0023] For example, such nucleic acids can be used to encode at least one antigen intended to generate an immune response in a mammal. Such immunogenic polypeptide constructs are peptide or protein antigens derived from pathogens associated with infectious diseases, including viruses, bacteria, parasites and fungi. The immunogenic polypeptide constructs can include one or more isolated immunogenic polypeptides, including variant coronavirus spike proteins that are variants of native coronavirus spike proteins or fragments thereof, that induce an immune response. In a specific embodiment, the immunogenic polypeptide construct is a variant coronavirus spike protein construct, and the variant coronavirus spike protein construct can include one or more immunogenic peptide sequences that induce an immune response.

[0024] Conditions and / or diseases that may be treated and / or prevented by such nucleic acid and / or peptide or polypeptide compositions include, but are not limited to, conditions and / or diseases caused and / or affected by viral infections, including, but not limited to, coronaviruses (such as Severe Acute Respiratory Syndrome virus (SARS) - e.g., SARS-CoV-2 or Middle East Respiratory Syndrome (MERS) virus).

[0025] The variant coronavirus spike protein construct can be based on any spike protein sequence from any Coronaviridae family virus. Coronaviridae is a family of enveloped, positive-sense, single-stranded RNA viruses. Coronavirus is the common name for the family Coronaviridae and the subfamily Orthocoronavirinae (also called Coronavirinae). The family Coronaviridae is organized in two subfamilies, five genera, 23 subgenera, and approximately 40 species. These are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid with helical symmetry.

[0026] Some coronaviruses use animals as their primary host and have also evolved to infect humans. There are four main subgroupings of coronaviruses, known as alpha, beta, gamma and delta; seven coronaviruses can infect humans. The four most common coronaviruses use humans as their natural host and include 229E (alphacoronavirus); NL63 (alphacoronavirus); OC43 (betacoronavirus); and HKU1 (betacoronavirus). Three other human coronaviruses are MERS-CoV (betacoronavirus that causes MERS); SARS-CoV (betacoronavirus that causes SARS); and SARS-CoV-2 (a novel coronavirus that causes coronavirus disease 2019 or COVID-19).

[0027] Coronaviruses have characteristic club-shaped spikes protruding from their surface, which in electron microscope images give them an appearance reminiscent of the solar corona from which they are named. The average diameter of the virus particle is around 120 nm (.12 μm). The diameter of the envelope is approximately 80 nm (.08 μm) and the spikes are approximately 20 nm (.02 μm) long. Beneath the spiked exterior of the virus is a circular core, surrounded by the viral envelope. The core contains the genetic material that the virus can inject into a cell to infect it.

[0028] The viral envelope consists of a lipid bilayer to which the membrane (M), envelope (E) and spike (S) structural proteins are tethered. Inside the envelope is a helically symmetric nucleocapsid formed from multiple copies of the nucleocapsid (N) protein bound to a positive-sense single-stranded RNA genome in a continuous beads-on-a-string conformation. Coronavirus genome sizes range from approximately 26 to 32 kilobases. The coronavirus genome organization is 5'-leader-UTR-replicase / transcriptase-spike (S)-envelope (E)-membrane (M)-nucleocapsid (N)-3'UTR-poly(A) tail. Open reading frames 1a and 1b, which occupy the first two-thirds of the genome, code for the replicase / transcriptase polyprotein. The replicase / transcriptase polyprotein autocleaves to form nonstructural proteins. Subsequent open reading frames code for the four main structural proteins: spike, envelope, membrane and nucleocapsid. Interspersed among these open reading frames are open reading frames for accessory proteins. The number of accessory proteins and their functions are specific to the specific coronavirus.

[0029] The lipid bilayer envelope, membrane proteins and nucleocapsid protect the virus when outside the host cell. The spike protein extends from within the core to the surface of the virus, allowing the virus to recognize and bind to specific cells in the body. When the spike engages a receptor on the host cell, a cascade is triggered that leads to cell-virus merging, allowing the virus to release its genetic material, overtake the cellular processes and produce new viruses.

[0030] Infection begins when the viral spike (S) glycoprotein attaches to its complementary host cell receptor. After attachment, host cell proteases (e.g., ACE2) cleave and activate the receptor-attached spike protein. Depending on the available host cell proteases, cleavage and activation allows the virus to enter the host cell by endocytosis or direct fusion of the viral envelope with the host membrane (Figure 2).

[0031] When the S protein is in the RBD-up conformation, the receptor binding site is exposed, so that the binding of the S1 subunit of the S protein to the host cell receptor stabilizes the S protein in the "up" conformation, making the protein more vulnerable to cleavage by host cell proteases. Moreover, when the RBD is in the up conformation, neutralization-sensitive epitopes are exposed, and thus, in some embodiments of the variant coronavirus spike proteins disclosed herein, modifications are made to promote the adoption of the RBD-up conformation or inhibit the adoption of the RBD-down conformation. This conformation of the S protein is illustrated by the coronavirus crystal structure shown in Figures 2 and 3. Cleavage occurs between the S1 and S2 subunits of the S protein, and cleavage induces a conformational change by the S2 subunit to allow the insertion of the S2 subunit into the host cell membrane and mediate fusion between the virus and the host cell membrane.

[0032] Upon entry into a host cell, the virus particle uncoats and its genome enters the cell's cytoplasm. The coronavirus RNA genome has a 5' methylated cap and a 3' polyadenylated tail, which allows the RNA to attach to the host cell's ribosomes for translation. The host ribosome translates the initial overlapping open reading frames of the viral genome to form a long polyprotein. The polyprotein has its own protease, which cleaves the polyprotein into multiple nonstructural proteins.

[0033] Viral entry is followed by viral replication. Several nonstructural proteins coalesce to form a multiprotein replicase-transcriptase complex (RTC). The major replicase-transcriptase protein is the RNA-dependent RNA polymerase (RdRp). It is directly involved in the replication and transcription of RNA from an RNA strand. Other nonstructural proteins in the complex aid in the replication and transcription process. Exoribonuclease nonstructural proteins provide extra fidelity to replication, for example, by providing a proofreading function that RNA-dependent RNA polymerase lacks. One of the main functions of the complex is to replicate the viral genome. RdRp directly mediates the synthesis of negative-sense genomic RNA from the positive-sense genomic RNA. This is followed by the replication of positive-sense genomic RNA from the negative-sense genomic RNA. Another important function of the complex is to transcribe the viral genome. RdRp directly mediates the synthesis of negative-sense subgenomic RNA molecules from the positive-sense genomic RNA. This is followed by transcription of such negative-sense subgenomic RNA molecules into their corresponding positive-sense mRNAs.

[0034] The replicated positive-sense genomic RNA becomes the genome of the progeny virus. The mRNA is the gene transcript of the last third of the viral genome after the initial overlapping reading frame. Such mRNA is translated by the host ribosomes into structural proteins and some accessory proteins. RNA translation occurs inside the endoplasmic reticulum. The viral structural proteins S, E and M travel along the secretory pathway into the intermediate Golgi compartment. There, the M protein directs most of the protein-protein interactions required for viral assembly after its binding to the nucleocapsid. The progeny virus is then released from the host cell by exocytosis through secretory vesicles.

[0035] The interaction of coronavirus spike proteins with their complementary host cell receptors is central in determining the tissue tropism, infectivity and species range of the virus. Coronaviruses primarily target epithelial cell receptors. Coronaviruses can be transmitted, for example, by aerosol, fomites or fecal routes. Human coronaviruses infect epithelial cells of the respiratory tract. For example, human coronaviruses can infect human epithelial cells of the lungs by the aerosol route through binding of the spike protein receptor-binding domain (RBD) to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell surface.

[0036] The WHO reported that the two groups at highest risk of experiencing severe illness from coronavirus infection are adults over 65 years of age and people with other underlying health conditions, including chronic lung disease, serious heart conditions, severe obesity, compromised immune systems, or diabetes. In humans, coronaviruses typically cause respiratory infections with mild to severe flu-like symptoms, although the exact symptoms vary depending on the coronavirus type. Four common human coronaviruses can cause people to develop runny nose, headache, cough, sore throat, and fever. In a subset of individuals, including those with cardiopulmonary disease or weakened immune systems, the viral infection can progress to more severe lower respiratory infections, such as pneumonia or bronchitis. In comparison, severe MERS and SARS infections often progress to pneumonia. Other symptoms of MERS include fever, cough, and shortness of breath, while SARS can cause fever, chills, and body aches.

[0037] SARS-CoV-2 causes symptoms similar to those of other coronaviruses, inducing fever, cough and shortness of breath in most patients. More unusual symptoms include dizziness, fatigue, aches, chills, sore throat, loss of smell, loss of taste, headache, nausea, vomiting and diarrhea. Emergency signs or symptoms may include difficulty breathing, persistent chest pain or pressure, new confusion, and / or blue lips or face. Complications of SARS-CoV-2 infection may include pneumonia, organ failure, respiratory failure, blood clots, cardiac conditions such as cardiomyopathy, acute kidney injury, and / or further viral and bacterial infections.

[0038] The present disclosure encompasses the treatment or prevention of a disease or condition caused by infection with any virus in the Coronaviridae family. In certain embodiments, the methods and compositions treat or prevent or reduce the symptoms or severity of COVID-19, caused by infection with SARS-CoV-2. In certain embodiments, the present disclosure encompasses the treatment or prevention of infection with any virus in the Coronaviridae subfamily and including the four genera Alpha, Beta, Gamma, and Deltacoronavirus. In specific embodiments, the present disclosure encompasses the treatment or prevention of infection with any virus in the genus of Betacoronavirus, including the subgeneus Sarbecovirus and Severe Acute Respiratory Syndrome-related coronavirus species; the subgeneus Embecovirus and Human coronavirus HKU1 species; and Betacoronavirus 1 species. In specific embodiments, the disclosure encompasses the treatment or prevention of infection with any virus in the severe acute respiratory syndrome-associated coronavirus species, including severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the virus that causes COVID-19) strains. The disclosure encompasses the treatment or prevention of infection with any isolate, strain, type (including types A, B, and C; Forster et al., 2020, PNAS, available on the World Wide Web at doi.org / 10.1073 / pnas.2004999117), cluster, or subcluster of the severe acute respiratory syndrome-associated coronavirus species, including at least SARS-CoV-2. In specific embodiments, the virus has a genome length of between 29,000-30,000, between 29,100-29,900, between 29,200-29,900, between 29,300-29,900, between 29,400-29,900, between 29,500-29,900, between 29,600-29,900, between 29,700-29,900, between 29,800-29,900, or between 29,780-29,900 base pairs in length.

[0039] Examples of specific SARS-CoV-2 viruses include those listed in the NCBI GenBank® database, and these GenBank® accession sequences are incorporated herein by reference in their entirety: (a) LC534419 and LC534418 and LC528233 and LC529905 (examples of different strains from Japan); (b) MT281577 and MT226610 and NC_045512 and MN996531 and MN908947 (examples of different strains from China); (c) MT281530 (Iran); (d) MT126808 (Brazil); (e) M T020781 (Finland); (f) MT093571 (Sweden); (g) MT263074 (Peru); (h) MT292582 and MT292581 and MT292580 and MT292579 (examples of different strains from Spain); (i) examples from the United States, e.g., MT276331 (TX); MT276330 (FL); MT276328 (OR) MT276327 (GA); MT276325 (WA); MT276324 (CA); MT276323 (RI); MT188341 (MN); and (j) MT276598 (Israel). In certain aspects, the disclosure encompasses the treatment or prevention of infection with any of these or similar viruses, including viruses whose genomes have at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8 or 99.9% identity to any of these viruses. In certain aspects, the disclosure encompasses the treatment or prevention of infection with any of these or similar viruses, including viruses whose genome has its entire sequence greater than 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8 or 99.9% identity to any of these viruses.In one embodiment, the disclosure includes a method of treating or preventing infection with a virus having the genomic sequence of GenBank® Accession No. NC_045512, and any virus having a genomic sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8 or 99.9% identity to said sequence.

[0040] III. Nucleic acids In certain embodiments, an immunogenic polypeptide construct, such as an isolated immunogenic polypeptide comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof, is intended to generate an immune response and is encoded by a nucleic acid. The nucleic acid encoding the immunogenic polypeptide construct can include one or more nucleotide sequences corresponding to one or more immunogenic peptide sequences that elicit an immune response.

[0041] The nucleic acid sequence can be present in various examples, such as an isolated segment; a recombinant vector of an incorporated sequence or recombinant polynucleotide encoding a polypeptide such as an antigen or one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof; a polynucleotide sufficient for use as a hybridization probe, PCR primer, or sequencing primer to identify, analyze, mutate, or amplify a polynucleotide; an antisense nucleic acid for inhibiting expression of a polynucleotide; an mRNA; a saRNA; and a complementary sequence of the foregoing as described herein. The nucleic acid can encode an antigen or epitope to which an antibody can bind. Nucleic acids encoding fusion proteins comprising the antigen or epitope are also provided. The nucleic acid can be single-stranded or double-stranded and can include RNA and / or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids).

[0042] The term "polynucleotide" refers to a nucleic acid molecule, which may be recombinant or isolated from total genomic nucleic acid. Recombinant vectors, including oligonucleotides (nucleic acids of 100 residues or less in length), such as plasmids, cosmids, phages, viruses, etc., are included within the term "polynucleotide". A polynucleotide, in certain embodiments, includes a regulatory sequence, isolated substantially away from its naturally occurring gene or protein coding sequence. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.

[0043] In this regard, the term "gene" is used to refer to a nucleic acid that encodes a protein, polypeptide or peptide (including any sequence required for proper transcription, post-translational modification or localization). As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences and smaller engineered nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins and mutants. A nucleic acid that encodes all or a portion of a polypeptide can contain contiguous nucleic acid sequences that encode all or a portion of such a polypeptide. It is also considered that a particular polypeptide can be encoded by a nucleic acid that contains variants that have slightly different nucleic acid sequences but nevertheless encode the same or substantially similar polypeptide.

[0044] In certain aspects, there are polynucleotide variants with substantial identity to the sequences disclosed herein; polynucleotide variants that comprise sequence identity equal to, at least any one of, at most any one of, or between any two of, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more compared to the polynucleotide sequences provided herein using methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence that encodes a polypeptide having at least 90% identity to the amino acid sequence described herein over the entire length of the sequence; or a nucleotide sequence that is complementary to the isolated polynucleotide. In some aspects, the isolated polynucleotide will comprise a nucleotide sequence that encodes a polypeptide having at least 95% identity to the amino acid sequence described herein over the entire length of the sequence; or a nucleotide sequence that is complementary to the isolated polynucleotide.

[0045] Regardless of the length of the coding sequence itself, nucleic acid segments may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., which may cause their overall length to vary considerably. Nucleic acids may be of any length. The nucleic acid can be, for example, equal to, at least one of, at most one of, or between any two of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides in length, and / or can include one or more additional sequences, e.g., regulatory sequences, and / or can be part of a larger nucleic acid, e.g., a vector. Thus, it is considered that almost any length of nucleic acid fragment can be used, and the total length is limited by the ease of preparation and use in the intended recombinant nucleic acid protocol.In some cases, the nucleic acid sequence can code a polypeptide sequence with additional heterologous coding sequences, for example, to allow purification, transportation, secretion, post-translational modification of the polypeptide, or for therapeutic and / or prophylactic benefits, such as targeting or efficacy.As described above, a tag or other heterologous polypeptide can be added to the sequence that codes for the modified polypeptide, and "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0046] In some embodiments, one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof are encoded by a nucleic acid and elicit an immune response. The immune response can be against an immunogenic variant coronavirus spike protein construct and / or an immunogenic native coronavirus spike protein sequence.

[0047] Immunogenic variant coronavirus spike protein constructs may be combined with nucleic acid encoding the native coronavirus spike protein or cDNA encoding the native coronavirus spike host protein in the following proportions: 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any derivable range therein), at least one of them, at most one of them, or similar, identical or homologous to some degree between any two of them, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or 100% (or any de 6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, The sequence may include contiguous nucleotides equal to, at least one of, at most one of, or between any two of 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more.

[0048] The nucleic acids of the present disclosure that encode an isolated immunogenic polypeptide comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof, may have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more amino acid residues in a sequence encoding an immunogenic variant coronavirus spike protein construct compared to a nucleic acid encoding an unmodified native immunogenic coronavirus spike protein construct. , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 or 54 mutations.The portion of the nucleic acid of the disclosure that encodes an immunogenic variant coronavirus spike protein construct may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 1 0%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% (or any derivable range therein), be equal to, be at least one of, be at most one of, or be similar, identical or homologous to some degree between any two of them.

[0049] A. Substitution Changes can be introduced by substitution in a nucleic acid, resulting in changes in the amino acid sequence of a polypeptide (e.g., an antigen or antibody or antibody derivative) that it encodes. Substitutions can be introduced using any technique known in the art. In one embodiment, one or more specific amino acid residues are altered, for example, using a site-directed mutagenesis protocol.

[0050] In some cases, substitutions can be introduced into a nucleic acid without significantly altering the biological activity of the polypeptide it encodes. For example, nucleotide substitutions can be made that result in amino acid substitutions at non-essential amino acid residues. Alternatively, one or more substitutions can be introduced into a nucleic acid that selectively alter the biological activity of the polypeptide it encodes. See, for example, Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, a substitution can quantitatively or qualitatively alter biological activity. Examples of quantitative changes include increasing, reducing or eliminating activity. Examples of qualitative changes include altering the antigen specificity of an antibody.

[0051] IV. Polypeptides The isolated immunogenic polypeptide construct may comprise one or more isolated immunogenic polypeptides, including variant coronavirus spike proteins that are variants of native coronavirus spike proteins or fragments thereof that induce an immune response. Thus, in certain embodiments, the isolated immunogenic polypeptide construct is a peptide or protein antigen derived from a pathogen associated with an infectious disease, including a coronavirus. In certain embodiments, the immunogenic polypeptide construct is a peptide or protein antigen derived from a coronavirus spike protein.

[0052] In certain embodiments, the peptide or protein can be present in various instances, such as an isolated or recombinant polypeptide, or a fragment, functional derivative, mutein or variant thereof, a peptide or protein sufficient for use as a hybridization probe, a peptide or protein for inhibiting expression of a polynucleotide, and the aforementioned complementary amino acid sequences described herein. The peptide or protein can be an epitope to which an antibody can bind. The peptide or protein can include RNA and / or DNA nucleotides (e.g., peptide nucleic acid).

[0053] In certain aspects, the size of a protein or peptide or derivative of the corresponding amino acid sequence described or referenced herein is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 110, 111, 112, 113, 2, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100 , 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 amino acid residues in length, or greater than or equal to, at least any one of, at most any one of, or between any two of, any one of the above. It is contemplated that a protein or peptide can be mutated by truncation to make it shorter than its corresponding native or wild-type form, and can also be altered by fusing or conjugating heterologous protein or peptide sequences having a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term "domain" refers to any distinct functional or structural unit of a protein or peptide, and generally refers to a sequence of amino acids having a structure or function recognizable by one of skill in the art.

[0054] In some embodiments, the one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof, comprise at least an RBD. The RBD may or may not be modified relative to the RBD of the native coronavirus spike protein. In some embodiments, the RBD is modified relative to the RBD of the native coronavirus spike protein.

[0055] In some embodiments, the one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof, comprise a leader sequence. In some embodiments, the leader sequence has an amino acid sequence that has a sequence identity equal to, at least, at most, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more compared to the leader sequence of the native coronavirus spike protein sequence. In some embodiments, the leader sequence has an amino acid sequence that is at least 80% identical to the leader sequence of the native coronavirus spike protein sequence. In some embodiments, the inclusion of a leader sequence as part of the isolated immunogenic polypeptide sequence inhibits disulfide scrambling.

[0056] In some embodiments, the one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof, comprise a transmembrane sequence. In some embodiments, the transmembrane sequence has an amino acid sequence that has a sequence identity equal to, at least, at most, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more with respect to the transmembrane sequence of the native coronavirus spike protein sequence. In some embodiments, the transmembrane sequence has an amino acid sequence that is at least 80% identical to the transmembrane sequence of the native coronavirus spike protein sequence. In some embodiments, the inclusion of a transmembrane sequence as part of the isolated immunogenic polypeptide sequence extends the half-life of the isolated immunogenic polypeptide.

[0057] In some embodiments, the one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof do not include an endoplasmic reticulum (ER) signal sequence. In some embodiments, the omission of the ER signal sequence improves localization of the variant coronavirus spike protein to the host cell membrane.

[0058] In some embodiments, the one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof, comprise a trimerization domain. In some embodiments, the trimerization domain is a foldon trimerization domain. In some embodiments, the trimerization domain sequence has an amino acid sequence that has a sequence identity equal to, at least, at most, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more with the trimerization domain sequence of the foldon trimerization domain of T4 fibritin. In some embodiments, the trimerization domain sequence has an amino acid sequence that is at least 80% identical to the trimerization domain sequence of the foldon trimerization domain of T4 fibritin.

[0059] In some embodiments, one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof are encoded by a nucleic acid and elicit an immune response. The immune response can be against the immunogenic variant coronavirus spike protein construct and / or the native coronavirus spike protein. The immunogenic variant coronavirus spike protein construct and the native coronavirus spike protein are 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 14 It may be equal to, be at least one of, be at most one of, or be similar, identical or homologous to some degree between any two of 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0060] In some embodiments, the variant coronavirus spike protein construct has a similar structure to the native coronavirus spike protein by 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein), or at least one of them, or at most one of them, or similar, identical, or homologous to some degree between any two of them, , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more contiguous amino acids.

[0061] The immunogenic variant coronavirus spike protein construct can include amino acid substitutions equal to, at least one of, at most one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 disclosed herein, as compared to a native, unmodified immunogenic polypeptide construct. The immunogenic variant coronavirus spike protein construct can include amino acid substitutions equal to, at least one of, at most one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 6%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 9 It may be equal to, be at least one of, be at most one of, or be somewhat similar, identical or homologous between any two of 9.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% (or any derivable range therein).

[0062] Nucleotide sequences of various genes as well as protein, polypeptide and peptide sequences have been disclosed before and can be found in recognized computerized databases.Two commonly used databases are the Genbank and GenPept databases of the National Center for Biotechnology Information (on the World Wide Web at ncbi.nlm.nih.gov) and the Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org) of the National Center for Biotechnology Information.The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or known to those skilled in the art.

[0063] V. Coronavirus spike protein modification The immunogenic native coronavirus spike protein constructs and / or nucleic acids encoding immunogenic native coronavirus spike protein constructs of the present disclosure can be modified to be substantially identical to the immunogenic variant coronavirus spike protein constructs and / or nucleic acids encoding immunogenic variant coronavirus spike protein constructs included in the immunogenic compositions described herein. In some embodiments, the immunogenic variant coronavirus spike protein constructs and / or nucleic acids encoding immunogenic variant coronavirus spike proteins continue to be bound by antibodies to elicit an immune response.

[0064] In certain embodiments, it is the peptide sequence of the native coronavirus spike protein and / or the nucleic acid encoding the peptide sequence that is modified. Thus, in some embodiments, the variant coronavirus spike protein comprises a sequence identity equal to, at least one of, at most one of, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more compared to the native coronavirus spike protein sequence. In some embodiments, the variant coronavirus spike protein can comprise an amino acid sequence that is at least 70% identical to the amino acid sequence of the native coronavirus spike protein. In some embodiments, the variant coronavirus spike protein can comprise an amino acid sequence that is at least 80% identical to the amino acid sequence of the native coronavirus spike protein. In some embodiments, the variant coronavirus spike protein can comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of the native coronavirus spike protein.

[0065] Polypeptide and / or nucleic acid sequences are "substantially identical" when optimally aligned using programs such as Clustal Omega, IGBLAST, GAP or BESTFIT using default gap weights, and share at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity or at least 99% sequence identity, or any range therein.

[0066] Immunogenic variant coronavirus spike protein constructs of the present disclosure and / or nucleic acids encoding immunogenic variant coronavirus spike protein constructs can include amino acid substitutions equal to, at least one of, at most one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 disclosed herein, and / or a native, unmodified immunogenic coronavirus of the present disclosure. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more, or at least one of them 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.It may be equal to, at least one of, at most one of, or similar, identical or homologous to, any one of 1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.

[0067] In some embodiments, the immunogenic variant coronavirus spike protein construct and / or nucleic acid encoding the immunogenic variant coronavirus spike protein construct is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109, 104, 105, 106, 107, 108, 109, 109, 109, 6%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47 %, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% , 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any derivable range therein), at least one of them, at most one of them, or similar, identical or homologous to some degree between any two of them, , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 6, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150,The amino acid sequence may include contiguous amino acids equal to, at least one of, at most one of, or between any two of 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more.

[0068] Since modifications and / or changes can be made in the sequence and / or structure of the polynucleotides and / or proteins of the present disclosure while obtaining molecules with similar or improved characteristics (e.g., maintenance of antibody binding and immune response, and reduced cross-reactivity with endogenously expressed host proteins), such biologically functional equivalents of the immunogenic polypeptide constructs and / or nucleic acids encoding the immunogenic polypeptide constructs are also encompassed within the present invention.

[0069] Immunogenic variant coronavirus spike protein constructs and / or nucleic acids encoding immunogenic variant coronavirus spike protein constructs can include polynucleotides engineered to contain distinct sequences while simultaneously retaining the ability to encode a "wild-type" or standard protein or peptide or an "altered" or "variant" protein or peptide. This can be accomplished by the degeneracy of the genetic code, i.e., the presence of multiple codons that code for the same amino acid. The term "functionally equivalent codon" is used herein to refer to a codon that codes for the same amino acid, such as the six different codons for arginine. "Neutral substitutions" or "neutral mutations," which refer to changes in a codon or codons that code for biologically equivalent amino acids, are also contemplated. In one example, one skilled in the art may wish to introduce mutations into a polynucleotide to reduce cross-reactivity between endogenously expressed host proteins and the protein encoded by the polynucleotide, without disrupting the ability of the polynucleotide to encode a protein that undergoes antibody binding and elicits an immune response.

[0070] In terms of functional equivalents, it is well understood by those skilled in the art that inherent in the definition of a "biologically functional equivalent" protein and / or polynucleotide is the concept that there is a limit to the number of changes that can be made within a defined portion of a molecule while still retaining the molecule with an acceptable level of equivalent biological activity. Thus, biologically functional equivalents are defined herein as proteins (and polynucleotides) with substitutions or mutations at selected amino acids (or codons) and / or proteins (and polynucleotides) with substitutions or mutations at selected amino acids (or codons) that retain the ability to be bound by an antibody and to elicit an immune response.

[0071] In one example, polynucleotides can result in (and encode) biologically functional equivalents with significant changes: certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, substrate molecules, binding sites in receptors, and the like.

[0072] A. Coronavirus spike protein The following Table 1 includes the amino acid sequences of spike proteins of seven coronaviruses known to infect humans. The amino acid sequences were obtained from the UniProt database, accessible via the World Wide Web at uniprot.org, or the GenBank database, accessible via the World Wide Web at ncbi.nlm.nih.gov, and the UniProt or GenBank database accession number for each spike protein sequence is included in Table 1. These amino acid sequences correspond to the amino acid sequences of native coronavirus spike proteins. In some embodiments, the amino acid sequences of native coronavirus spike proteins can be modified as described herein to produce isolated immunogenic polypeptides that include variant coronavirus spike proteins that are modifications of native coronavirus spike proteins or fragments thereof. For example, in some embodiments, the amino acid sequences of native coronavirus spike proteins are substituted as described herein to produce isolated immunogenic polypeptides that include variant coronavirus spike proteins that are modifications of native coronavirus spike proteins or fragments thereof.

[0073] Also included in Table 1 below are amino acid sequences of spike proteins of SARS-CoV-2 variants, including alpha, beta, gamma, delta and omicron variants (including omicron BA1 and BA2). As with the amino acid sequences of native coronavirus spike proteins, the amino acid sequences of spike proteins of these SARS-CoV-2 variants can be modified at corresponding positions as described herein to produce isolated immunogenic polypeptides that include variant coronavirus spike proteins that are modifications of native variant coronavirus spike proteins or fragments thereof. For example, in some embodiments, the amino acid sequences of spike proteins of these SARS-CoV-2 variants are substituted as described herein to produce isolated immunogenic polypeptides that include variant coronavirus spike proteins that are modifications of variant coronavirus spike proteins or fragments thereof. Additional variants not specifically listed below are also contemplated. For example, any variant coronavirus spike protein having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to a native coronavirus spike protein sequence can be modified (e.g., substituted) at the corresponding positions as described herein to produce an isolated immunogenic polypeptide comprising a variant coronavirus spike protein that is a modification of a native coronavirus spike protein or a fragment thereof.

[0074] [Table 1-1]

[0075] [Table 1-2]

[0076] [Table 1-3]

[0077]

Table 1-4

[0078]

Table 1-5

[0079]

Table 1-6

[0080]

Table 1-7

[0081]

Table 1-8

[0082]

Table 1-9

[0083]

Table 1-10

[0084]

Table 1-11

[0085]

Table 1-12

[0086]

Table 1-13

[0087] [Table 1-14]

[0088] A schematic diagram of coronavirus spike protein with S1 and S2 subunits is shown in FIG. 1. The S1 subunit contains a leader or signal sequence (SS), an N-terminal domain (NTD) and a receptor binding domain (RBD). The S2 subunit contains a heptad repeat region (HR1 and HR2) and a transmembrane domain (TM). Modifications to the spike protein sequence can be made anywhere within the sequence described herein, but in some embodiments, the modifications are made to the sequence linking the NTD or the RBD or the NTD and the RBD. In some embodiments, the modifications are made to the amino acid sequence linking the first heptad repeat region to the second heptad repeat region. In some embodiments, the modifications are made to the amino acid sequence at the interface of the S1 and S2 subunits. For example, in some embodiments, amino acid substitutions are made in the NTD and / or in the RBD and / or in the sequence linking the NTD and RBD, and / or amino acid substitutions are made in the amino acid sequence linking a first heptad repeat region to a second heptad repeat region, and / or amino acid substitutions are made in the amino acid sequence at the interface of the S1 and S2 subunits.

[0089] B. Specific Modifications and Combinations The following Table 2 lists various combinations of modifications that can be made to amino acids in the coronavirus spike protein sequences disclosed above to produce isolated immunogenic polypeptides that include variant coronavirus spike proteins that are variants of native coronavirus spike proteins or fragments thereof. The "+" symbol indicates the inclusion of the specified modification in a particular variant coronavirus spike protein. In some examples, the variant contains any combination of modifications in Table 2 below. The amino acid positions correspond to the amino acid positions in SEQ ID NO: 1 and can be used to determine the corresponding amino acids in other coronavirus spike proteins.

[0090] [Table 2-1]

[0091] [Table 2-2]

[0092] [Table 2-3]

[0093] [Table 2-4]

[0094] [Table 2-5]

[0095] The amino acid numbers in Table 2 above correspond to amino acid positions in the sequence of the SARS-CoV-2 Omicron BA1 variant spike protein (SEQ ID NO:1) and identify the positions of modifications that can be made to any coronavirus spike protein. As an example, the amino acid in SARS-CoV corresponding to the amino acid at position 326 of SEQ ID NO:1 can be modified to produce an isolated immunogenic polypeptide that includes a variant coronavirus spike protein. As another example, the amino acid in MERS-CoV corresponding to the amino acid at position 326 of SEQ ID NO:1 can be modified to produce an isolated immunogenic polypeptide that includes a variant coronavirus spike protein. The amino acids in each human coronavirus spike protein sequence and their corresponding positions with respect to SEQ ID NO:1 can be determined based on alignment of the protein sequences. Table 3 below is an alignment of human coronavirus spike protein sequences (e.g., spike protein sequences of NL63-CoV, 229E-CoV, OC43-CoV, HKU1-CoV, MERS-CoV, SARS-CoV and SARS-CoV-2) with SEQ ID NO:1 (Omicron, corresponding to the SARS-CoV-2 BA1 Omicron variant spike protein). The highlighted positions in the alignment below correspond to the positions of the amino acids to be modified identified in Table 2 above.

[0096] [Table 3-1]

[0097] [Table 3-2]

[0098] [Table 3-3]

[0099] [Table 3-4]

[0100] [Table 3-5]

[0101] [Table 3-6]

[0102] [Table 3-7]

[0103] [Table 3-8]

[0104] In some embodiments, the amino acid corresponding to the amino acid at position 326 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 326 in SEQ ID NO:1 can be substituted with a serine residue to produce a variant coronavirus spike protein. The substitution with a serine residue at 326 may be referred to herein as 326S.

[0105] In some embodiments, the amino acid corresponding to the amino acid at position 364 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 364 in SEQ ID NO:1 can be substituted with a phenylalanine residue to produce a variant coronavirus spike protein. The substitution with a phenylalanine residue at 364 may be referred to herein as 364F.

[0106] In some embodiments, the amino acid corresponding to the amino acid at position 567 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 567 in SEQ ID NO:1 can be substituted with a cysteine ​​residue to produce a variant coronavirus spike protein. The substitution with a cysteine ​​residue at 567 may be referred to herein as 567C.

[0107] In some embodiments, the amino acid corresponding to the amino acid at position 611 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 611 in SEQ ID NO:1 can be substituted with a glycine residue to produce a variant coronavirus spike protein. The substitution with a glycine residue at 611 may be referred to herein as 611G.

[0108] In some embodiments, the amino acid corresponding to the amino acid at position 814 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 814 in SEQ ID NO:1 can be substituted with a phenylalanine residue to produce a variant coronavirus spike protein. The substitution with a phenylalanine residue at 814 may be referred to herein as 814P.

[0109] In some embodiments, the amino acid corresponding to the amino acid at position 840 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 840 in SEQ ID NO:1 can be substituted with an asparagine residue to produce a variant coronavirus spike protein. The substitution with an asparagine residue at 840 may be referred to herein as 840N.

[0110] In some embodiments, the amino acid corresponding to the amino acid at position 851 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 851 in SEQ ID NO:1 can be substituted with a phenylalanine residue to produce a variant coronavirus spike protein. The substitution with a phenylalanine residue at 851 may be referred to herein as 851F.

[0111] In some embodiments, the amino acid corresponding to the amino acid at position 889 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 889 in SEQ ID NO:1 can be substituted with a proline residue to produce a variant coronavirus spike protein. The substitution with a proline residue at 889 may be referred to herein as 889P.

[0112] In some embodiments, the amino acid corresponding to the amino acid at position 896 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 896 in SEQ ID NO:1 can be substituted with a proline residue to produce a variant coronavirus spike protein. The substitution with a proline residue at 896 may be referred to herein as 896P.

[0113] In some embodiments, the amino acid corresponding to the amino acid at position 939 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 939 in SEQ ID NO:1 can be substituted with a proline residue to produce a variant coronavirus spike protein. The substitution with a proline residue at 939 may be referred to herein as 939P.

[0114] In some embodiments, the amino acid corresponding to the amino acid at position 957 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 957 in SEQ ID NO:1 can be substituted with a cysteine ​​residue to produce a variant coronavirus spike protein. The substitution with a cysteine ​​residue at 957 may be referred to herein as 957C.

[0115] In some embodiments, the amino acid corresponding to the amino acid at position 977 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 977 in SEQ ID NO:1 can be substituted with a cysteine ​​residue to produce a variant coronavirus spike protein. The substitution with a cysteine ​​residue at 977 may be referred to herein as 977C.

[0116] In some embodiments, the amino acid corresponding to the amino acid at position 981 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 981 in SEQ ID NO:1 can be substituted with a cysteine ​​residue to produce a variant coronavirus spike protein. The substitution with a cysteine ​​residue at 981 may be referred to herein as 981C.

[0117] In some embodiments, the amino acid corresponding to the amino acid at position 982 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 982 in SEQ ID NO:1 can be substituted with a proline residue to produce a variant coronavirus spike protein. The substitution with a proline residue at 982 may be referred to herein as 982P.

[0118] In some embodiments, the amino acid corresponding to the amino acid at position 983 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 983 in SEQ ID NO:1 can be substituted with a proline residue to produce a variant coronavirus spike protein. The substitution with a proline residue at 983 may be referred to herein as 983P.

[0119] In some embodiments, the amino acid corresponding to the amino acid at position 984 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 984 in SEQ ID NO:1 can be substituted with a proline residue to produce a variant coronavirus spike protein. The substitution at 983 with a proline residue may be referred to herein as 984P.

[0120] In some embodiments, the amino acid corresponding to the amino acid at position 986 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 986 in SEQ ID NO:1 can be substituted with a cysteine ​​residue to produce a variant coronavirus spike protein. The substitution with a cysteine ​​residue at 986 may be referred to herein as 986C.

[0121] In some embodiments, the amino acid corresponding to the amino acid at position 989 in SEQ ID NO:1 can be substituted to produce a variant coronavirus spike protein. In some embodiments, the amino acid corresponding to the amino acid at position 989 in SEQ ID NO:1 can be substituted with a cysteine ​​residue to produce a variant coronavirus spike protein. The substitution with a cysteine ​​residue at 989 may be referred to herein as 989C.

[0122] In some embodiments, the variant spike protein comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or seventh of the following modifications at positions 326, 364, 567, 611, 814, 840, 851, 889, 896, 939, 957, 977, 981, 982, 983, 984, 986, or 989 as set forth in the SARS-CoV-2 Omicron (BA1, formerly B.1.1.529) spike protein, UniProt Accession No. UFO69279.1: and / or has at least or at most 13, 14, 15, 16, 17, and / or 18, or the corresponding amino acids in another coronavirus spike protein, and in some embodiments, the modifications at or corresponding positions are: serine at 326, phenylalanine at 364, cysteine ​​at 567, glycine at 611, proline at 814, asparagine at 840, phenylalanine at 851, proline at 889, proline at 896, proline at 939, cysteine ​​at 957, cysteine ​​at 977, cysteine ​​at 981, proline at 982, proline at 983, proline at 984, cysteine ​​at 986, and cysteine ​​at 989; and in further embodiments, the variant spike protein is a SARS-CoV-2 omicron nucleotide. The coronavirus spike protein has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the native coronavirus spike protein (BA1, formerly B.1.1.529) spike protein, UniProt accession number UFO69279.1. In some examples, the modifications described herein can be applied alone or in combination with any one or more additional modifications described herein to produce an isolated immunogenic polypeptide that includes a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof.In some embodiments, these modifications can (a) increase recruitment by the RBD of the variant coronavirus spike protein of the RBD-up conformation to expose more neutralization-sensitive epitopes on the spike protein, (b) decrease recruitment by the RBD of the variant coronavirus spike protein of the RBD-down conformation, (c) increase expression of the variant coronavirus spike protein compared to the native coronavirus spike protein, (d) increase adoption of the prefusion conformation, (e) decrease shedding of the S1 subunit of the variant coronavirus spike protein, and / or (f) improve localization of the variant coronavirus spike protein to the host cell membrane.

[0123] In some embodiments, the variant spike protein comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, and / or eighteen of the following alterations at positions 326, 364, 567, 611, 814, 840, 851, 889, 896, 939, 957, 977, 981, 982, 983, 984, 986, 989 as set forth in UniProt Accession No. UFO69279.1, or a spike protein of another coronavirus. In some embodiments, the modifications at the following positions or corresponding positions are: 326 any amino acid except phenylalanine; 364 any amino acid except valine; 567 any amino acid except alanine; 611 any amino acid except glycine; 814 any amino acid except phenylalanine; 840 any amino acid except aspartic acid; 851 any amino acid except lysine; 889 any amino acid except alanine; 896 any amino acid except alanine; 939 any amino acid except alanine; 957 any amino acid except asparagine; 977 any amino acid except isoleucine; 981 any amino acid except leucine; 982 any amino acid except aspartic acid; 983 any amino acid except lysine; and 984 any amino acid except valine. and at 986 is any amino acid except alanine and at 989 is any amino acid except glutamine, in a further embodiment the variant spike protein is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.10%, 99.11%, 99.12%, 99.13%, 99.14%, 99.15%, 99.16%, 99.17%, 99.18%, 99.19 ...3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity. In some examples, the modifications described herein can be applied alone or in combination with any one or more additional modifications described herein to produce an isolated immunogenic polypeptide comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof. In some embodiments, these modifications can (a) increase the recruitment by the RBD of the variant coronavirus spike protein of the RBD-up conformation to expose more neutralization-sensitive epitopes in the spike protein, (b) decrease the recruitment by the RBD of the variant coronavirus spike protein of the RBD-down conformation, (c) increase the expression of the variant coronavirus spike protein compared to the native coronavirus spike protein, (d) increase the adoption of the prefusion conformation, (e) decrease the shedding of the S1 subunit of the variant coronavirus spike protein, and / or (f) improve the localization of the variant coronavirus spike protein to the host cell membrane.

[0124] In some examples, the variant spike protein is truncated compared to SEQ ID NO: 1. The truncated variant may be a transmembrane RBD trimer in some examples. The truncated variant may contain an RBD domain in some examples, and may further include a leader sequence, a transmembrane domain, and a trimerization domain. In some examples, the truncated variant does not contain an ER signal sequence. In some examples, the truncated variant contains a substitution at an amino acid position corresponding to amino acid position 364 of the SARS-CoV-2 Omicron (BA1, formerly B.1.1.529) spike protein, UniProt accession number UFO69279.1. In some examples, the substitution is a replacement with a phenylalanine residue. In some examples, the leader sequence has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the leader sequence of the SARS-CoV-2 Omicron (BA1, formerly B.1.1.529) spike protein, UniProt accession number UFO69279.1. In some examples, the transmembrane domain has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the transmembrane domain of the SARS-CoV-2 Omicron (BA1, formerly B.1.1.529) spike protein, UniProt accession number UFO69279.1.In some examples, the trimerization domain has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the trimerization domain of the SARS-CoV-2 Omicron (BA1, formerly B.1.1.529) spike protein, UniProt accession number UFO69279.1. In some examples, the truncated variant has the amino acid sequence of RBDT3 shown in Table 4.

[0125] [Table 4]

[0126] C. Altered amino acids As used herein, "amino molecule" refers to any amino acid, amino acid derivative, or amino acid mimetic, as known to those skilled in the art. In certain embodiments, the residues of a peptide or protein are consecutive, and no non-amino molecule interrupts the sequence of amino molecule residues. In other embodiments, the sequence may include one or more non-amino molecule moieties. In certain embodiments, the sequence of residues of a proteinaceous molecule may be interrupted by one or more non-amino molecule moieties. Peptides and proteins contain the 20 "natural" amino acids and their post-translational modifications. However, in vitro peptide synthesis allows the use of modified and / or unusual amino acids.

[0127] Thus, the terms "protein," "peptide," or "polypeptide" encompass amino acid sequences that contain at least one of the 20 common amino acids found in naturally synthesized proteins, or at least one modified or unusual amino acid, including, but not limited to, those shown in Table 5 below.

[0128] [Table 5]

[0129] VI. EMBODIMENTS FOR OBTAINING THE ENCODED POLYPEPTIDES In some aspects, there is a nucleic acid molecule that encodes a peptide of interest, e.g., an antigen. Such nucleic acids can be produced by methods known in the art.

[0130] A. Expression The nucleic acid molecules described herein can be used to express large amounts of a polypeptide of interest, e.g., an antigen, e.g., a variant coronavirus spike protein.

[0131] 1. Nucleic acid synthesis In some embodiments, isolated nucleic acid molecules are contemplated, including nucleic acid molecules that encode a polypeptide of a desired sequence or a portion thereof (e.g., one or more polypeptides or fragments containing an antigen). In some embodiments, the nucleic acid molecules can encode antigens, fusion proteins, modified antibodies, antibody fragments, and probes thereof. In addition to control sequences that govern transcription and translation, nucleic acid molecules can contain nucleic acid sequences that serve other functions as well.

[0132] In some embodiments, a nucleic acid molecule is an analog and can include modifications, particularly modifications that increase nuclease resistance, improve binding affinity, and / or improve binding specificity. For example, if the sugar moiety of a nucleoside or nucleotide is replaced by a carbocyclic moiety, it is no longer a sugar. Furthermore, if other substitutions are made, such as substitutions for intersugar phosphodiester linkages, the resulting material is no longer a true species. All such compounds are considered to be analogs. Throughout this specification, reference to the sugar moiety of a nucleic acid species shall be understood to refer to either the true sugar or a species that adopts the structural arrangement of the sugar of a wild-type nucleic acid. Furthermore, reference to the intersugar linkage shall be interpreted to include a moiety that functions to link the sugar or sugar analog moiety in the manner of a wild-type nucleic acid.

[0133] Modified oligonucleotides and oligonucleotide analogs can show increased chemical and / or enzymatic stability compared to their naturally occurring counterparts.Extracellular and intracellular nucleases generally do not recognize and therefore do not bind to backbone-modified compounds.When present as protonated acid form, the lack of negatively charged backbone can facilitate cell penetration.

[0134] The modified internucleoside linkages are intended to replace the naturally occurring phosphodiester-5'-methylene linkage with a four atom linking group to confer nuclease resistance and enhanced cellular uptake to the resulting compounds.

[0135] The modification can be accomplished using a solid support that can be manually manipulated or used in conjunction with a nucleic acid synthesizer using methodologies generally known to those skilled in the art of nucleic acid synthesis. In general, the procedure involves functionalization of the sugar moieties of two nucleosides that will be adjacent to each other in a selected sequence. In the 5' to 3' sense, the "upstream" synthon, e.g., structure H, is modified at its terminal 3' site, while the "downstream" synthon, e.g., structure H1, is modified at its terminal 5' site.

[0136] Oligonucleosides linked by hydrazine, hydroxylamine and other linking groups can be protected by a dimethoxytrityl group at the 5'-hydroxyl and activated for coupling at the 3'-hydroxyl by a cyanoethyldiisopropyl-phosphite moiety. These compounds can be inserted into any desired sequence by standard, solid-phase, automated nucleic acid synthesis techniques. One of the most common processes is the phosphoramidite technique. Oligonucleotides containing uniform backbone linkages can be synthesized by using a CPG-solid support and standard nucleic acid synthesis machines, such as Applied Biosystems Inc. 380B and 394 and Milligen / Biosearch 7500 and 8800s. The initial nucleotide (number 1 at the 3' end) is attached to a solid support, such as controlled pore glass. In a sequence-specific order, each new nucleotide is attached by manual manipulation or by an automated synthesizer system.

[0137] Free amino groups can be alkylated, for example, with acetone and sodium cyanoborohydride in acetic acid. The alkylation step can be used to introduce other useful functional molecules onto the macromolecule. Such useful functional molecules include, but are not limited to, reporter molecules, cleavage groups, groups for improving the pharmacokinetic properties of oligonucleotides, and groups for improving the pharmacodynamic properties of oligonucleotides. Such molecules can be attached or conjugated to the macromolecule by attachment to a nitrogen atom in the backbone linkage. Alternatively, such molecules can be attached to a pendant group extending from the hydroxyl group of one or more sugar moieties of the nucleotide. Examples of such other useful functional groups are provided by WO1993007883, which is incorporated herein by reference, and in other of the above-referenced patent applications.

[0138] The solid support may comprise any of the solid supports known in the art for polynucleotide synthesis, including controlled pore glass (CPG), oxalyl controlled pore glass, TentaGel Support - an amino polyethylene glycol derivatized support, or Poros - a copolymer of polystyrene / divinylbenzene. Attachment and cleavage of nucleotides and oligonucleotides may be effected by standard procedures. As used herein, the term solid support further includes any linker (e.g., long chain alkylamines and succinyl residues) used to attach growing oligonucleosides to stationary phases such as CPG. In some embodiments, the oligonucleotide may be further defined as having one or more locked nucleotides, ethylene bridged nucleotides, peptide nucleic acids, or 5'(E)-vinyl-phosphonate (VP) modifications. In some embodiments, the oligonucleotide has one or more phosphorothioated nucleobases.

[0139] 2. Expression system There are numerous expression systems that contain at least some or all of the proteins, peptides or nucleic acid molecules described above. Prokaryotic and / or eukaryotic based or cell-free systems can be used for use according to the embodiments to produce proteins, peptides, nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Commercially available and widely used systems include, but are not limited to, bacterial, mammalian, yeast, insect cells and cell-free systems. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. Appropriate cell lines, host systems or expression systems can be chosen to ensure correct modification and processing of the expressed nucleic acid or polypeptide(s). Those skilled in the art can use appropriate expression systems to express vectors to produce nucleic acid sequences or their cognate polypeptides, proteins or peptides.

[0140] In some aspects, the immunogenic variant coronavirus spike protein constructs and / or nucleic acids encoding the immunogenic variant coronavirus spike protein constructs of the present disclosure are achieved by operably linking the nucleic acid encoding the immunogenic variant coronavirus spike protein construct to a promoter, incorporating the construct into an expression vector, and incorporating it into a cell for expression. The vector may be suitable for replication in eukaryotes and, in some cases, integration. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0141] In certain embodiments, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, and other virology and molecular biology manuals.

[0142] Several virus-based systems have been developed for gene transfer into mammalian cells. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses (including self-inactivating lentiviral vectors). For example, adenoviruses provide a convenient platform for gene delivery systems. Using techniques known in the art, a selected gene can be inserted into a vector and packaged into retroviral particles. The recombinant virus can then be isolated and delivered to cells of a subject either in vivo or ex vivo. Thus, in some aspects, the nucleic acid encoding the immunogenic polypeptide construct of the present disclosure is introduced into cells using a recombinant vector, such as a viral vector, including, for example, lentivirus, retrovirus, gamma-retrovirus, adeno-associated virus (AAV), herpes virus, or adenovirus.

[0143] Those of skill in the art will be well able to construct vectors containing one or more desired polynucleotide sequences by standard recombinant techniques (see, e.g., Maniatis et al., 1988 and Ausubel et al., 1994, both of which are specifically incorporated by reference in their entireties).

[0144] Vectors can also contain other components or functionalities that further modulate gene delivery and / or gene expression or that otherwise provide beneficial properties to targeted cells. Such other components include, for example, components that affect binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that affect uptake of vector nucleic acid by cells; components that affect localization of polynucleotides within cells after uptake (such as agents that mediate nuclear localization); and components that affect expression of polynucleotides.

[0145] Such components may also include markers, such as detectable and / or selectable markers that can be used to detect or select cells that have taken up and expressed the nucleic acid delivered by the vector. Such components may be provided as natural features of the vector (such as the use of certain viral vectors that have components or functionality that mediate binding and uptake), or the vector may be modified to provide such functionality. A wide variety of such vectors are known in the art and are generally available. When the vector is maintained in the host cell, it may be stably replicated by the cell during mitosis as an autonomous structure, may be incorporated into the genome of the host cell, or may be maintained in the nucleus or cytoplasm of the host cell.

[0146] The eukaryotic expression cassette contained in the vector contains, inter alia, (in the 5' to 3' direction) regulatory elements including a eukaryotic transcriptional promoter operably linked to the protein coding sequence, a splice signal with intervening sequences, a transcription termination / polyadenylation sequence, a post-transcriptional regulatory element, and an origin of replication.

[0147] 3. Nucleic Acid Delivery Methods Methods suitable for nucleic acid delivery to result in expression of the composition are expected to include virtually any method capable of introducing a particular nucleic acid (e.g., DNA) into a cell, tissue or organism, as described herein or known to those of skill in the art.Such methods include direct delivery of nucleic acids, e.g., by injection, including microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5,789,215, incorporated herein by reference) (U.S. Patent Nos. 5,994,624; 5,981,274; 5,945,100; 5,780,448; 5,736,524; 5,702,932; 5,656,610; 5,589,466; and 5,580,859, each of which is incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonication (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by particle bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128, each of which is incorporated herein by reference; U.S. Patent Nos. 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,610,042; and 5,710,042. Nos. 5,388,77 and 5,538,880); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each of which is incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); or by desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1985).Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

[0148] One illustrative delivery vehicle is lipids and / or liposomes. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo or in vivo). In another embodiment, the nucleic acid can be associated with lipids. The nucleic acid associated with lipids can be encapsulated in the aqueous interior of the liposome, can be interspersed within the lipid bilayer of the liposome, can be attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, can be entrapped in the liposome, can be complexed with the liposome, can be dispersed in a solution containing lipids, can be mixed with lipids, can be combined with lipids, can be contained as a suspension in lipids, can be contained in or complexed with micelles, or can be otherwise associated with lipids. The lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or with a "collapsed" structure. They may simply be interspersed in the solution, forming aggregates that are perhaps not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include the lipid droplets that naturally occur in the cytoplasm, as well as the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols and aldehydes.

[0149] In certain embodiments, the nucleic acid may be entrapped in a lipid complex, such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Multilamellar liposomes form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before the formation of a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). The amount of liposomes used may vary depending on the nature of the liposomes and the cells used, for example, about 5 to about 20 μg vector DNA per one million to ten million cells may be considered.

[0150] Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980).

[0151] In certain embodiments, the liposome can be complexed with hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the liposome can be complexed or used in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, the liposome can be complexed or used in conjunction with both HVJ and HMG-1. In other embodiments, the delivery vehicle can include a ligand and a liposome.

[0152] In various embodiments, lipids suitable for use can be obtained from commercial sources. For example, lipofectamine can be obtained from Thermo Fisher Scientific, Waltham, Mass.; dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform can be used as the only solvent because it evaporates more easily than methanol. "Liposome" is a generic term that encompasses a variety of single and multilamellar lipid vehicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Multilamellar liposomes form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before the formation of a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al. (1991) Glycobiology 5:505-510). However, compositions with in-solution structures that differ from the usual vesicular structure are also encompassed. For example, lipids can assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0153] In certain embodiments, the nucleic acid is introduced into the cells by electroporation. Electroporation involves exposure of a suspension of cells and DNA to a high voltage discharge. Recipient cells can be made more susceptible to transformation by mechanical wounding. The amount of vector used can also vary depending on the nature of the cells used, for example, about 5 to about 20 μg vector DNA per 1 to 10 million cells can be considered.

[0154] Transfection of eukaryotic cells using electroporation has been fairly successful: in this manner, mouse pre-B lymphocytes have been transfected with the human kappa-immunoglobulin gene (Potter et al., 1984) and rat hepatocytes with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986).

[0155] In other embodiments, nucleic acid is introduced into cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA using this technique (Graham and Van Der Eb, 1973). Similarly, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells have been transfected with the neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes have been transfected with various marker genes (Rippe et al., 1990).

[0156] In another embodiment, the nucleic acid is delivered to cells using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids have been introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).

[0157] 4.Host cells In another embodiment, the use of a host cell into which a nucleic acid molecule has been introduced is considered. The nucleic acid can be transfected into the cell according to various methods known in the art. The nucleic acid can be introduced into a prokaryotic or eukaryotic cell by conventional transformation or transfection techniques. Some nucleic acids can use control sequences that allow the nucleic acid to be replicated and / or expressed in both prokaryotic and eukaryotic cells. In certain embodiments, the expression construct of the polypeptide of interest or the nucleic acid replicase can be placed under the control of a promoter associated with T cell activation, such as those controlled by NFAT-1 or NF-κB, both of which are transcription factors that can be activated after T cell activation. Control of expression allows T cells, e.g., tumor-targeting T cells, to sense their surroundings and perform real-time modulation of cytokine signaling, both in the T cell itself and in the surrounding endogenous immune cells. Those skilled in the art will understand the conditions for incubating and maintaining the host cell and allowing the replication of the nucleic acid molecule. The techniques and conditions which will allow for large-scale production of nucleic acid molecules, as well as the production of the nucleic acid encoded by the vector, and its cognate polypeptide, protein, or peptide, are also understood and known.

[0158] It is known that for mammalian cell transfection, depending on the nucleic acid and transfection technique used, a small proportion of cells can integrate exogenous nucleic acid into themselves. Regardless of the method used to introduce exogenous nucleic acid into host cells, various assays can be carried out to identify and select such integrants. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as immunological means (ELISA and Western blot) or the assays described herein to identify agents that fall within the scope of the present disclosure, for example, detection of the presence or absence of specific peptides.

[0159] In certain embodiments, cells containing exogenous nucleic acid can be identified in vitro or in vivo by including a marker in the expression vector or exogenous nucleic acid. Such a marker will confer an identifiable change to the cell, allowing easy identification of cells containing the expression vector. In general, a selection marker can be a marker that confers a property that allows selection. A positive selection marker can be a marker whose presence allows its selection, while a negative selection marker is a marker whose presence prevents its selection. An example of a positive selection marker is a drug resistance marker.

[0160] In addition to markers that confer a phenotype that allows for the identification of transformants based on the implementation of conditions, other types of markers are also considered, including screenable markers such as GFP, whose basis is colorimetric analysis. Alternatively, screenable enzymes as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT), can be utilized. Those skilled in the art will also know how to use immunological markers, perhaps in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are known to those skilled in the art.

[0161] Selectable markers can include types of reporter genes used in laboratory microbiology, molecular biology and genetic engineering to indicate the success of transfection or other procedures intended to introduce foreign DNA into cells. Selectable markers are often antibiotic resistance genes; cells subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and cells that are able to grow have successfully taken up and expressed the introduced genetic material. Examples of selectable markers include the Abicr gene, or the Neo gene from Tn5, which confers antibiotic resistance to geneticin.

[0162] Screenable markers can include reporter genes that allow researchers to distinguish between desired and undesired cells. Certain aspects of the present invention utilize reporter genes to indicate specific cell lineages. For example, reporter genes can be placed within expression elements and under the control of ventricular or atrial selective regulatory elements that are normally associated with the coding regions of ventricular or atrial selective genes for co-expression. Reporters allow cells of specific lineages to be isolated without being placed under drug or other selection pressure or otherwise risking cell viability.

[0163] Examples of such reporters include genes encoding cell surface proteins (e.g., CD4, HA epitope), fluorescent proteins, antigenic determinants and enzymes (e.g., β-galactosidase). The vector containing cells can be isolated by FACS using, for example, fluorescently tagged antibodies against cell surface proteins or substrates that can be converted to fluorescent products by vector-encoded enzymes.

[0164] In a specific embodiment, the reporter gene is a fluorescent protein. A wide range of fluorescent protein genetic variants have been developed featuring fluorescence emission spectral profiles spanning almost the entire visible light spectrum (see Table 1 for non-limiting examples). Mutagenesis attempts in the original Aequorea victoria jellyfish green fluorescent protein have resulted in new fluorescent probes with colors ranging from blue to yellow that are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins emitting in the orange and red spectral regions have been developed from marine anemones, Discosoma striata, and reef corals belonging to the class Anthozoa. Still other species have been unearthed to produce similar proteins with cyan, green, yellow, orange, and crimson fluorescence emissions. Developmental research efforts are ongoing to improve the brightness and stability of fluorescent proteins and thus their overall utility.

[0165] In certain embodiments, the cells of the present disclosure may be specifically formulated and / or cultured in a particular medium. The cells may be formulated in such a manner that they are suitable for delivery to a recipient without adverse effects.

[0166] In a specific embodiment, the medium can be prepared using a medium used for culturing animal cells as its base medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640 and Fisher's medium, and any combination thereof, but the medium may not be particularly limited thereto, so long as it can be used for culturing animal cells. In particular, the medium can be xeno-free or chemically defined.

[0167] The medium may be serum-containing or serum-free or xeno-free. In an embodiment to prevent contamination with foreign animal-derived components, the serum may be derived from the same animal as the stem cell(s). Serum-free medium refers to a medium that does not contain raw or unpurified serum, and thus may include a medium with purified blood-derived components or animal tissue-derived components (such as growth factors).

[0168] The medium may or may not contain any serum replacement. Serum replacement may include materials that suitably contain albumin (lipid-rich albumin, bovine albumin, albumin substitutes such as recombinant or humanized albumin, vegetable starch, dextran, and protein hydrolysates, etc.), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolgiycerol, or their equivalents. Serum replacement may be prepared, for example, by the methods disclosed in WO 98 / 30679, which is incorporated by reference in its entirety. Alternatively, for greater convenience, any commercially available material may be used. Commercially available materials include knockout serum replacement (KSR), chemically defined concentrated lipids (Gibco), and Glutamax (Gibco).

[0169] In certain embodiments, the medium may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more of the following: vitamins, e.g., biotin; DL alpha tocopherol acetate; DL alpha-tocopherol; vitamin A (acetate); proteins, e.g., BSA (bovine serum albumin) or human albumin, fatty acid free fraction V; catalase; human recombinant insulin; human transferrin; superoxide dismutase; other components, e.g., corticosterone; D-galactose; ethanolamine HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and / or T3 (triodo-I-thyronine). In specific embodiments, one or more of these may be specifically excluded.

[0170] In some embodiments, the medium further comprises vitamins. In some embodiments, the medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium comprises combinations thereof or salts thereof. In some embodiments, the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol and vitamin B12. In some embodiments, the vitamin comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some embodiments, the medium further comprises a protein. In some embodiments, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some embodiments, the medium further comprises one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or combinations thereof. In some embodiments, the medium comprises one or more of the following: B-27® supplement, xeno-free B-27® supplement, GS21™ supplement, or combinations thereof. In some embodiments, the medium comprises or further comprises amino acids, monosaccharides, inorganic ions, In some embodiments, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof.In some embodiments, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some embodiments, the medium further includes one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain embodiments, the medium comprises or consists essentially of one or more vitamins described herein and / or one or more proteins described herein and / or one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite or triiodo-I-thyronine, B-27® supplement, xeno-free B-27® supplement, GS21™ supplement, amino acids (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In specific embodiments, one or more of these may be specifically excluded.

[0171] The medium may also contain one or more exogenously added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffering agents, and / or inorganic salts, of which, in specific embodiments, one or more may be specifically excluded.

[0172] One or more of the medium components can be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / mL, μg / mL, mg / mL, or any range derivable therein.

[0173] In a specific embodiment, the cells of the present disclosure are specifically formulated. The cells of the present disclosure may or may not be formulated as a cell suspension. In a specific case, the cells of the present disclosure are formulated in a single dose form. The cells of the present disclosure may be formulated for systemic or local administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation may include one or more cryopreservatives, such as DMSO (e.g., in 5% DMSO). The cell formulation may include albumin, including human albumin, and a specific formulation includes 2.5% human albumin. The cells may be specifically formulated for intravenous administration; for example, the cells are formulated for intravenous administration for less than 1 hour. In a specific embodiment, the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from the time of thawing.

[0174] B. Isolation The nucleic acid molecules disclosed herein can be obtained from any source that produces nucleic acids. Methods for isolating nucleic acids are well known in the art.

[0175] VII. Immune Responses and Assays As described herein, the present disclosure relates to eliciting or inducing an immune response in a subject against a coronavirus protein, e.g., a native or variant coronavirus spike protein. In one aspect, the immune response can protect against infection or related disease, particularly infection or related disease associated with coronavirus, or treat a subject having, suspected of having, or at risk of developing an infection or related disease, particularly infection or related disease associated with coronavirus. One use of the immunogenic composition of the present disclosure is to prevent coronavirus infection by inoculating a subject.

[0176] A. Immunoassays The present disclosure includes carrying out serological assays to assess whether and to what extent an immune response is induced or elicited by the compositions of the present disclosure. There are many types of immunoassays that can be carried out. Immunoassays encompassed by the present disclosure include, but are not limited to, those described in U.S. Patent No. 4,367,110 (double monoclonal antibody sandwich assay) and U.S. Patent No. 4,452,901 (Western blot). Other assays include immunoprecipitation and immunocytochemistry of labeled ligands, both in vitro and in vivo.

[0177] Immunoassays are generally binding assays. Certain preferred immunoassays are the various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. In one example, an antibody or antigen is immobilized on a selected surface, such as a well in a polystyrene microtiter plate, a dipstick, or a column support. A test composition suspected of containing the desired antigen or antibody, such as a clinical sample, is then added to the well. After binding and washing to remove non-specifically bound immune complexes, the bound antigen or antibody can be detected. Detection is generally achieved by the addition of another antibody specific for the desired antigen or antibody, linked to a detectable label. This type of ELISA is known as a "sandwich ELISA." Detection can also be achieved by the addition of a second antibody specific for the desired antigen, followed by the addition of a third antibody that has binding affinity for the second antibody and is linked to a detectable label. Competitive ELISA is also a possible implementation in which the test sample competes with a known amount of labeled antigen or antibody for binding. The amount of reactive species in an unknown sample is determined by mixing the sample with a known labeled species before or during incubation with the coated well. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well, thus reducing the final signal. Regardless of the format used, ELISAs have certain features in common, such as coating, incubation or binding, washing to remove non-specifically bound species, and detection of bound immune complexes.

[0178] The antigen or antibody may be linked to a solid support, such as in the form of a plate, bead, dipstick, membrane or column matrix, and the sample to be analyzed is applied to the immobilized antigen or antibody. In coating a plate with either an antigen or antibody, the wells of the plate are generally incubated with a solution of the antigen or antibody, either overnight or for a specified period of time. The wells of the plate are then washed to remove incompletely adsorbed material. All remaining available well surfaces are then "coated" with a non-specific protein that is antigenically neutral with respect to the test antiserum. Such non-specific proteins include bovine serum albumin (BSA), casein, and a solution of milk powder. The coating allows for blocking of non-specific adsorption sites on the immobilizing surface, thus reducing the background caused by non-specific binding of the antiserum to the surface.

[0179] B. Diagnosis of Coronavirus Infection In addition to the use of the proteins, polypeptides and / or peptides to treat, prevent or reduce the severity of disease due to infection as described above, the present disclosure contemplates the use of these polypeptides, proteins and / or peptides in a variety of ways, including detecting the presence of coronavirus to diagnose infection. In accordance with the present disclosure, a preferred method of detecting the presence of infection involves obtaining a sample from an individual suspected of being infected with one or more coronavirus strains, such as a sample taken from the individual's blood, saliva, tissue, bone, muscle, cartilage or skin. After isolation of the sample, a diagnostic assay utilizing the polypeptides, proteins and / or peptides of the present disclosure can be performed to detect the presence of coronavirus, and such assay techniques for determining such presence in a sample are well known to those of skill in the art and include methods such as radioimmunoassay, Western blot analysis and ELISA assay.

[0180] Generally, in accordance with the present disclosure, a method of diagnosing an infection is contemplated, in which a sample suspected of being infected with a coronavirus is spiked with a polypeptide, protein or peptide in accordance with the present disclosure, and a coronavirus is indicated by an antibody in the sample that binds to the polypeptide, protein and / or peptide, or a polypeptide, protein and / or peptide that binds to the antibody.

[0181] Thus, the polypeptides, proteins and / or peptides according to the present disclosure can be used to treat, prevent, or reduce the severity of infectious disease caused by coronavirus infection (i.e., active or passive immunization), or for use as research tools.

[0182] Any of the polypeptides, proteins and / or peptides described above can be directly labeled with a detectable label for coronavirus identification and quantification. Labels for use in immunoassays are generally known to those of skill in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold or latex beads. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISAs).

[0183] C. Protective immunity In some aspects of the present disclosure, the proteinaceous composition confers protective immunity to a subject. Protective immunity refers to the body's ability to mount a specific immune response that protects the subject from developing a particular disease or condition involving a pathogen against which an immune response exists. An immunogenic effective amount is capable of conferring protective immunity to a subject.

[0184] As used herein, in this specification and in the following claims section, the term polypeptide or peptide refers to a sequence of amino acids covalently linked between them by peptide bonds. Different polypeptides have different functionalities according to the present invention. According to one embodiment, the polypeptide is derived from an immunogen designed to induce an active immune response in a recipient, whereas according to another embodiment of the present invention, the polypeptide is derived from an antibody that arises after eliciting an active immune response in an animal, for example, and can function to induce a passive immune response in a recipient. However, in both cases, the polypeptide is encoded by a polynucleotide according to any possible codon usage.

[0185] As used herein, the phrase "immune response" or its equivalent "immunological response" refers to the development of a humoral (antibody-mediated), cellular (mediated by antigen-specific T cells or their secretory products), or both humoral and cellular response to a protein, peptide, carbohydrate, or polypeptide of the invention in a recipient patient. Such a response can be an active response induced by administration of an immunogen, or a passive response induced by administration of an antibody, antibody-containing material, or primed T cells. A cellular immune response is elicited by the presentation of a polypeptide epitope in association with a class I or class II MHC molecule to activate antigen-specific CD4(+) T helper cells and / or CD8(+) cytotoxic T cells. The response may also involve the activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglial cells, eosinophils, or other components of innate immunity. As used herein, "active immunity" refers to any immunity conferred in a subject by administration of an antigen.

[0186] As used herein, "passive immunity" refers to any immunity conferred in a subject without administration of an antigen to the subject. Thus, "passive immunity" includes, but is not limited to, administration of activated immune effectors, including cellular or protein mediators of the immune response (e.g., monoclonal and / or polyclonal antibodies). Monoclonal or polyclonal antibody compositions can be used in passive immunization to treat, prevent, or reduce the severity of disease caused by infection with an organism carrying the antigen recognized by the antibody. The antibody composition can include antibodies that bind to a variety of antigens, which in turn can be associated with a variety of organisms. The antibody component can be a polyclonal antiserum. In certain embodiments, the antibody(ies) have been affinity purified from an animal or a second subject that has been challenged with the antigen(s). Alternatively, an antibody mixture can be used, which is a mixture of monoclonal and / or polyclonal antibodies against antigens present in the same, closely related, or different microorganisms or organisms, such as viruses, including but not limited to coronaviruses.

[0187] Passive immunity can be conferred to a patient or subject by administering to the patient immunoglobulin (Ig) and / or other immune factors obtained from a donor or other non-patient source with known immune reactivity. In other embodiments, the immunogenic composition of the present disclosure can be administered to a subject, who then acts as a source or donor of globulins ("hyperimmune globulins") produced in response to challenge with the immunogenic composition that contain antibodies against coronavirus or other organisms. A subject thus treated will donate plasma from which hyperimmune globulins can then be obtained by conventional plasma fractionation methodologies and administered to another subject to confer resistance to or treat coronavirus infection.

[0188] For purposes of this specification and the appended claims, the terms "epitope" and "antigenic determinant" are used interchangeably to refer to a site on an antigen to which B and / or T cells respond or recognize. B cell epitopes can be formed from both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols (1996). Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen. T cells recognize continuous epitopes of about 9 amino acids for CD8 cells or about 13-15 amino acids for CD4 cells. T cells that recognize an epitope are expressed by primed T cells that have responded to the epitope. 3 It can be identified by in vitro assays measuring antigen-dependent proliferation as determined by H-thymidine incorporation (Burke et al., 1994), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., 1996), or by cytokine secretion. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4(+) T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contribution of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating IgG and T cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

[0189] As used herein, the terms "antibody" or "immunoglobulin" are used interchangeably and refer to any of several classes of structurally related proteins that function as part of an animal's or recipient's immune response, including IgG, IgD, IgE, IgA, IgM and related proteins. Under normal physiological conditions, antibodies are found in plasma and other body fluids and in the membranes of certain cells and are produced by lymphocytes of a type designated B cells or their functional equivalents.

[0190] As used herein, the terms "immunogenic substance" or "immunogen" or "antigen" are used interchangeably to refer to a molecule that is capable of inducing an immunological response against itself upon administration to a recipient, either alone, in conjunction with an adjuvant, or presented in a display medium.

[0191] VIII. Composition In one aspect, the present disclosure relates to an immunogenic composition for administration to a host. In some aspects, the host is human. In other aspects, the host is non-human.

[0192] In some embodiments, the composition comprises an immunogenic polypeptide construct. In some embodiments, the immunogenic polypeptide construct of the composition is an isolated immunogenic polypeptide comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof, and / or a nucleic acid encoding an isolated immunogenic polypeptide comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof. In some embodiments, the composition comprises a nucleic acid encoding a variant coronavirus spike protein construct and / or a modified variant coronavirus spike protein construct. A variant coronavirus spike protein construct may differ from a native, unmodified coronavirus spike protein construct in one or more amino acids. In some embodiments, a variant coronavirus spike protein construct has at least about 50% amino acid sequence identity with a native, unmodified coronavirus spike protein construct.

[0193] A. Vaccines In some examples, the compositions described herein are immunogenic compositions. In some examples, the compositions described herein comprise at least one isolated nucleic acid or polypeptide molecule described herein. In some examples, the compositions described herein are vaccines. In specific embodiments, the immunogenic composition comprises a nucleic acid, and the immunogenic composition is a nucleic acid vaccine. In other embodiments, the immunogenic composition comprises DNA, and the vaccine is a DNA vaccine. In yet other embodiments, the immunogenic composition comprises a polypeptide, and the vaccine is a polypeptide vaccine. Conditions and / or diseases that can be treated with nucleic acid and / or peptide or polypeptide compositions include, but are not limited to, conditions and / or diseases caused and / or affected by infection, cancer, rare diseases, and other diseases or conditions caused by overproduction, underproduction, or inappropriate production of proteins or nucleic acids.

[0194] In some embodiments, a composition comprises a nucleic acid or polypeptide molecule that is substantially free of one or more impurities or contaminants, e.g., equal to, at least one of, at most any one of, or to some degree between any two of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure; at least 98% pure, or at least 99% pure.

[0195] The present invention includes methods for preventing or ameliorating coronavirus infection. Thus, the present invention contemplates vaccines for use in both active and passive immunization aspects. Immunogenic compositions proposed to be suitable for use as vaccines can be prepared from native or variant coronavirus polypeptide(s), such as native or variant coronavirus spike proteins. In other aspects, coronavirus spike proteins can be used in combination with other secreted virulence proteins, surface proteins, or immunogenic fragments thereof. In certain aspects, the antigenic material is extensively dialyzed to remove undesired small molecular weight molecules and / or lyophilized for more rapid formulation into the desired medium.

[0196] The preparation of vaccines containing nucleic acids and / or peptides or polypeptides as active ingredients is generally well understood in the art, as exemplified by U.S. Patent Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all of which are incorporated herein by reference. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in liquid prior to injection can also be prepared. Preparations may be emulsified. The active immunogenic ingredient is often mixed with an excipient that is pharma- ceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the vaccine can contain amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccine. In specific embodiments, the vaccine is formulated with a combination of substances as described in U.S. Patent Nos. 6,793,923 and 6,733,754, which are incorporated herein by reference.

[0197] Vaccines can be administered parenterally, by injection, for example, either subcutaneously or intramuscularly, in conventional manner. Additional formulations suitable for other administration mechanisms include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers can include, for example, polyalkalene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1% to about 2%. Oral formulations contain commonly employed excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Such compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain about 10% to about 95% of the active ingredient, preferably about 25% to about 70%.

[0198] The polypeptides and polypeptide-encoding nucleic acid constructs can be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like.

[0199] Typically, the vaccine is administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective and immunogenic. The amount to be administered depends on the subject to be treated, including the capacity of the individual's immune system to synthesize antibodies and the degree of protection desired. The exact amount of active ingredient required to be administered depends on the judgment of the practitioner. However, suitable dosage ranges are on the order of several hundred micrograms of active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.

[0200] The mode of application can vary widely. Any of the conventional methods for administration of vaccines are applicable. Such methods are contemplated to include oral application in a solid physiologically acceptable base or in a physiologically acceptable dispersion system, parenterally, by injection, etc. The dosage of the vaccine will depend on the route of administration and will vary according to the size and health of the subject.

[0201] In certain instances, it will be desirable to have multiple administrations of the vaccine, e.g., 2, 3, 4, 5, 6 or more administrations. Vaccinations may be at intervals of 1, 2, 3, 4, 5, 6, 7, 8 to 5, 6, 7, 8, 9, 10, 11, 12 (twelve) weeks (including all ranges therebetween). Periodic boosters at intervals of 1 to 5 years may be desirable to maintain protective levels of antibodies. The progress of immunization can be followed by assays for antibodies to the antigen, as described in U.S. Patent Nos. 3,791,932; 4,174,384 and 3,949,064.

[0202] 1. Carrier A given composition may vary in its immunogenicity. Therefore, it is often necessary to boost the host immune system, as can be achieved by coupling peptides or polypeptides to carriers. Exemplary carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins, such as ovalbumin, mouse serum albumin or rabbit serum albumin, can also be used as carriers. Means for conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

[0203] 2. Adjuvants The immunogenicity of a polypeptide or peptide composition can be enhanced by the use of non-specific stimulators of immune response, known as adjuvants. Suitable adjuvants include all acceptable immune stimulating compounds, such as cytokines, toxins or synthetic compositions. Some adjuvants can be used to enhance the antibody response to variant SpA polypeptide or coagulase, or any other bacterial protein or combination considered herein. Adjuvants can (1) trap antigens in the body and cause slow release; (2) attract cells involved in immune response to the administration site; (3) induce proliferation or activation of immune system cells; or (4) improve the diffusion of antigens throughout the subject's body.

[0204] Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides, and natural substances.Specific adjuvants that can be used include IL-1, IL-2, IL-4, IL-7, IL-12, □-interferon, GMCSP, BCG, aluminum salts, such as aluminum hydroxide or other aluminum compounds, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).RIBI contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS), in a 2% squalene / Tween 80 emulsion.MHC antigens may be used. Other adjuvants or methods are illustrated in US Pat. Nos. 6,814,971, 5,084,269, and 6,656,462, each of which is incorporated herein by reference.

[0205] Various methods of achieving adjuvant affect for vaccines include the use of agents such as aluminum hydroxide or aluminum phosphate (Alum), which are commonly used as about 0.05 to about 0.1% solutions in phosphate buffer solutions, mixtures with synthetic polymers of sugars (Carbopol®) used as about 0.25% solutions, and aggregation of proteins in the vaccine by heat treatment at temperatures ranging between about 70°C to about 101°C for periods of 30 seconds to 2 minutes, respectively. Adjuvant effects can also be produced using agglutination by reactivation with pepsin-treated (Fab) antibodies to albumin; mixtures with bacterial cells (e.g., C. parvum), endotoxins or lipopolysaccharide components of gram-negative bacteria; emulsions in physiologically acceptable oil vehicles (e.g., mannide mono-oleate (Aracel A)); or emulsions with a 20% solution of perfluorocarbon (Fluosol-DA®) used as a block substitute.

[0206] Examples of often preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide.

[0207] In some embodiments, adjuvants are preferably selected to be preferential inducers of either Th1 or Th2-type responses: high levels of Th1-type cytokines tend to favor the induction of a cell-mediated immune response to a given antigen, while high levels of Th2-type cytokines tend to favor the induction of a humoral immune response to the antigen.

[0208] The distinction between Th1 and Th2 type immune responses is not absolute. In reality, an individual will support an immune response that is described as predominantly Th1 or predominantly Th2. However, it is often convenient to consider the family of cytokines in terms of those described in mouse CD4+ T cell clones by Mosmann and Coffman (Mosmann and Coffman, 1989). Traditionally, Th1 type responses are associated with the production of INF-γ and IL-2 cytokines by T lymphocytes. Other cytokines often directly associated with the induction of Th1 type immune responses are not produced by T cells, such as IL-12. In contrast, Th2 type responses are associated with the secretion of IL-4, IL-5, IL-6, IL-10.

[0209] In addition to adjuvants, it may be desirable to co-administer a biological response modifier (BRM) to enhance the immune response. BRMs have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include cimetidine (CIM; 1200 mg / d) (Smith / Kline, PA); or low-dose cyclophosphamide (CYP; 300 mg / m 2) (Johnson / Mead, NJ) and genes encoding proteins involved in immune helper function, such as cytokines, e.g., □-interferon, IL-2 or IL-12, or B-7.

[0210] B. Lipid Components and Molecules In some embodiments, the composition further comprises a lipid-based delivery system (e.g., a lipid-based vaccine) that can deliver the nucleic acid molecule to the interior of a cell where it can then replicate, inhibit expression of a protein of interest, and / or express the encoded polypeptide of interest. The delivery system can have an adjuvant effect that enhances the immunogenicity of the encoded antigen.

[0211] Thus, in certain embodiments, the present invention relates to compositions comprising one or more lipids associated with nucleic acid or polypeptide / peptide.Lipids are substances that are insoluble in water and can be extracted with organic solvents.Compounds other than those specifically described herein are understood by those skilled in the art as lipids and are encompassed by the compositions and methods of the present invention.Lipid components and non-lipids can be attached to each other by either covalent or non-covalent bonds.

[0212] Lipids can be naturally occurring lipids or synthetic lipids.However, lipids are usually biological substances.Biological lipids are well known in the art and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, sphingoglycolipids, glycolipids, sulfatides, lipids with ether- and ester-linked fatty acids and polymerizable lipids, and combinations thereof.

[0213] In some embodiments, the composition further comprises a neutral lipid, a cationic lipid, cholesterol, and polyethylene glycol (PEG) to form a nanoparticle that contains or encapsulates the nucleic acid molecule. In some embodiments, the composition further comprises any one of a cationic lipid, a liposome, a lipid nanoparticle, a polyplex, a cochleate, a virosome, an immune stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsome, a polycationic peptide, and a cationic nanoemulsion. In some embodiments, the nucleic acid molecule is encapsulated in, bound to, or adsorbed to any one of cationic lipids, liposomes, lipid nanoparticles, polyplexes, cochleates, virosomes, immune stimulating complexes, microparticles, microspheres, nanospheres, unilamellar vesicles, multilamellar vesicles, oil-in-water emulsions, water-in-oil emulsions, emulsomes, polycationic peptides, and cationic nanoemulsions, or combinations thereof.As used herein, "encapsulate", "encapsulated", "encapsulation" and grammatically comparable variants thereof mean that at least a portion of a substance is enclosed or surrounded by another material or substance in a composition.In some embodiments, a substance such as a nucleic acid may be completely encapsulated or surrounded by another material or substance in a composition, such as a lipid.

[0214] In certain embodiments, the composition comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, approx. 31%, approx. 32%, approx. 33%, approx. 34%, approx. 35%, approx. 36%, approx. 37%, approx. 38%, approx. 39%, approx. 40%, approx. 41%, approx. 42%, approx. 43%, approx. 44%, approx. 45%, approx. 46%, approx. 47%, approx. 48%, approx. 49%, approx. 50%, approx. 51%, approx. 52%, approx. 53%, approx. 54%, approx. 55%, approx. 56%, approx. 57%, approx. 58%, approx. 59%, approx. 60% , about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or any range therebetween of a particular lipid, lipid type, or non-lipid component, such as an adjuvant, antigen, peptide, polypeptide, sugar, nucleic acid, or other material disclosed herein or that would be known to one of skill in the art.

[0215] C. General Pharmaceutical Compositions In some embodiments, the composition further comprises one or more stabilizing agents and one or more buffers. The nucleic acid molecule, e.g., naked or encapsulated nucleic acid or polypeptide disclosed herein, may be included in a solution comprising one or more stabilizing agents and one or more buffers. In some embodiments, the stabilizing agent comprises sucrose, mannose, sorbitol, raffinose, trehalose, mannitol, inositol, sodium chloride, arginine, lactose, hydroxyethyl starch, dextran, polyvinylpyrolidone, glycine, or a combination thereof. In some embodiments, the stabilizing agent is a disaccharide or sugar. In one embodiment, the stabilizing agent is sucrose. In another embodiment, the stabilizing agent is trehalose. In a further embodiment, the stabilizing agent is a combination of sucrose and trehalose. In some embodiments, the total concentration of the stabilizing agent(s) in the composition is about 5% to about 10% w / v. For example, the total concentration of the stabilizer can be equal to, at least one of, at most one of, or between any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% w / v, or any range or value derivable therein. In a specific embodiment, the total concentration of the stabilizer(s) in the composition is 10% w / v. In a specific embodiment, the amino acid concentration is 5% w / v.

[0216] Examples of buffering agents include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, amino-sulfonate buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof. In some embodiments, the buffer is a HEPES buffer, a Tris buffer or a PBS buffer. In one embodiment, the buffer is a Tris buffer. In another embodiment, the buffer is a HEPES buffer. In a further embodiment, the buffer is a PBS buffer. In some embodiments, the concentration of the buffer in the composition is about 10 mM. For example, the buffer concentration can be equal to, at least one of, at most one of, or between any two of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM or 20 mM, or any range or value derivable therein. In a specific embodiment, the buffer concentration is 10 mM. The buffer may be neutral pH, pH 6.5-8.5, pH 7.0-pH 8.0 or pH 7.2-pH 7.6.For example, the buffer can be at pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4 or 8.5, or any range or value derivable therein. In a specific embodiment, the buffer is at pH 7.4.

[0217] The composition may further comprise one or more salts, and / or one or more pharma- ceutically acceptable surface active agents, preservatives, carriers, diluents and / or excipients in some cases. In some embodiments, the composition further comprises a pharma- ceutically acceptable vehicle. In some embodiments, each of the buffer, stabilizer, salt, surface active agent, preservative and excipient is included in the composition. In other embodiments, any one or more of the buffer, stabilizer, salt, surface active agent, preservative, excipient, carrier, diluent or vehicle may be excluded from the composition.

[0218] Examples of salts include, but are not limited to, sodium salts and / or potassium salts. In some embodiments, the sodium salt comprises sodium chloride. In some embodiments, the potassium salt comprises potassium chloride. The concentration of the salt in the composition can be about 70 mM to about 140 mM. For example, the salt concentration can be equal to, at least one of, at most one of, or between any two of 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM, or any range or value derivable therein. In a specific embodiment, the salt concentration is 70 mM. In a specific embodiment, the salt concentration is 140 mM. The salt may be at neutral pH, pH 6.5-8.5, pH 7.0-pH 8.0, or pH 7.2-pH 7.6. For example, the salt may be at a pH equal to, at least one of, at most one of, or between any two of 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein.

[0219] Examples of excipients, which refer to ingredients in a composition that are not active ingredients, include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavorings, disintegrants, coatings, plasticizers, compression agents, wet granulation agents, or colorants. Preservatives for use in the compositions disclosed herein include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal. As used herein, "pharmaceutically acceptable carriers" include any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, and the like), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweeteners, flavoring agents, dyes, fluid and nutrient replenishers, such similar materials, and combinations thereof, known to those of skill in the art. Diluents, or diluting or thinning agents, include, but are not limited to, ethanol, glycerol, water, sugars such as lactose, sucrose, mannitol and sorbitol, and starches derived from wheat, corn, rice and potato; and celluloses such as microcrystalline cellulose. The amount of diluent in the composition can range from about 10% to about 90% by weight, about 25% to about 75% by weight, about 30% to about 60%, or about 12% to about 60% by weight of the total composition.

[0220] The pH and exact concentration of various components in pharmaceutical composition are adjusted according to well-known parameters.The use of such media and agents for pharmaceutical active substances is well-known in the art.Unless any conventional media or agent is incompatible with active ingredient, its use in immunogenic, preventive and / or therapeutic compositions is considered.

[0221] D. Combination Therapy The compositions and related methods of the present invention, particularly the administration of coronavirus proteins, including isolated polypeptides comprising native or variant coronavirus spike proteins or fragments thereof, can also be used in combination with the administration of traditional therapies. Such include, but are not limited to, the administration of antiviral therapies, such as nirmatrelvir / ritonavir, remdesivir, or various combinations of antiviral drugs. Also included are the administration of corticosteroids, such as steroids including dexamethasone, anti-inflammatory drugs including acetaminophen or ibuprofen, or various combinations thereof.

[0222] In one embodiment, it is contemplated that the vaccine and / or therapy is used in conjunction with an antiviral treatment. Alternatively, the therapy may precede or follow the other agent treatment by an interval ranging from minutes to weeks. In embodiments in which the other agent and / or vaccine is administered separately, one will generally ensure that no significant period of time expires between each delivery time point, so that the agent and the immunogenic composition can still exert their advantageously combined effect in the subject. In such instances, it is contemplated that both modalities can be administered within about 12-24 hours of each other or within about 6-12 hours of each other. In some circumstances, it may be desirable to significantly extend the period for administration, where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) pass between each administration.

[0223] Various combinations can be used, for example, an antiviral therapy is "A" and an immunogenic polypeptide given as part of an immunotherapy regimen is "B": A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0224] Administration of the immunogenic composition of the present invention to a patient / subject will follow the general protocol for administration of such compounds, taking into account the toxicity, if any, of the coronavirus spike protein composition or other compositions described herein. It is expected that treatment cycles will be repeated as necessary. It is also considered that various standard therapies, such as hydration, can be applied in combination with the described treatments.

[0225] E. Administration Administration of the compositions described herein can be carried out by any of the accepted administration mechanisms of drugs that serve similar utilities. Pharmaceutical compositions can be formulated into preparations in solid, semi-solid, liquid or gas form, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions described herein are formulated so that the active ingredient contained therein is bioavailable after administration of the composition to a patient. The composition to be administered to a subject or patient takes the form of one or more dosage units, where, for example, a tablet can be a single dosage unit and a container of the compound in aerosol form can hold multiple dosage units. The composition to be administered will, in each case, contain a therapeutically and / or prophylactically effective amount of a compound within the scope of the present disclosure, or a pharma- ceutically acceptable salt thereof, for treatment of the disease or condition of interest, in accordance with the teachings set forth herein.

[0226] Pharmaceutical compositions within the scope of this disclosure can be in solid or liquid form. In one aspect, the carrier(s) are particulate, so that the composition is, for example, in tablet or powder form. The carrier(s) can be liquid, and the composition is, for example, oral syrup, injectable liquid, or aerosol, for example, useful in inhaler administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, in which case semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered herein as either solid or liquid. As a solid composition for oral administration, the pharmaceutical composition can be formulated into powder, granule, compressed tablet, pill, capsule, chewing gum, wafer, or similar form. Such solid compositions will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present or excluded: binders, such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth or gelatin; excipients, such as starch, lactose or dextrin; disintegrants, such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants, such as magnesium stearate or Sterotex; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; flavorings, such as peppermint, methyl salicylate or orange flavoring; and colorings. When the pharmaceutical composition is in the form of a capsule, such as a gelatin capsule, it can contain a liquid carrier, such as polyethylene glycol or oil, in addition to materials of the above type. The pharmaceutical composition can be in the form of a liquid, such as an elixir, syrup, solution, emulsion or suspension. The liquid can be for oral administration or delivery by injection, to name just two examples. When intended for oral administration, preferred compositions contain, in addition to the compound, one or more of a sweetener, a preservative, a dye / colorant, and a flavor enhancer. In a composition intended to be administered by injection, one or more of a surface active agent, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent may be included or excluded.

[0227] Liquid pharmaceutical compositions, whether in solution, suspension or other similar form, may contain or exclude one or more of the following adjuvants: sterile diluents, such as water for injection, saline solution, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic mono- or diglycerides, polyethylene glycols, glycerin, propylene glycol, or other solvents that can function as solvents or suspension media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and agents for adjusting osmolarity, such as sodium chloride or dextrose; agents for acting as cryoprotectants, such as sucrose or trehalose. Parenteral preparations may be enclosed in glass or plastic ampoules, disposable syringes or multiple dose vials. Saline is the preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0228] A liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of the compound such that a suitable dosage will be obtained.

[0229] Pharmaceutical compositions can be prepared by methodology well known in the pharmaceutical art. For example, pharmaceutical compositions intended to be administered by injection can be prepared by combining nucleic acid or polypeptide with sterile, distilled water or other carriers to form a solution. Surfactants can be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with compounds consistent with the teachings herein to facilitate the dissolution or homogeneous suspension of the compound in aqueous delivery systems.

[0230] Compositions within the scope of the present disclosure are administered in therapeutically and / or prophylactically effective amounts, which will vary depending on a variety of factors, including: the activity of the particular therapeutic and / or prophylactic agent being used; the metabolic stability of the therapeutic and / or prophylactic agent and its length of action; the age, weight, general health, sex, and diet of the patient; the mechanism and time of administration; rate of excretion; drug combination; the severity of the particular disorder or condition; and therapies the subject is undergoing.

[0231] IX. METHODS OF VARIANT PROTEIN SELECTION AND USE The methods of the present disclosure include treatments for diseases or conditions caused by coronavirus. The immunogenic polypeptides of the present disclosure can be provided to induce an immune response in individuals infected with coronavirus or suspected of exposure to coronavirus. The immunogenic polypeptides of the present disclosure can be provided to induce an immune response in individuals at risk of coronavirus infection or suspected of exposure to coronavirus. The methods can be used with individuals who have tested positive for exposure to coronavirus or are believed to be at risk of infection based on possible exposure.

[0232] In particular, the present disclosure encompasses methods of treating, preventing, or reducing the severity of infection caused by coronavirus. In some embodiments, the treatment is administered in the absence of an adjuvant or carrier or other coronavirus antigen. In some embodiments, the treatment is administered in the presence of an adjuvant or carrier or other coronavirus antigen. In addition, in some examples, the treatment includes administration of other agents commonly used against viral infections, such as one or more antiviral drugs.

[0233] In one aspect, the disclosure relates to methods for producing immunogenic compositions (e.g., vaccines) comprising immunogenic polypeptide constructs and / or nucleic acids encoding immunogenic polypeptide constructs (e.g., coronavirus spike proteins or fragments thereof). The method can include one or both of the following steps: (1) identifying at least one modification to be made to the native, unmodified coronavirus spike protein that will (a) increase recruitment by the RBD of the variant coronavirus spike protein in an RBD-up conformation to expose more neutralization-sensitive epitopes in the spike protein, (b) decrease recruitment by the RBD of the variant coronavirus spike protein in an RBD-down conformation, (c) increase expression of the variant coronavirus spike protein compared to the native coronavirus spike protein, (d) increase adoption of a prefusion conformation, (e) decrease shedding of the S1 subunit of the variant coronavirus spike protein, and / or (f) improve localization of the variant coronavirus spike protein to a host cell membrane; and (2) modifying the antigenic peptide sequence of the native, unmodified coronavirus spike protein according to the identified modification to form a variant coronavirus spike protein construct and / or a nucleic acid encoding a variant coronavirus spike protein construct.

[0234] In some aspects, the method includes both the steps of: (1) identifying at least one modification to be made to a native, unmodified coronavirus spike protein that will (a) increase recruitment by the RBD of the variant coronavirus spike protein of an RBD-up conformation to expose more neutralization-sensitive epitopes in the spike protein, (b) decrease recruitment by the RBD of the variant coronavirus spike protein of an RBD-down conformation, (c) increase expression of the variant coronavirus spike protein compared to the native coronavirus spike protein, (d) increase adoption of a prefusion conformation, (e) decrease shedding of the S1 subunit of the variant coronavirus spike protein, and / or (f) improve localization of the variant coronavirus spike protein to a host cell membrane; and (2) modifying the antigenic peptide sequence of the native, unmodified coronavirus spike protein according to the identified modification to form a variant coronavirus spike protein construct and / or a nucleic acid encoding a variant coronavirus spike protein construct.

[0235] In some embodiments, modifications to a native coronavirus spike protein to produce a variant coronavirus spike protein that will (a) increase recruitment by the RBD of the variant coronavirus spike protein of the RBD-up conformation exposing more neutralization-sensitive epitopes in the spike protein, (b) decrease recruitment by the RBD of the variant coronavirus spike protein of the RBD-down conformation, (c) increase expression of the variant coronavirus spike protein compared to the native coronavirus spike protein, (d) increase adoption of a prefusion conformation, (e) decrease shedding of the S1 subunit of the variant coronavirus spike protein, and / or (f) improve localization of the variant coronavirus spike protein to the host cell membrane can be identified using deep learning methods that utilize coronavirus genome sequences compiled by the GISAID database (found on the world wide web at GSAID.org). Deep learning methods can analyze genotype-phenotype relationships between genomic sequences; perform deep mutational scans of the RBDs of the sequences to assess antigenic drift, including analysis of ACE2 binding and / or antibody escape mutations; and / or perform structural modeling of variant spike proteins. In some embodiments, deep learning methods can be used to model and predict variant coronavirus strains such that isolated immunogenic polypeptides comprising the spike proteins of these identified variants can be generated for use in treating, preventing or reducing the severity of infections caused by the coronaviruses described herein.

[0236] In some embodiments, a population of isolated immunogenic polypeptides comprising variant coronavirus spike proteins that are variants of native coronavirus spike proteins or fragments thereof of the present disclosure comprises a plurality of variant coronavirus spike proteins in an RBD-up conformation. In some embodiments, the population of variant coronavirus spike proteins in an RBD-up conformation is greater than the population of native coronavirus spike proteins and / or alternative variant coronavirus spike proteins. In some embodiments, the population of variant coronavirus spike proteins of the present disclosure in an RBD-up conformation is greater than the population of native coronavirus spike proteins and / or alternative variant coronavirus spike proteins. In some embodiments, the population of variant coronavirus spike proteins of the present disclosure in an RBD-up conformation is greater than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 150 ... about 95% or more, about 1 fold or more, about 2 fold or more, about 3 fold or more, about 4 fold or more, about 5 fold or more, about 10 fold or more, about 20 fold or more, about 30 fold or more, about 40 fold or more, about 50 fold or more, about 100 fold or more, about 200 fold or more, about 300 fold or more, about 400 fold or more, about 500 fold or more, about 1000 fold or more, about 2000 fold or more, about 3000 fold or more, about 4000 fold or more, about 5000 fold or more, or about 10000 fold or more, or to some degree greater than any one of them.

[0237] In some embodiments, the population of variant coronavirus spike proteins in the RBD-down conformation is reduced as compared to the population of native coronavirus spike proteins and / or alternative variant coronavirus spike proteins. In some embodiments, the population of variant coronavirus spike proteins of the present disclosure in the RBD-down conformation is reduced as compared to the population of native coronavirus spike proteins and / or alternative variant coronavirus spike proteins by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 1 / 2 or more. a decrease of at least one of, at most one of, or some degree between any two of, about 1, about 2, about 3, about 4, about 5, about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, about 1000, about 2000, about 3000, about 4000, about 5000, or about 10000;

[0238] In some aspects, expression is increased in a population of isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or fragment thereof of the present disclosure. In some aspects, expression of a population of variant coronavirus spike proteins of the present disclosure is increased compared to expression of a population of native coronavirus spike proteins and / or alternative variant coronavirus spike proteins. In some aspects, expression of a population of variant coronavirus spike proteins of the present disclosure is increased by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more compared to a population of native coronavirus spike proteins and / or alternative variant coronavirus spike proteins. , about 1 fold or more, about 2 fold or more, about 3 fold or more, about 4 fold or more, about 5 fold or more, about 10 fold or more, about 20 fold or more, about 30 fold or more, about 40 fold or more, about 50 fold or more, about 100 fold or more, about 200 fold or more, about 300 fold or more, about 400 fold or more, about 500 fold or more, about 1000 fold or more, about 2000 fold or more, about 3000 fold or more, about 4000 fold or more, about 5000 fold or more, or about 10000 fold or more, or to some extent greater than any one of them.

[0239] In some embodiments, the population of isolated immunogenic polypeptides comprising variant coronavirus spike proteins that are variants of native coronavirus spike proteins or fragments thereof of the present disclosure are stabilized in a prefusion conformation. In some embodiments, the population of variant coronavirus spike proteins in a stabilized prefusion conformation is greater than the population of native coronavirus spike proteins and / or alternative variant coronavirus spike proteins. In some embodiments, the population of variant coronavirus spike proteins in a stabilized prefusion conformation is greater than the population of native coronavirus spike proteins and / or alternative variant coronavirus spike proteins. In some embodiments, the population of variant coronavirus spike proteins in a stabilized prefusion conformation is greater than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more, about 1 fold or more, about 2 fold or more, about 3 fold or more, about 4 fold or more, about 5 fold or more, about 10 fold or more, about 20 fold or more, about 30 fold or more, about 40 fold or more, about 50 fold or more, about 100 fold or more, about 200 fold or more, about 300 fold or more, about 400 fold or more, about 500 fold or more, about 1000 fold or more, about 2000 fold or more, about 3000 fold or more, about 4000 fold or more, about 5000 fold or more, or about 10000 fold or more, or to some degree greater than any one of them.

[0240] In some embodiments, shedding of the S1 subunit by a population of isolated immunogenic polypeptides comprising a variant coronavirus spike protein of the present disclosure that is a variant of a native coronavirus spike protein or a fragment thereof is reduced, hi some embodiments, shedding of the S1 subunit by a population of variant coronavirus spike proteins of the present disclosure is reduced compared to a population of native coronavirus spike proteins and / or alternative variant coronavirus spike proteins. In some embodiments, the shedding of the S1 subunit by a population of variant coronavirus spike proteins of the disclosure is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 9 ...15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more, about 15% or more a reduction of at least one of, at most one of, or some degree between any two of, about one-half, about one-third, about one-quarter, about one-fifth, about one-tenth, about one-twentieth, about one-thirtieth, about one-fortieth, about one-fiftieth, about one-hundredth, about one-hundredth, about one-hundredth, about one-hundredth, about one-hundredth, about one-hundredth, about one-hundredth, about one-hundredth, about one-thirtyth, about one-hundredth, about one-thirtyth, about one-thirtyth, about one-hundredth, about one-hundredth, about one-thirtyth, about one-thirtyth, about one-thirtyth, about one-thirtyth, about one-hundred ...

[0241] In some aspects, the localization of a population of isolated immunogenic polypeptides comprising a variant coronavirus spike protein of the present disclosure that is a variant of a native coronavirus spike protein or fragment thereof to a host cell membrane is improved. In some aspects, the localization of a population of variant coronavirus spike proteins of the present disclosure to a host cell membrane is improved as compared to a population of native coronavirus spike proteins and / or alternative variant coronavirus spike proteins. In some aspects, the localization of a population of variant coronavirus spike proteins of the present disclosure to a host cell membrane is improved as compared to a population of native coronavirus spike proteins and / or alternative variant coronavirus spike proteins. In some aspects, the localization of a population of variant coronavirus spike proteins of the present disclosure to a host cell membrane is improved as compared to a population of native coronavirus spike proteins and / or alternative variant coronavirus spike proteins by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90 ... The improvement is equal to, at least one of, at most one of, or between any two of the following: 5% or more, about 1 fold or more, about 2 fold or more, about 3 fold or more, about 4 fold or more, about 5 fold or more, about 10 fold or more, about 20 fold or more, about 30 fold or more, about 40 fold or more, about 50 fold or more, about 100 fold or more, about 200 fold or more, about 300 fold or more, about 400 fold or more, about 500 fold or more, about 1000 fold or more, about 2000 fold or more, about 3000 fold or more, about 4000 fold or more, about 5000 fold or more, or about 10000 fold or more.

[0242] In some aspects, isolated immunogenic polypeptides and / or compositions thereof comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or fragment thereof of the present disclosure produced by the methods of the present disclosure have efficacy, intracellular delivery and / or immunogenicity that is equivalent to or greater than the efficacy, intracellular delivery and / or immunogenicity of a native, unmodified coronavirus spike protein and / or a native, unmodified coronavirus spike protein construct and / or composition thereof.

[0243] In some aspects, a population of isolated immunogenic polypeptides comprising variant coronavirus spike proteins that are variants of native coronavirus spike proteins or fragments thereof of the present disclosure have efficacy, intracellular delivery and / or immunogenicity that is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75 ... The antibody has high efficacy, intracellular delivery and / or immunogenicity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 100 fold, at least about 200 fold, at least about 300 fold, at least about 400 fold, at least about 500 fold, at least about 1000 fold, at least about 2000 fold, at least about 3000 fold, at least about 4000 fold, at least about 5000 fold or at least about 10000 fold.

[0244] In one aspect, the disclosure relates to methods for producing immunogenic compositions comprising immunogenic polypeptide constructs and / or nucleic acids encoding immunogenic polypeptide constructs. The method can include one or both of the following steps: (1) identifying at least one modification to be made to the native, unmodified coronavirus spike protein that will (a) increase recruitment by the RBD of the variant coronavirus spike protein in an RBD-up conformation to expose more neutralization-sensitive epitopes in the spike protein, (b) decrease recruitment by the RBD of the variant coronavirus spike protein in an RBD-down conformation, (c) increase expression of the variant coronavirus spike protein compared to the native coronavirus spike protein, (d) increase adoption of a prefusion conformation, (e) decrease shedding of the S1 subunit of the variant coronavirus spike protein, and / or (f) improve localization of the variant coronavirus spike protein to a host cell membrane; and (2) modifying the antigenic peptide sequence of the native, unmodified coronavirus spike protein according to the identified modification to form a variant coronavirus spike protein construct and / or a nucleic acid encoding a variant coronavirus spike protein construct.

[0245] In another aspect, the disclosure relates to a method of inducing an immune response in a subject, the method comprising administering to a mammalian cell an effective amount of a composition described herein.

[0246] In another aspect, the present disclosure relates to a method of vaccinating a subject, the method comprising administering to a subject in need thereof an effective amount of a composition described herein.

[0247] In another aspect, the present disclosure relates to a method of treating or preventing or reducing the severity of an infectious disease, comprising administering to a subject an effective amount of a composition described herein.

[0248] In another aspect, the disclosure relates to a method for treating or preventing an infectious disease in a subject or reducing its severity, for example by inducing an immune response against the infectious disease in the subject. In some aspects, the method comprises administering a priming composition comprising an effective amount of a composition as described herein and administering a booster composition comprising an effective amount of an adenoviral vector encoding an antigen. In another aspect, the method comprises administering a priming composition comprising an effective amount of an adenoviral vector encoding an antigen and administering a booster composition comprising an effective amount of a composition as described herein. In some aspects, the composition induces an immune response, including an antibody response. In some aspects, the composition induces an immune response, including a T cell response.

[0249] In another aspect, the disclosure relates to a method of treating, preventing, or reducing the severity of a coronavirus infection and / or disease caused by a coronavirus, the method comprising administering to a subject an effective amount of a composition described herein.

[0250] In another aspect, the disclosure relates to a method of treating or preventing or reducing the severity of a coronavirus infection and / or disease caused by a coronavirus in a subject, for example, by inducing an immune response to the coronavirus infection in the subject. In some aspects, the method includes administering a priming composition comprising an effective amount of a composition described herein and administering a booster composition comprising an effective amount of an adenoviral vector encoding an antigen. In another aspect, the method includes administering a priming composition comprising an effective amount of an adenoviral vector encoding an antigen and administering a booster composition comprising an effective amount of a composition described herein. In some aspects, the composition elicits an immune response including an antibody response. In some aspects, the composition elicits an immune response including a T cell response.

[0251] In another aspect, the disclosure relates to the use of an isolated immunogenic polypeptide described herein in the manufacture of an immunogenic composition for the treatment or prevention of an infectious disease. In another aspect, the disclosure relates to the use of an isolated immunogenic polypeptide described herein in the manufacture of an immunogenic composition for the treatment or prevention of a coronavirus infection. EXAMPLES

[0252] The following examples are included to demonstrate aspects of the present disclosure. It should be appreciated by those skilled in the art that the techniques disclosed in the examples below represent techniques discovered by the inventors to function well in the practice of the present disclosure, and thus may be considered to constitute preferred mechanisms for its practice. However, those skilled in the art should appreciate, in light of the present disclosure, that many changes can be made to the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the present disclosure.

[0253] Example 1 * The amino acid positions in Example 1, Table 6 and their associated figures are derived from the SARS-CoV-2 spike protein sequence found in UniProt Accession No. P0DTC2-1 sequence.

[0254] The strategy used to identify, produce and test variant coronavirus spike protein constructs is illustrated in Figure 4. In particular, in some embodiments, variants are selected based at least in part on their ability to increase the RBD-up conformation of the spike protein, improve the expression of the spike protein, and / or increase the adoption of the prefusion conformation. Cells are transiently transfected with an expression construct encoding the selected variant, the variant is expressed by the cells, and the expressed variant is purified. Supernatants collected during purification of the variants are analyzed to determine the amount of variant expression, octet titer, and S1 subunit shedding. ACE2 and mAb stress tests, Tm, and negative staining are performed to further characterize the variants. Finally, in vivo testing with the variants is performed to evaluate the immunogenicity and tolerability of the variants.

[0255] The 87 variants examined and referenced in FIG. 4 are shown in Table 6 below. In Table 6, the numbering of the mutant variants is under the column "Code". Each mutant variant contains the mutations listed in the respective row. As an example, mutant 66 is represented in the "066" code row, which has the following mutations according to the amino acid positions from the SARS-CoV-2 spike protein sequence found in the UniProt accession number P0DTC2-1 sequence: D614G, F817P, A892P, A899P, A942P, V987P, I980C-Q992C, and RRAR(682-685)→GSAS. As another example, mutant 70 is represented in the "070" column, which has the following mutations according to the amino acid positions from the SARS-CoV-2 spike protein sequence found in UniProt accession number P0DTC2-1 sequence: D614G, F817P, A892P, A899P, A942P, D985P, V987P, RRAR(682-685)→GSAS, and F329S.

[0256] [Table 6-1]

[0257] [Table 6-2]

[0258] [Table 6-3]

[0259] [Table 6-4]

[0260] [Table 6-5]

[0261] [Table 6-6]

[0262] At least some variants were selected for their ability to increase the RBD-up conformation of the spike protein and / or enhance the stability of the spike protein prior to fusion with a host cell membrane. For example, Figure 5 illustrates the conformation of the coronavirus spike protein prior to fusion with a host cell membrane and after fusion with a host cell membrane. Three amino acid residues were selected for modification. These amino acids are located at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, * 982, * 983 and / or * This corresponds to the amino acid residue at 984.

[0263] Figure 6 shows the amino acid positions of the SARS-CoV-2 spike protein. * 982, * 983 and / or *We demonstrate that these modifications at the amino acid position corresponding to 984 can improve antigen expression and stabilize the prefusion conformation of the variant coronavirus spike protein.

[0264] Additional variants selected for at least their ability to increase the RBD-up conformation of the spike protein and / or increase the adoption of a prefusion conformation include those illustrated by Figure 7. Figure 7 illustrates the conformations of the coronavirus spike protein before (top, Figure 7A) and after (bottom, Figure 7B) fusion with the host cell membrane. Location of SARS-CoV-2 spike protein sequence * 982, * 983 and / or * The position of the SARS-CoV-2 spike protein sequence in addition to the amino acid residue corresponding to the amino acid residue at 984 * The amino acid residue corresponding to amino acid residue at 326 was selected for modification.

[0265] Figure 7 shows the amino acid positions of the SARS-CoV-2 spike protein. * 326, * 982, * 983 and / or * We also demonstrate that these modifications at the amino acid position corresponding to 984 can affect the conformation assumed by the RBD of the variant coronavirus spike protein, resulting in a higher number of RBDs in the RBD-up conformation.

[0266] In some embodiments, the selected variants are screened for functional characteristics including protein expression, ACE2 binding and 3022 binding levels. Figure 8 provides protein expression, ACE2 binding and 3022 binding levels of variant coronavirus spike proteins according to some embodiments disclosed herein. ACE2 is a key epitope in coronavirus spike proteins and 3022 corresponds to a highly conserved cryptic epitope in the receptor binding domain of SARS-CoV-2 and SARS-CoV that is exposed when the RBD of the spike protein is in the RBD-up conformation (Figure 2, Figure 3). In some embodiments, these measurements are utilized to select variant coronavirus spike proteins for in vivo evaluation. The results of functional characteristics including protein expression, ACE2 binding and 3022 binding levels obtained from the 87 mutants listed in Table 6 are shown in Table 7 below.

[0267] [Table 7-1]

[0268] [Table 7-2]

[0269] Figure 9 provides the specific modifications made to some of the variant spike proteins.

[0270] Mutants 70, 41, 84, 57, 75, 50 and 85 from Tables 6 and 7 were selected for in vivo immunogenicity testing. Mutant 50 showed the greatest immunogenicity in the group tested.

[0271] FIG. 10 describes additional variants selected for in vivo immunogenicity and tolerability studies. These variants have sequence scaffolds with the modifications described in FIG. 9. Thus, in some embodiments, amino acid residues in addition to those described in FIG. 9 are modified to produce additional variant spike proteins, such as some of the variants in FIG. 10. These modifications include the amino acid residues listed in the "Mutation" column in FIG. 10 and refer to amino acids at positions corresponding to amino acids in the SARS-CoV-2 spike protein. These effects of these modifications in combination with those described in FIG. 9 on protein expression, 3022 binding and ACE2 binding by additional variant coronavirus spike proteins are listed in FIG. 10 under the "Protein Expression" ("CR3022 Response" and "ACE2 Response" columns, respectively.

[0272] * D985P, * V987P, * F817P, * A892P, * A899P and * The P6' (P6-prime) construct containing the A942P mutation shows increased protein expression and increased ACE2 receptor binding over the P6 spike protein.

[0273] Additional mutants were designed with different combinations of interprotomer disulfide bonds that introduced cysteines at the corresponding residue pair positions (such as those listed below) and proline substitutions. Mutants with any one of the following five interprotomer disulfide bonds were prepared: (i) A570C-N960C; (ii) A570C-K964C; (iii) A570C-S967C; (iv) T547C-N978C; and (v) T547C-S982C.

[0274] Exemplary mutants designed include those listed in Table 8.

[0275] [Table 8]

[0276] Immunogenicity studies were conducted to test designs 100-112, listed in Table 8. Designs 109 and 110 in Table 8 are preferred embodiments.

[0277] Example 2 Immunogenicity of variants This study was conducted to evaluate the immunogenicity of new modified mRNA construct designs of protein mutants listed in Table 6 and / or Table 8. The modRNAs contained N1-methylpseudouracil and were encapsulated in lipid nanoparticles (LNPs). Groups containing 10 mice each were immunized with the LNP formulations in a two-dose series. Serum was collected on days 29 and 49 for neutralizing antibody responses. The interprotomer A570C-N960C was immunogenic.

[0278] [Table 9]

[0279] [Table 10]

[0280] All of the methods disclosed and claimed herein can be made and carried out without undue experimentation in light of this disclosure.Although the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to one skilled in the art that variations can be applied to the methods described herein and in the steps or sequence of steps of the methods without departing from the concept, spirit and scope of this disclosure.More specifically, it will be apparent that certain agents that are both chemically and physiologically related can be substituted for the agents described herein while achieving the same or similar results.All such similar substitutions and modifications that are apparent to one skilled in the art are deemed to be within the spirit, scope and concept of this disclosure as defined by the appended claims.

[0281] [Table 11-1]

[0282] [Table 11-2]

[0283] [Table 11-3]

[0284] [Table 11-4]

[0285] [Table 11-5]

[0286] [Table 11-6]

[0287] [Table 11-7]

[0288]

Table 11-8

[0289]

Table 11-9

[0290]

Table 11-10

[0291]

Table 11-11

[0292]

Table 11-12

[0293]

Table 11-13

[0294]

Table 11-14

[0295]

Table 11-15

[0296]

Table 11-16

[0297]

Table 11-17

[0298] [Table 11-18]

[0299] [Table 11-19]

[0300] [Table 11-20]

[0301] Embodiment The foregoing describes embodiments of the present invention along with possible alternatives. However, these embodiments are merely for illustrative purposes and the present invention is not limited thereto. 1. An isolated immunogenic polypeptide comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof, wherein the variant coronavirus spike protein is (a) increasing recruitment by the receptor binding domain (RBD) of the up-conformational variant coronavirus spike protein; and / or (b) a down-conformation variant that reduces recruitment by the RBD of the coronavirus spike protein; and / or (c) increasing the expression of a variant coronavirus spike protein relative to a native coronavirus spike protein; and / or (d) increasing the adoption of the prefusion conformation; and / or (e) reducing shedding of the S1 subunit of the variant coronavirus spike protein; and / or (f) improving localization of the variant coronavirus spike protein to the host cell membrane; An isolated immunogenic polypeptide having one or more modifications compared to a native coronavirus spike protein. 2. The isolated immunogenic polypeptide of embodiment 1, wherein the variant coronavirus spike protein comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of a native coronavirus spike protein. 3. The isolated immunogenic polypeptide of embodiment 1 or embodiment 2, wherein the variant coronavirus spike protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a native coronavirus spike protein. 4. The isolated immunogenic polypeptide of any one of embodiments 1-3, wherein the variant coronavirus spike protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of a native coronavirus spike protein. 5. The isolated immunogenic polypeptide of any one of embodiments 1-4, wherein the variant coronavirus spike protein comprises at least the RBD. 6. The isolated immunogenic polypeptide of any one of embodiments 1-5, wherein the variant coronavirus spike protein has one or more modifications that increase recruitment by the RBD of the variant coronavirus spike protein of the up conformation. 7. The isolated immunogenic polypeptide of embodiment 6, wherein increased recruitment by the RBD of the up conformational variant coronavirus spike protein increases ACE2 receptor binding by the RBD compared to the native coronavirus spike protein prior to fusion with a host cell membrane. 8. The isolated immunogenic polypeptide of embodiment 6 or embodiment 7, wherein increased recruitment by the RBD of the variant coronavirus spike protein of a pre-fusion up conformation with the host cell membrane stabilizes the variant coronavirus spike protein in a pre-fusion conformation. 9. The isolated immunogenic polypeptide of any one of embodiments 6 to 8, wherein increased recruitment by the RBD of the variant coronavirus spike protein of the up conformation prior to fusion with the host cell membrane reduces shedding of the S1 subunit of the variant coronavirus spike protein. 10. The isolated immunogenic polypeptide of any one of embodiments 6-9, wherein the one or more modifications comprise one or more amino acid substitutions in the S1 subunit of a variant coronavirus spike protein. 11. The isolated immunogenic polypeptide of any one of embodiments 6-10, wherein the one or more modifications comprise one or more amino acid substitutions in the amino acid sequence linking the N-terminal domain of the variant coronavirus spike protein to the RBD. 12. The isolated immunogenic polypeptide of any one of embodiments 6 to 11, wherein the one or more modifications in the variant coronavirus spike protein comprise an amino acid substitution at the amino acid corresponding to amino acid position 326 of SEQ ID NO:1. 13. The isolated immunogenic polypeptide of any one of embodiments 6 to 12, wherein the one or more modifications in the variant coronavirus spike protein comprise a substitution of the amino acid residue at the amino acid corresponding to amino acid position 326 of SEQ ID NO:1 with a serine residue. 14. The isolated immunogenic polypeptide of any one of embodiments 6-13, wherein the one or more modifications comprise one or more amino acid substitutions in the RBD of a variant coronavirus spike protein. 15. The isolated immunogenic polypeptide of any one of embodiments 6 to 14, wherein the one or more modifications of the variant coronavirus spike protein comprise an amino acid substitution at the amino acid corresponding to amino acid position 364 of SEQ ID NO:1. 16. The isolated immunogenic polypeptide of any one of embodiments 6 to 15, wherein the one or more modifications in the variant coronavirus spike protein comprise a substitution of the amino acid residue at the amino acid corresponding to amino acid position 364 of SEQ ID NO:1 with a phenylalanine residue. 17. The isolated immunogenic polypeptide of any one of embodiments 6 to 16, wherein the one or more modifications comprise one or more amino acid substitutions in the S2 subunit of a variant coronavirus spike protein. 18. The isolated immunogenic polypeptide of any one of embodiments 6 to 17, wherein the one or more modifications comprise one or more amino acid substitutions in the amino acid sequence linking the first heptad repeat region to the second heptad repeat region of the variant coronavirus spike protein. 19. One or more modifications of the variant coronavirus spike protein are (a) an amino acid substitution at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1; and / or (b) an amino acid substitution at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1; and / or (c) an amino acid substitution at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1; and / or (d) an amino acid substitution at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1; and / or (e) an amino acid substitution at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1; and / or (f) an amino acid substitution at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1; and / or (g) an amino acid substitution at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1; 19. The isolated immunogenic polypeptide according to any one of embodiments 6 to 18, comprising: 20. One or more modifications of the variant coronavirus spike protein are (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and / or (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and / or (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and / or (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and / or (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and / or (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and / or (g) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 20. The isolated immunogenic polypeptide of any one of embodiments 6 to 19, comprising: 21. The isolated immunogenic polypeptide of any one of embodiments 6 to 20, wherein the one or more modifications comprise one or more amino acid substitutions at the interface of the S1 and S2 subunits of the variant coronavirus spike protein. 22. The isolated immunogenic polypeptide of any one of embodiments 6 to 21, wherein the one or more modifications of the variant coronavirus spike protein comprise an amino acid substitution at the amino acid corresponding to amino acid position 611 of SEQ ID NO:1. 23. The isolated immunogenic polypeptide of any one of embodiments 6 to 22, wherein the one or more modifications in the variant coronavirus spike protein comprise a substitution of the amino acid residue at the amino acid corresponding to amino acid position 611 of SEQ ID NO:1 with a glycine residue. 24. The isolated immunogenic polypeptide of any one of embodiments 1 to 23, wherein the variant coronavirus spike protein has one or more modifications that reduce recruitment by the RBD of the variant coronavirus spike protein of the down conformation. 25. The isolated immunogenic polypeptide of embodiment 24, wherein reduced recruitment by the RBD of the variant coronavirus spike protein of a down conformation prior to fusion with the host cell membrane increases ACE2 receptor binding by the RBD compared to the native coronavirus spike protein. 26. The isolated immunogenic polypeptide of embodiment 24 or embodiment 25, wherein reduced recruitment by the RBD of the variant coronavirus spike protein of a pre-fusion down conformation with the host cell membrane stabilizes the variant coronavirus spike protein in a pre-fusion conformation. 27. The isolated immunogenic polypeptide of any one of embodiments 24 to 26, wherein the reduced recruitment by the RBD of the variant coronavirus spike protein of a down conformation prior to fusion with the host cell membrane reduces shedding of the S1 subunit of the variant coronavirus spike protein. 28. The isolated immunogenic polypeptide of any one of embodiments 24-27, wherein the one or more modifications comprise one or more amino acid substitutions in the S1 subunit of a variant coronavirus spike protein. 29. The isolated immunogenic polypeptide of any one of embodiments 24-28, wherein the one or more modifications comprise one or more amino acid substitutions in the amino acid sequence linking the N-terminal domain of the variant coronavirus spike protein to the RBD. 30. The isolated immunogenic polypeptide of any one of embodiments 24 to 29, wherein the one or more modifications of the variant coronavirus spike protein comprises an amino acid substitution at the amino acid corresponding to amino acid position 326 of SEQ ID NO:1. 31. The isolated immunogenic polypeptide of any one of embodiments 24 to 30, wherein the one or more modifications of the variant coronavirus spike protein comprise a substitution of the amino acid residue at the amino acid corresponding to amino acid position 326 of SEQ ID NO:1 with a serine residue. 32. The isolated immunogenic polypeptide of any one of embodiments 24-31, wherein the one or more modifications comprise one or more amino acid substitutions in the RBD of a variant coronavirus spike protein. 33. The isolated immunogenic polypeptide of any one of embodiments 24 to 32, wherein the one or more modifications of the variant coronavirus spike protein comprise an amino acid substitution at the amino acid corresponding to amino acid position 364 of SEQ ID NO:1. 34. The isolated immunogenic polypeptide of any one of embodiments 24 to 33, wherein the one or more modifications in the variant coronavirus spike protein comprise a substitution of the amino acid residue at the amino acid corresponding to amino acid position 364 of SEQ ID NO:1 with a phenylalanine residue. 35. The isolated immunogenic polypeptide of any one of embodiments 24-34, wherein the one or more modifications comprise one or more amino acid substitutions in the S2 subunit of a variant coronavirus spike protein. 36. The isolated protein of any one of embodiments 24 to 35, wherein the one or more modifications comprise one or more amino acid substitutions in the amino acid sequence linking the first heptad repeat region to the second heptad repeat region of the variant coronavirus spike protein. 37. One or more modifications of the variant coronavirus spike protein are (a) an amino acid substitution at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1; and / or (b) an amino acid substitution at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1; and / or (c) an amino acid substitution at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1; and / or (d) an amino acid substitution at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1; and / or (e) an amino acid substitution at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1; and / or (f) an amino acid substitution at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1; and / or (g) an amino acid substitution at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1; 37. The isolated immunogenic polypeptide according to any one of embodiments 24 to 36, comprising: 38. One or more modifications of the variant coronavirus spike protein are (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and / or (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and / or (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and / or (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and / or (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and / or (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and / or (g) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 38. The isolated immunogenic polypeptide of any one of embodiments 24 to 37, comprising: 39. The isolated immunogenic polypeptide of any one of embodiments 24-38, wherein the one or more modifications comprise one or more amino acid substitutions at the interface of the S1 and S2 subunits of the variant coronavirus spike protein. 40. The isolated immunogenic polypeptide of any one of embodiments 24 to 39, wherein the one or more modifications of the variant coronavirus spike protein comprise an amino acid substitution at the amino acid corresponding to amino acid position 611 of SEQ ID NO:1. 41. The isolated immunogenic polypeptide of any one of embodiments 24 to 40, wherein the one or more modifications of the variant coronavirus spike protein comprise a substitution of the amino acid residue at the amino acid corresponding to amino acid position 611 of SEQ ID NO:1 with a glycine residue. 42. The isolated immunogenic polypeptide of any one of embodiments 1 to 41, wherein the variant coronavirus spike protein has one or more modifications that increase expression of the variant coronavirus spike protein compared to a native coronavirus spike protein. 43. The isolated immunogenic polypeptide of embodiment 42, wherein the one or more modifications comprise one or more amino acid substitutions in the S1 subunit of a variant coronavirus spike protein. 44. The isolated immunogenic polypeptide of embodiment 42 or embodiment 43, wherein the one or more modifications comprise one or more amino acid substitutions in the RBD of a variant coronavirus spike protein. 45. The isolated immunogenic polypeptide of any one of embodiments 42 to 44, wherein the one or more modifications of the variant coronavirus spike protein comprise an amino acid substitution at the amino acid corresponding to amino acid position 364 of SEQ ID NO:1. 46. ​​The isolated immunogenic polypeptide of any one of embodiments 42 to 45, wherein the one or more modifications in the variant coronavirus spike protein comprise a substitution of the amino acid residue at the amino acid corresponding to amino acid position 364 of SEQ ID NO:1 with a phenylalanine residue. 47. The isolated immunogenic polypeptide of any one of embodiments 42-46, wherein the one or more modifications comprise one or more amino acid substitutions in the S2 subunit of a variant coronavirus spike protein. 48. The isolated protein of any one of embodiments 42-47, wherein the one or more modifications comprise one or more amino acid substitutions in the amino acid sequence linking the first heptad repeat region to the second heptad repeat region of the variant coronavirus spike protein. 49. One or more modifications of the variant coronavirus spike protein are (a) an amino acid substitution at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1; and / or (b) an amino acid substitution at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1; and / or (c) an amino acid substitution at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1; and / or (d) an amino acid substitution at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1; and / or (e) an amino acid substitution at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1; and / or (f) an amino acid substitution at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1; and / or (g) an amino acid substitution at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1; 49. The isolated immunogenic polypeptide of any one of embodiments 42 to 48, comprising: 50. One or more modifications of the variant coronavirus spike protein are (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and / or (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and / or (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and / or (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and / or (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and / or (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and / or (g) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 50. The isolated immunogenic polypeptide of any one of embodiments 42 to 49, comprising: 51. The isolated immunogenic polypeptide of any one of embodiments 1 to 50, wherein the variant coronavirus spike protein has one or more modifications that increase the adoption of the prefusion conformation. 52. The isolated immunogenic polypeptide of embodiment 50, wherein stabilization of the variant coronavirus spike protein in a prefusion conformation increases ACE2 receptor binding by the RBD compared to the native coronavirus spike protein. 53. The isolated immunogenic polypeptide of embodiment 50 or embodiment 52, wherein stabilization of the variant coronavirus spike protein in a prefusion conformation reduces shedding of the S1 subunit of the variant coronavirus spike protein. 54. The isolated immunogenic polypeptide of any one of embodiments 51-53, wherein the one or more modifications comprise one or more amino acid substitutions in the S2 subunit of a variant coronavirus spike protein. 55. The isolated protein of any one of embodiments 51 to 54, wherein the one or more modifications comprise one or more amino acid substitutions in an amino acid sequence linking a first heptad repeat region to a second heptad repeat region of the variant coronavirus spike protein. 56. One or more modifications of the variant coronavirus spike protein are (a) an amino acid substitution at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1; and / or (b) an amino acid substitution at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1; and / or (c) an amino acid substitution at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1; and / or (d) an amino acid substitution at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1; and / or (e) an amino acid substitution at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1; and / or (f) an amino acid substitution at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1; and / or (g) an amino acid substitution at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1; 56. The isolated immunogenic polypeptide of any one of embodiments 51 to 55, comprising: 57. One or more modifications of the variant coronavirus spike protein are (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and / or (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and / or (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and / or (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and / or (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and / or (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and / or (g) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 57. The isolated immunogenic polypeptide of any one of embodiments 51 to 56, comprising: 58. The isolated immunogenic polypeptide of any one of embodiments 1 to 57, wherein the variant coronavirus spike protein has one or more modifications that reduce shedding of the S1 subunit of the variant coronavirus spike protein. 59. The isolated immunogenic polypeptide of embodiment 58, wherein reduced shedding of the S1 subunit of the variant coronavirus spike protein increases ACE2 receptor binding by the RBD compared to the native coronavirus spike protein. 60. The isolated immunogenic polypeptide of embodiment 58 or embodiment 59, wherein reduced shedding of the S1 subunit of the variant coronavirus spike protein stabilizes the variant coronavirus spike protein in a prefusion conformation. 61. The isolated immunogenic polypeptide of any one of embodiments 58-60, wherein the one or more modifications comprise one or more amino acid substitutions in the S2 subunit of a variant coronavirus spike protein. 62. The isolated protein of any one of embodiments 58-61, wherein the one or more modifications comprise one or more amino acid substitutions in the amino acid sequence linking the first heptad repeat region to the second heptad repeat region of the variant coronavirus spike protein. 63. One or more modifications of the variant coronavirus spike protein are (a) an amino acid substitution at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1; and / or (b) an amino acid substitution at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1; and / or (c) an amino acid substitution at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1; and / or (d) an amino acid substitution at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1; and / or (e) an amino acid substitution at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1; and / or (f) an amino acid substitution at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1; and / or (g) an amino acid substitution at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1; 63. The isolated immunogenic polypeptide of any one of embodiments 58 to 62, comprising: 64. One or more modifications of the variant coronavirus spike protein are (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and / or (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and / or (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and / or (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and / or (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and / or (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and / or (g) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 64. The isolated immunogenic polypeptide of any one of embodiments 58 to 63, comprising: 65. An isolated immunogenic polypeptide comprising a variant coronavirus spike protein, which is a variant of a native coronavirus spike protein or a portion thereof according to any one of embodiments 1 to 64, wherein the variant coronavirus spike protein has one or more modifications compared to the native coronavirus spike protein, and the one or more modifications are: (a) one or more amino acid substitutions in the S1 subunit of a native coronavirus spike protein; and / or (b) one or more amino acid substitutions in the amino acid sequence linking the N-terminal domain of a native coronavirus spike protein to the RBD; and / or (c) one or more amino acid substitutions in the RBD of a native coronavirus spike protein; and / or (d) one or more amino acid substitutions or insertions in the S2 subunit of a native coronavirus spike protein; and / or (e) one or more amino acid substitutions or insertions in the amino acid sequence linking the first heptad repeat region to the second heptad repeat region of the native coronavirus spike protein; and / or (f) one or more amino acid substitutions at the interface between the S1 and S2 subunits of the native coronavirus spike protein; 1. An isolated immunogenic polypeptide comprising: 66.(a) One or more amino acid substitutions in the S1 subunit of a native coronavirus spike protein i. an amino acid substitution at the amino acid corresponding to amino acid position 326 of SEQ ID NO:1; and / or ii. comprises an amino acid substitution at the amino acid corresponding to amino acid position 364 of SEQ ID NO:1; and / or (b) the one or more amino acid substitutions in the amino acid sequence linking the N-terminal domain of a native coronavirus spike protein to the RBD include an amino acid substitution at an amino acid corresponding to amino acid position 326 of SEQ ID NO:1; and / or (c) the one or more amino acid substitutions in the RBD of a native coronavirus spike protein include an amino acid substitution at an amino acid corresponding to amino acid position 364 of SEQ ID NO:1; and / or (d) one or more amino acid substitutions or insertions in the S2 subunit of a native coronavirus spike protein, i. an amino acid substitution at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1; and / or ii. an amino acid substitution at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1; and / or iii. an amino acid substitution at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1; and / or iv. an amino acid substitution at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1; and / or v. an amino acid substitution at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1; and / or vi. an amino acid substitution at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1; and / or vii. comprises an amino acid substitution at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1; and / or (e) one or more amino acid substitutions or insertions in the amino acid sequence linking the first heptad repeat region to the second heptad repeat region of a native coronavirus spike protein, i. an amino acid substitution at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1; and / or ii. an amino acid substitution at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1; and / or iii. an amino acid substitution at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1; and / or iv. an amino acid substitution at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1; and / or v. an amino acid substitution at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1; and / or vi. an amino acid substitution at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1; and / or vii. comprises an amino acid substitution at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1; and / or (f) the one or more amino acid substitutions at the interface of the S1 and S2 subunits of the native coronavirus spike protein include an amino acid substitution at an amino acid corresponding to amino acid position 611 of SEQ ID NO:1; 66. An isolated immunogenic polypeptide according to embodiment 65. 67.(a) One or more amino acid substitutions in the S1 subunit of a native coronavirus spike protein i. Substitution of the amino acid residue at the amino acid corresponding to amino acid position 326 of SEQ ID NO:1 with a serine residue; and / or ii. comprising a substitution of the amino acid residue at the amino acid corresponding to amino acid position 364 of SEQ ID NO: 1 with a phenylalanine residue; and / or (b) the one or more amino acid substitutions in the amino acid sequence linking the N-terminal domain of a native coronavirus spike protein to the RBD comprise a substitution of an amino acid residue at the amino acid corresponding to amino acid position 326 of SEQ ID NO:1 with a serine residue; and / or (c) the one or more amino acid substitutions in the RBD of a native coronavirus spike protein include a substitution of an amino acid residue at the amino acid corresponding to amino acid position 364 of SEQ ID NO:1 with a phenylalanine residue; and / or (d) one or more amino acid substitutions or insertions in the S2 subunit of a native coronavirus spike protein, i. Substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and / or ii. Substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and / or iii. Substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and / or iv. Substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and / or v. Substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and / or vi. Substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and / or vii. comprising a substitution of the amino acid residue at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1 with a proline residue; and / or (e) one or more amino acid substitutions or insertions in the amino acid sequence linking the first heptad repeat region to the second heptad repeat region of a native coronavirus spike protein, i. Substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and / or ii. Substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and / or iii. Substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and / or iv. Substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and / or v. Substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and / or vi. Substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and / or vii. comprising a substitution of the amino acid residue at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1 with a proline residue; and / or (f) the one or more amino acid substitutions at the interface of the S1 and S2 subunits of the native coronavirus spike protein include a substitution of the amino acid residue at the amino acid corresponding to amino acid position 611 with a glycine residue; 67. An isolated immunogenic polypeptide according to embodiment 66. 68. An isolated immunogenic polypeptide comprising a variant coronavirus spike protein, which is a variant of a native coronavirus spike protein or a portion thereof according to any one of embodiments 1 to 67, wherein the variant coronavirus spike protein has one or more modifications compared to the native coronavirus spike protein, and the one or more modifications are: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 326 of SEQ ID NO:1 with a serine residue; and / or (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 364 of SEQ ID NO:1 with a phenylalanine residue; and / or (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 567 of SEQ ID NO:1 with a cysteine ​​residue; and / or (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 611 of SEQ ID NO:1 with a glycine residue; and / or (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and / or (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 840 of SEQ ID NO:1 with an asparagine residue; and / or (g) substitution of the amino acid residue at the amino acid corresponding to amino acid position 851 of SEQ ID NO:1 with a phenylalanine residue; and / or (h) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and / or (i) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and / or (j) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and / or (k) substitution of the amino acid residue at the amino acid corresponding to amino acid position 957 of SEQ ID NO:1 with a cysteine ​​residue; and / or (l) substitution of the amino acid residue at the amino acid corresponding to amino acid position 977 of SEQ ID NO:1 with a cysteine ​​residue; and / or (m) substitution of the amino acid residue at the amino acid corresponding to amino acid position 981 of SEQ ID NO:1 with a cysteine ​​residue; and / or (n) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and / or (o) substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and / or (p) substitution of the amino acid residue at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1 with a proline residue; and / or (q) substitution of the amino acid residue at the amino acid corresponding to amino acid position 986 of SEQ ID NO:1 with a cysteine ​​residue; and / or (r) substitution of an amino acid residue at amino acid position 989 of SEQ ID NO:1 with a cysteine ​​residue 1. An isolated immunogenic polypeptide comprising: 69. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 326 of SEQ ID NO:1 with a serine residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 364 of SEQ ID NO:1 with a phenylalanine residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 567 of SEQ ID NO:1 with a cysteine ​​residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 611 of SEQ ID NO:1 with a glycine residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 840 of SEQ ID NO:1 with an asparagine residue; and (g) substitution of the amino acid residue at the amino acid corresponding to amino acid position 851 of SEQ ID NO:1 with a phenylalanine residue; and (h) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (i) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (j) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (k) substitution of the amino acid residue at the amino acid corresponding to amino acid position 957 of SEQ ID NO:1 with a cysteine ​​residue; and (l) substitution of the amino acid residue at the amino acid corresponding to amino acid position 977 of SEQ ID NO:1 with a cysteine ​​residue; and (m) substitution of an amino acid residue at the amino acid corresponding to amino acid position 981 of SEQ ID NO:1 with a cysteine ​​residue; and (n) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and (o) substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and (p) substitution of the amino acid residue at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1 with a proline residue; and (q) substitution of the amino acid residue at the amino acid corresponding to amino acid position 986 of SEQ ID NO:1 with a cysteine ​​residue; and (r) substitution of an amino acid residue at amino acid position 989 of SEQ ID NO:1 with a cysteine ​​residue 69. The isolated immunogenic polypeptide of embodiment 68, comprising: 70. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and (b) Substitution of the amino acid residue at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 71. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 72. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and (f) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 73. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and (f) substitution of the amino acid residue at amino acid position 983 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 74. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and (g) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 75. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 326 of SEQ ID NO:1 with a serine residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and (c) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 76. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 611 of SEQ ID NO:1 with a glycine residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and (c) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 77. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 611 of SEQ ID NO:1 with a glycine residue; and (b) a substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and a substitution of the amino acid residue at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 78. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 611 of SEQ ID NO:1 with a glycine residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and (g) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 79. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 611 of SEQ ID NO:1 with a glycine residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and (g) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 80. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 326 of SEQ ID NO:1 with a serine residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and (g) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 81. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 981 of SEQ ID NO:1 with a cysteine ​​residue; and (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and (g) substitution of the amino acid residue at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1 with a proline residue; and (h) substitution of the amino acid residue at amino acid position 986 of SEQ ID NO:1 with a cysteine ​​residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 82. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 977 of SEQ ID NO:1 with a cysteine ​​residue; and (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and (g) substitution of the amino acid residue at the amino acid corresponding to amino acid position 984 of SEQ ID NO:1 with a proline residue; and (h) substitution of the amino acid residue at amino acid position 989 of SEQ ID NO:1 with a cysteine ​​residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 83. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 840 of SEQ ID NO:1 with an asparagine residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and (g) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 84. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 840 of SEQ ID NO:1 with an asparagine residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (f) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 85. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 851 of SEQ ID NO:1 with a phenylalanine residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 982 of SEQ ID NO:1 with a proline residue; and (g) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 86. One or more modifications: (a) substitution of the amino acid residue at the amino acid corresponding to amino acid position 570 of SEQ ID NO:1 with a cysteine ​​residue; and (b) substitution of the amino acid residue at the amino acid corresponding to amino acid position 814 of SEQ ID NO:1 with a proline residue; and (c) substitution of the amino acid residue at the amino acid corresponding to amino acid position 889 of SEQ ID NO:1 with a proline residue; and (d) substitution of the amino acid residue at the amino acid corresponding to amino acid position 896 of SEQ ID NO:1 with a proline residue; and (e) substitution of the amino acid residue at the amino acid corresponding to amino acid position 939 of SEQ ID NO:1 with a proline residue; and (f) substitution of the amino acid residue at the amino acid corresponding to amino acid position 957 of SEQ ID NO:1 with a cysteine ​​residue; and (g) substitution of the amino acid residue at the amino acid corresponding to amino acid position 983 of SEQ ID NO:1 with a proline residue; and (h) substitution of the amino acid residue at amino acid position 984 of SEQ ID NO:1 with a proline residue. 70. The isolated immunogenic polypeptide of embodiment 68 or embodiment 69, comprising: 87. The variant coronavirus spike protein is a variant of SEQ ID NO:1 or a fragment thereof, wherein the variant coronavirus spike protein has one or more modifications compared to SEQ ID NO:1, and the one or more modifications of the variant coronavirus spike protein are: (a) one or more amino acid substitutions in an amino acid sequence corresponding to the S1 subunit of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1; and / or (b) one or more amino acid substitutions in an amino acid sequence corresponding to the amino acid sequence linking the N-terminal domain of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1 to the RBD; and / or (c) one or more amino acid substitutions in an amino acid sequence corresponding to the RBD of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1; and / or (d) one or more amino acid substitutions in an amino acid sequence corresponding to the S2 subunit of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1; and / or (e) one or more amino acid substitutions in an amino acid sequence corresponding to the amino acid sequence linking a first heptad repeat region to a second heptad repeat region of a coronavirus spike protein having the amino acid sequence of SEQ ID NO:1; and / or (f) one or more amino acid substitutions in the amino acid sequence corresponding to the interface between the S1 subunit and the S2 subunit of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1; 68. The isolated immunogenic polypeptide of any one of embodiments 1 to 67, comprising: 88. (a) one or more amino acid substitutions in an amino acid sequence corresponding to the S1 subunit of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1 i. an amino acid substitution at amino acid position 326 of SEQ ID NO:1; and / or ii. comprises an amino acid substitution at amino acid position 364 of SEQ ID NO:1; and / or (b) the one or more amino acid substitutions in the amino acid sequence corresponding to the amino acid sequence linking the N-terminal domain to the RBD of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1 include an amino acid substitution at amino acid position 326 of SEQ ID NO:1; and / or (c) the one or more amino acid substitutions in the amino acid sequence corresponding to the RBD of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1 include an amino acid substitution at amino acid position 364 of SEQ ID NO:1; and / or (d) one or more amino acid substitutions or insertions in an amino acid sequence corresponding to the S2 subunit of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1, i. an amino acid substitution at amino acid position 814 of SEQ ID NO:1; and / or ii. an amino acid substitution at amino acid position 889 of SEQ ID NO:1; and / or iii. an amino acid substitution at amino acid position 896 of SEQ ID NO:1; and / or iv. an amino acid substitution at amino acid position 939 of SEQ ID NO:1; and / or v. an amino acid substitution at amino acid position 982 of SEQ ID NO:1; and / or vi. an amino acid substitution at amino acid position 983 of SEQ ID NO:1; and / or vii. comprises an amino acid substitution at amino acid position 984 of SEQ ID NO:1; and / or (e) one or more amino acid substitutions or insertions in an amino acid sequence corresponding to the amino acid sequence linking a first heptad repeat region to a second heptad repeat region of a coronavirus spike protein having the amino acid sequence of SEQ ID NO:1, i. an amino acid substitution at amino acid position 814 of SEQ ID NO:1; and / or ii. an amino acid substitution at amino acid position 889 of SEQ ID NO:1; and / or iii. an amino acid substitution at amino acid position 896 of SEQ ID NO:1; and / or iv. an amino acid substitution at amino acid position 939 of SEQ ID NO:1; and / or v. an amino acid substitution at amino acid position 982 of SEQ ID NO:1; and / or vi. an amino acid substitution at amino acid position 983 of SEQ ID NO:1; and / or vii. comprises an amino acid substitution at amino acid position 984 of SEQ ID NO:1; and / or (f) the one or more amino acid substitutions in the amino acid sequence corresponding to the interface of the S1 and S2 subunits of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1 include an amino acid substitution at amino acid position 611 of SEQ ID NO:1; 88. An isolated immunogenic polypeptide according to embodiment 87. 89. (a) one or more amino acid substitutions in an amino acid sequence corresponding to the S1 subunit of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1 i. Substitution of amino acid position 326 of SEQ ID NO:1 with a serine residue; and / or ii. comprising a substitution at amino acid position 364 of SEQ ID NO: 1 with a phenylalanine residue; and / or (b) one or more amino acid substitutions in an amino acid sequence corresponding to the amino acid sequence linking the N-terminal domain of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1 to the RBD include a substitution of a serine residue at amino acid position 326 of SEQ ID NO:1; and / or (c) the one or more amino acid substitutions in the amino acid sequence corresponding to the RBD of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1 include a substitution of a phenylalanine residue at amino acid position 364 of SEQ ID NO:1; and / or (d) one or more amino acid substitutions or insertions in an amino acid sequence corresponding to the S2 subunit of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1, i. Substitution of amino acid position 814 of SEQ ID NO:1 with a proline residue; and / or ii. Substitution of amino acid position 889 of SEQ ID NO:1 with a proline residue; and / or iii. Substitution of amino acid position 896 of SEQ ID NO:1 with a proline residue; and / or iv. Substitution of amino acid position 939 of SEQ ID NO:1 with a proline residue; and / or v. Substitution of amino acid position 982 of SEQ ID NO:1 with a proline residue; and / or vi. Substitution of amino acid position 983 of SEQ ID NO:1 with a proline residue; and / or vii. comprising a substitution of amino acid position 984 of SEQ ID NO:1 with a proline residue; and / or (e) one or more amino acid substitutions or insertions in an amino acid sequence corresponding to the amino acid sequence linking a first heptad repeat region to a second heptad repeat region of a coronavirus spike protein having the amino acid sequence of SEQ ID NO:1, i. Substitution of amino acid position 814 of SEQ ID NO:1 with a proline residue; and / or ii. Substitution of amino acid position 889 of SEQ ID NO:1 with a proline residue; and / or iii. Substitution of amino acid position 896 of SEQ ID NO:1 with a proline residue; and / or iv. Substitution of amino acid position 939 of SEQ ID NO:1 with a proline residue; and / or v. Substitution of amino acid position 982 of SEQ ID NO:1 with a proline residue; and / or vi. Substitution of amino acid position 983 of SEQ ID NO:1 with a proline residue; and / or vii. comprising a substitution of amino acid position 984 of SEQ ID NO:1 with a proline residue; and / or (f) one or more amino acid substitutions in an amino acid sequence corresponding to the interface of the S1 and S2 subunits of the coronavirus spike protein having the amino acid sequence of SEQ ID NO:1, comprising a substitution of a glycine residue at amino acid position 611 of SEQ ID NO:1; 89. An isolated immunogenic polypeptide according to embodiment 88. 90. The isolated immunogenic polypeptide of any one of embodiments 1 to 89, further comprising a leader sequence having an amino acid sequence that is at least 80% identical to the leader sequence of a native coronavirus spike protein. 91. The isolated immunogenic polypeptide of embodiment 90, wherein the leader sequence inhibits disulfide scrambling. 92. The isolated immunogenic polypeptide of any one of embodiments 1 to 91, further comprising a transmembrane sequence having an amino acid sequence that is at least 80% identical to the transmembrane sequence of a native coronavirus spike protein. 93. The isolated immunogenic polypeptide according to embodiment 92, wherein the transmembrane sequence extends the half-life of the isolated immunogenic polypeptide. 94. The isolated immunogenic polypeptide according to any one of embodiments 1 to 93, which does not contain an endoplasmic reticulum (ER) signal sequence. 95. The isolated immunogenic polypeptide of embodiment 94, wherein the absence of an ER signal sequence improves localization of the variant coronavirus spike protein to the host cell membrane. 96. The isolated immunogenic polypeptide of any one of embodiments 1 to 95, further comprising a trimerization domain. 97. The isolated immunogenic polypeptide of embodiment 96, wherein the trimerization domain is a Foldon trimerization domain. 98. Variant coronavirus spike protein, (a) a leader sequence having an amino acid sequence that is at least 80% identical to the leader sequence of a native coronavirus spike protein; (b) a transmembrane sequence having an amino acid sequence that is at least 80% identical to the transmembrane sequence of a native coronavirus spike protein; (c) a trimerization domain; and (d) RBD domain 98. The isolated immunogenic polypeptide of any one of embodiments 1 to 97, wherein the variant coronavirus spike protein does not comprise an ER signal sequence. 99. Variant coronavirus spike protein, (a) a leader sequence having an amino acid sequence that is at least 80% identical to the leader sequence of a native coronavirus spike protein; (b) a transmembrane sequence having an amino acid sequence that is at least 80% identical to the transmembrane sequence of a native coronavirus spike protein; (c) trimerization domain; (d) an RBD domain; and (e) Substitution of amino acid position 364 of SEQ ID NO:1 with a phenylalanine residue 99. The isolated immunogenic polypeptide of any one of embodiments 1 to 98, wherein the variant coronavirus spike protein does not comprise an ER signal sequence. 100. Variant coronavirus spike protein, (a) a leader sequence having an amino acid sequence that is at least 80% identical to the leader sequence of a native coronavirus spike protein; (b) a transmembrane sequence having an amino acid sequence that is at least 80% identical to the transmembrane sequence of a native coronavirus spike protein; (c) trimerization domain; (d) an RBD domain; and (e) an amino acid substitution at the amino acid corresponding to amino acid position 364 of SEQ ID NO:1; 100. The isolated immunogenic polypeptide of any one of embodiments 1 to 99, wherein the variant coronavirus spike protein does not comprise an ER signal sequence. In a preferred embodiment, the isolated nucleic acid does not encode a polypeptide comprising the amino acid sequence of SEQ ID NO:8 with the following substitutions: D985P, V987P, F817P, A892P, A899P, A942P, and RRAR(682-685) to GSAS. In a preferred embodiment, the isolated nucleic acid does not encode a polypeptide comprising the amino acid sequence SEQ ID NO:21. 101. A plurality of isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof, wherein the isolated immunogenic polypeptides constituting the plurality of isolated immunogenic polypeptides comprise an isolated immunogenic polypeptide according to any one of embodiments 1 to 100, and wherein at least about 25%, 30%, 35%, 40% or more than 50% of the variant coronavirus spike protein adopt an up conformation. 102. A plurality of isolated immunogenic polypeptides according to embodiment 101, wherein at least about 25% or more of the variant coronavirus spike protein adopts an up conformation. 103. A nucleic acid encoding an isolated immunogenic polypeptide according to any one of embodiments 1 to 100. 104. The nucleic acid according to embodiment 103, which is DNA. 105. A composition comprising an isolated immunogenic polypeptide comprising a variant of a native coronavirus spike protein according to any one of embodiments 1 to 100 or a nucleic acid encoding an isolated immunogenic polypeptide according to any one of embodiments 103 to 104 in a pharma- ceutically acceptable composition. 106. An immunogenic composition comprising an isolated immunogenic polypeptide comprising a variant of a native coronavirus spike protein according to any one of embodiments 1 to 100. 107. A vaccine comprising an isolated immunogenic polypeptide according to any one of embodiments 1 to 100. 108. A method for producing a vaccine, comprising mixing an isolated immunogenic polypeptide according to any one of embodiments 1 to 100 with a pharma- ceutically acceptable excipient to produce a composition according to any one of embodiments 105 or embodiment 106. 109. A method for preventing or treating a coronavirus infection, comprising administering the vaccine described in embodiment 107 to a subject in need thereof. 110. Use of an isolated immunogenic polypeptide according to any one of embodiments 1 to 100 or a composition according to any one of embodiments 105 or embodiment 106 in the manufacture of a vaccine for the treatment or prevention of a coronavirus infection. 111. Use of an isolated immunogenic polypeptide according to any one of embodiments 1 to 100, a nucleic acid according to any one of embodiments 103 to 104, or a composition according to any one of embodiments 105 or embodiment 106 for bioinformatics analysis. 112. An automated method for producing an isolated immunogenic polypeptide according to any one of embodiments 1 to 100, a nucleic acid according to any one of embodiments 103 to 104, or a composition according to any one of embodiments 105 or embodiment 106. 113. A method for synthesizing an isolated immunogenic polypeptide according to any one of embodiments 1 to 100, a nucleic acid according to any one of embodiments 103 to 104, or a composition according to any one of embodiments 105 or embodiment 106. 114. A formulation comprising an isolated immunogenic polypeptide according to any one of embodiments 1 to 100, a nucleic acid according to any one of embodiments 103 to 104, or a composition according to any one of embodiments 105 or embodiment 106. 115. A device for delivery of an isolated immunogenic polypeptide according to any one of embodiments 1 to 100, a nucleic acid according to any one of embodiments 103 to 104, or a composition according to any one of embodiments 105 or embodiment 106. 116. The device according to embodiment 115, comprising a syringe.

Claims

1. An isolated immunogenic polypeptide comprising an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 15-113.

2. 2. The isolated immunogenic polypeptide of claim 1, wherein the one or more modifications of the variant coronavirus spike protein comprise: (a) an amino acid substitution at the amino acid corresponding to amino acid position 817 of SEQ ID NO:8; and / or (b) an amino acid substitution at the amino acid corresponding to amino acid position 892 of SEQ ID NO:8; and / or (c) an amino acid substitution at the amino acid corresponding to amino acid position 899 of SEQ ID NO:8; and / or (d) an amino acid substitution at the amino acid corresponding to amino acid position 942 of SEQ ID NO:8; and / or (e) an amino acid substitution at the amino acid corresponding to amino acid position 986 of SEQ ID NO:8; and / or (f) an amino acid substitution at the amino acid corresponding to amino acid position 987 of SEQ ID NO:8; and / or (g) an amino acid substitution at the amino acid corresponding to amino acid position 943 of SEQ ID NO:8; and / or (h) an amino acid substitution at the amino acid corresponding to amino acid position 944 of SEQ ID NO:

8.

3. 3. The isolated immunogenic polypeptide of any one of claims 1 to 2, wherein the one or more modifications in the variant coronavirus spike protein comprise: (a) an amino acid substitution at the amino acid corresponding to amino acid position F817 of SEQ ID NO:8; and / or (b) an amino acid substitution at the amino acid corresponding to amino acid position A892 of SEQ ID NO:8; and / or (c) an amino acid substitution at the amino acid corresponding to amino acid position A899 of SEQ ID NO:8; and / or (d) an amino acid substitution at the amino acid corresponding to amino acid position A942 of SEQ ID NO:8; and / or (e) an amino acid substitution at the amino acid corresponding to amino acid position K986 of SEQ ID NO:8; and / or (f) an amino acid substitution at the amino acid corresponding to amino acid position V987 of SEQ ID NO:8; and / or (g) an amino acid substitution at the amino acid corresponding to amino acid position S943 of SEQ ID NO:8; and / or (h) an amino acid substitution at the amino acid corresponding to amino acid position A944 of SEQ ID NO:

8.

4. 4. The isolated immunogenic polypeptide of claim 1, wherein the substitution is with a proline residue.

5. 5. The isolated immunogenic polypeptide of claim 1, further comprising an amino acid substitution at the amino acid corresponding to amino acid position D614 of SEQ ID NO:

8.

6. 6. The isolated immunogenic polypeptide of any one of claims 1 to 5, further comprising amino acid substitutions at amino acids corresponding to amino acid positions 682-685 of SEQ ID NO:

8.

7. 7. The isolated immunogenic polypeptide of claim 6, wherein the substitution comprises a substitution of RRAR(682-685), corresponding to amino acid positions 682-685 of SEQ ID NO:8, with GSAS.

8. 6. The isolated immunogenic polypeptide of claim 1, further comprising any one of the following amino acid substitutions in SEQ ID NO:8: K854F; A609C and I692C; K304E; D571C and S967C; A570C and N960C.

9. 9. The isolated immunogenic polypeptide of claim 8, further comprising the A570C and N960C amino acid substitutions of SEQ ID NO:

8.

10. 10. The isolated immunogenic polypeptide of any one of claims 1 to 9, further comprising any one of the following amino acid substitutions in SEQ ID NO:8: A944P; S943P; N919Y; L981Y; V1061I; S975P; N969P; N955L; L938F; N856L; R815M; A845P; S746I; T734I and Y1007F; L948C-G1059C; F970C-G999C; A972C-Q992C; I980C-Q992C; S967C-S975C; L984C-A989C; L984C-E988C; Q755C-N969C; T791C-A879C; S884C-A893C.

11. 11. The isolated immunogenic polypeptide of any one of claims 1 to 10, further comprising any one of the following amino acid substitutions in SEQ ID NO:8: S383C-D985C; V367F; F329Y; F329S; D568N; E619Q; D830N; D839N; D843N; D848N; T827C-Q949C; S383C-D985C; A570C-K964C; A570C-S967C; A570C-K964C; A570C-S967C; T547C-N978C; T547C-S982C; T547C-N978C; T547C-S982C.

12. 2. The isolated immunogenic polypeptide of claim 1, comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:

110.

13. 2. The isolated immunogenic polypeptide of claim 1, comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:

111.

14. A composition comprising an isolated immunogenic polypeptide according to any one of claims 1 to 13.

15. A composition comprising an isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 13.

16. 16. The composition of claim 14, further comprising lipid nanoparticles.

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