Vaccine against coronavirus

Abstract

This disclosure relates to the field of vaccines and binding molecules, and their preparations and methods of use in the treatment and / or prevention of disease. Vaccines and binding molecules, including multivalent mRNA and nanoparticle vaccines, compositions containing them, and their use for treating or preventing coronavirus infection are described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Nos. 63 / 399,990, filed August 22, 2022; 63 / 400,334, filed August 23, 2022; 63 / 431,286, filed December 8, 2022; and European Patent Application No. 23315318.8, filed August 17, 2023, the entire contents of each of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present disclosure relates to the field of vaccines, and preparations and methods of their use in the treatment and / or prevention of disease. Vaccines, pharmaceutical compositions containing same (also referred to herein as immunogenic compositions), and their use for treating or preventing coronavirus infections and related viral infections, such as those caused by sarbecoviruses and merbecoviruses, are described. Government Assistance Clause This invention was made with government support under W81XWH-18-2-0040 awarded by the U.S. Army Medical Research and Development Command, and MI220230 awarded by the Military Infectious Disease Research Program. The government has certain rights in this invention. [Background technology]

[0003] The following discussion is provided solely to aid the reader in understanding the present disclosure and is not admitted to describe or constitute prior art against it. The emergence of SARS-CoV-2, also designated COVID-19 and sometimes referred to herein as SARS2 or SARS-2, marks the seventh coronavirus isolated from humans and the third to cause severe disease, after Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). The rapid spread and relatively high case fatality rate of SARS-CoV-2 have resulted in significant loss of life and prolonged morbidity involving millions of cases worldwide. The rapidly evolving epidemiological situation of the SARS-CoV-2 pandemic has highlighted the need to understand the molecular biology of this novel coronavirus, especially as new variants of the virus with different levels of pathogenicity and transmissibility continue to circulate.

[0004] Effective prophylactic vaccines against SARS-CoV-2 and other members of the Sarbecovirus subgenus are urgently needed to ensure protection against SARS-CoV-2 variants of concern, which continue to evolve and cause new pandemics. Currently available vaccines against SARS-CoV-2 include mRNA, vector-based, and recombinant protein vaccines targeting the SARS-CoV-2 spike protein derived from the parental beta and / or omicron strains. Despite these available vaccines, SARS-CoV-2 variants continue to circulate among both vaccinated and unvaccinated individuals. This highlights the public health need for vaccines that will be effective against current and future circulating SARS-CoV-2 variants. The development of vaccines that elicit broadly protective immune responses against sarbecoviruses could form the basis of next-generation prophylactic vaccines. Vaccines against MERS-CoV and other MERS-CoVs are also needed for pandemic prevention efforts. MERS-CoV continues to cause significant mortality and morbidity in the Arabian Peninsula, and despite its currently low growth rate, the potential for a major pandemic remains high. Vaccines capable of eliciting broad, protective immune responses against several or all β-coronaviruses, and ultimately a broad spectrum of several or all β-coronaviruses and / or sarbecoviruses, are of interest for pandemic preparedness against other future coronavirus zoonotic events. The present disclosure provides mRNA and nanoparticle vaccines that can be used to treat or prevent coronavirus infections and other related viral infections. Summary of the Invention

[0005] Described herein are immunogenic compositions and vaccines for the treatment and / or prevention of infections caused by coronaviruses, including merbecoviruses and sarbecoviruses, and methods and uses thereof.

[0006] According to one aspect, an immunogenic composition is provided comprising at least two antigenic coronavirus peptides, or one or more messenger RNA (mRNA) molecules encoding them, comprising at least a first antigenic coronavirus peptide and a second antigenic coronavirus peptide, wherein each antigenic coronavirus peptide is independently selected from a coronavirus receptor-binding domain (RBD or R), or a fragment or variant thereof, a coronavirus N-terminal domain (NTD), or a fragment or variant thereof, a coronavirus S1 domain, or a fragment or variant thereof, a coronavirus stabilized spike S-2P domain, or a fragment or variant thereof, a coronavirus stabilized spike S domain, or a fragment or variant thereof, and a coronavirus stabilized spike S trimer, or a fragment or variant thereof; The peptides include antigenic coronavirus peptides selected from the following combinations of virus strains: (i) two or more selected from Clade 1b; (ii) one or more selected from Clade 1b and one or more selected from Clade 1a; (iii) one or more selected from Clade 1b, one or more selected from Clade 1a and one or more selected from Clade 2; (iv) one or more selected from Clade 1b, one or more selected from Clade 1a and one or more selected from Clade 3; (v) one or more selected from Clade 1b, one or more selected from Clade 2, and one or more selected from Clade 3; and (v) one or more selected from Clade 1a, one or more selected from Clade 2, and one or more selected from Clade 3. In some embodiments, at least one of the antigenic coronavirus peptides is an S-2P peptide, and the S-2P peptide may comprise the amino acid sequence of any one of SEQ ID NOs: 536-543, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity thereto.Additionally or alternatively, in some embodiments, one or more of the antigenic coronavirus peptides are included in a fusion protein comprising the antigenic coronavirus peptide and a nanoparticle-forming protein, where two or more of the antigenic coronavirus peptides may be included in the same or different fusion proteins comprising the nanoparticle-forming protein.

[0007] According to another aspect, an immunogenic composition is provided comprising nanoparticles comprising at least two antigenic coronavirus peptides, comprising at least a first antigenic coronavirus peptide and a second antigenic coronavirus peptide, or one or more messenger RNA (mRNA) molecules encoding the at least two antigenic coronavirus peptides, wherein each antigenic coronavirus peptide is selected from the group consisting of: a receptor-binding domain (RBD or R) of a coronavirus, or a fragment or variant thereof; an N-terminal domain (NTD) of a coronavirus, or a fragment or variant thereof; an S1 domain of a coronavirus, or a fragment or variant thereof; a stabilized form of a coronavirus the antigenic coronavirus peptides are independently selected from an extracellular spike S-2P domain, or a fragment or variant thereof; a stabilized coronavirus extracellular spike S domain, or a fragment or variant thereof; and a stabilized coronavirus extracellular spike S trimer, or a fragment or variant thereof, wherein each antigenic coronavirus peptide is comprised in a fusion protein comprising the antigenic coronavirus peptide and a nanoparticle-forming protein, and the antigenic coronavirus peptides may be comprised in the same or different fusion proteins, and the composition comprises antigenic coronavirus peptides from at least two different coronavirus strains, or one or more mRNA molecules encoding them. In some embodiments, each antigenic coronavirus peptide is derived from a coronavirus strain independently selected from clade 1a, clade 1b, clade 2, clade 3, and Middle East respiratory syndrome-related coronavirus (MERS-CoV), wherein at least the first and second antigenic coronavirus peptides may be derived from coronavirus strains of different clades. In some embodiments, the at least first and second antigenic coronavirus peptides are derived from different coronavirus strains independently selected from WA-1, Beta, Omicron BQ.1.1, Omicron XBB.1.5, viral strains of SARS-CoV-1, BANAL20-247, Khosta-2, and MERS-CoV.In some embodiments, the antigenic coronavirus peptides include antigenic coronavirus peptides selected from a combination of the following viral strains: (i) two or more selected from WA-1, Beta, Omicron XBB.1.5, and Omicron BQ.1.1; (ii) one or more selected from WA-1, Beta, Omicron XBB.1.5, and Omicron BQ.1.1, and a viral strain of SARS-CoV-1; and (iii) selected from WA-1, Beta, Omicron XBB.1.5, and Omicron BQ.1.1. (iv) one or more selected from WA-1, Beta, Omicron XBB.1.5, and Omicron BQ.1.1, and one or more selected from SARS-CoV-1 virus strains, BANAL20-247, and Khosta-2, and MERS-CoV; and (v) two or more selected from SARS-CoV-1 virus strains, BANAL20-247, and Khosta-2.

[0008] In some embodiments of any of the preceding aspects, the antigenic coronavirus peptides include antigenic coronavirus peptides selected from the following viral strain combinations: WA-1, Beta, and Omicron BQ1.1; WA-1, Omicron BQ.1.1, and SARS-CoV-1; WA-1, SARS-CoV-1, and Khosta2; WA-1, SARS-CoV-1, and BANAL20-247; WA-1, SARS-CoV-1, and MERS-CoV; and SAR-CoV-1, Khosta-2, and BANAL20-247.

[0009] In some embodiments of any of the preceding aspects, the antigenic coronavirus peptides include antigenic coronavirus peptides selected from the following combinations of virus strains: (i) WA-1, beta, and omicron BQ.1.1 (or XBB.1.5), in which the antigenic coronavirus peptide is an RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(beta)FN, and R(BQ1.1)FN, or a spike antigen contained in S-2P (e.g., S(WA-1)-2P, S(beta)-2P, and S(BQ1.1)-2P) or SpFN fusion proteins Sp(WA-1)FN, Sp(beta)FN, and Sp(BQ1.1)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion protein, or any combination thereof; (ii) WA-1, Omicron BQ.1.1 (or XBB1.5), and SARS-CoV-1, wherein the antigenic coronavirus peptide is an RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(BQ1.1)FN, and R(SARS-CoV-1)FN, or a spike antigen contained in S-2P (e.g., S(WA-1)-2P, S(BQ1.1)-2P, and S(SARS-CoV-1)-2P) or the SpFN fusion proteins Sp(WA-1)FN, Sp(BQ1.1)FN, and Sp(SARS-CoV-1)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion protein, or any combination thereof; (iii) WA-1, SARS-CoV-1, and Khosta2, wherein the antigenic coronavirus peptide is an RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(SARS-CoV-1)FN, and R(Khosta2)FN, or a spike antigen contained in S-2P (e.g., S(WA-1)-2P, S(SARS-CoV-1)-2P, and S(Khosta2)-2P) or SpFN fusion proteins Sp(WA-1)FN, Sp(SARS-CoV-1)FN, and Sp(Khosta2)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion proteins, or any combination thereof; (iv) WA-1, SARS-CoV-1, and BANAL20-247, wherein the antigenic coronavirus peptide is an RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(SARS-CoV-1)FN, and R(BANAL20-247)FN, or a spike antigen contained in S-2P (e.g., S(WA-1)-2P, S(SARS-CoV-1)-2P, and S(BANAL20-247)-2P) or the SpFN fusion proteins Sp(WA-1)FN, Sp(SARS-CoV-1)FN, and Sp(BANAL20-247)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion proteins, or any combination thereof; (v) WA-1, SARS-CoV-1, and MERS-CoV, wherein the antigenic coronavirus peptide is an RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(SARS-CoV-1)FN, and R(MERS-CoV)FN, or a spike antigen contained in S-2P (e.g., S(WA-1)-2P, S(SARS-CoV-1)-2P, and S(MERS-CoV)-2P) or SpFN fusion proteins Sp(WA-1)FN, Sp(SARS-CoV-1)FN, and Sp(MERS-CoV)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion protein, or any combination thereof; (vi) SAR-CoV-1, Khosta-2, and BANAL20-247, wherein the antigenic coronavirus peptide is an RBD antigen contained in the RFN fusion proteins R(SARS-CoV-1)FN, R(Khosta2)FN, and R(BANAL20-247)FN, or a spike antigen contained in S-2P (e.g., S(SARS-CoV-1)-2P, S(Khosta2)-2P, and S(BANAL20-247)-2P) or the SpFN fusion proteins Sp(SARS-CoV-1)FN, Sp(Khosta2)FN, and Sp(BANAL20-247)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion proteins, or any combination thereof; (vii) beta, omicron BQ.1.1 (or XBB1.5), and SARS-CoV-1, wherein the antigenic coronavirus peptide is an RBD antigen contained in the RFN fusion proteins R(beta)FN, R(BQ1.1)FN, and R(SARS-CoV-1)FN, or a spike antigen contained in S-2P (e.g., S(beta)-2P, S(BQ1.1)-2P, and S(SARS-CoV-1)-2P) or the SpFN fusion proteins Sp(beta)FN, Sp(BQ1.1)FN, and Sp(SARS-CoV-1)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion protein, or any combination thereof; (viii) beta, omicron XBB.1.5, and SARS-CoV-1, wherein the antigenic coronavirus peptide is an RBD antigen contained in the RFN fusion proteins R(beta)FN, R(XBB1.5)FN, and R(SARS-CoV-1)FN, or a spike antigen contained in S-2P (e.g., S(beta)-2P, S(XBB1.5)-2P, and S(SARS-CoV-1)-2P) or SpFN fusion proteins Sp(beta)FN, Sp(XBB1.5)FN, and Sp(SARS-CoV-1)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion proteins, or any combination thereof; (ix) Omicron BQ.1.1, SARS-CoV-1, and Khosta2, wherein the antigenic coronavirus peptide is an RBD antigen contained in the RFN fusion proteins R(Omicron BQ.1.1)FN, R(SARS-CoV-1)FN, and R(Khosta2)FN, or a spike antigen contained in S-2P (e.g., S(Omicron BQ.1.1)-2P, S(SARS-CoV-1)-2P, and S(Khosta2)-2P) or SpFN fusion proteins Sp(Omicron BQ.1.1)FN, Sp(SARS-CoV-1)FN, and Sp(Khosta2)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion protein, or any combination thereof. ***

[0010] In some embodiments of any of the preceding aspects, the nanoparticle-forming peptide comprises or is a ferritin protein, or a fragment or variant thereof, hi some embodiments, the nanoparticle-forming peptide comprises or is Helicobacter pylori ferritin (Hpf), or a fragment or variant thereof. In some embodiments, the nanoparticle-forming peptide is ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 1), or a fragment or variant thereof, DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKA YEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 2) or a fragment or variant thereof, and SKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 3) or a fragment or variant thereof.

[0011] In another aspect, the present disclosure provides nanoparticles comprising a nanoparticle-forming peptide and a fusion protein comprising at least two antigenic coronavirus peptides selected from a coronavirus receptor-binding domain (RBD), or a fragment or variant thereof, a coronavirus N-terminal domain (NTD), or a fragment or variant thereof, a coronavirus S1 domain, or a fragment or variant thereof, a coronavirus stabilized extracellular spike S-2P domain, or a fragment or variant thereof, a coronavirus stabilized extracellular spike S domain, or a fragment or variant thereof, a coronavirus stabilized extracellular spike S trimer, or a fragment or variant thereof, and a mosaic coronavirus spike protein, wherein at least one domain of the mosaic coronavirus spike protein is substituted or added from a heterologous coronavirus strain or virus species.

[0012] In some embodiments, the nanoparticle-forming peptide comprises or is a ferritin protein or a fragment or variant thereof, hi some embodiments, the nanoparticle-forming peptide comprises or is Helicobacter pylori ferritin (Hpf) or a fragment or variant thereof. In some embodiments, the nanoparticle-forming peptide is ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 1), or a fragment or variant thereof, DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKA YEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 2) or a fragment or variant thereof, and SKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 3) or a fragment or variant thereof.

[0013] In some embodiments of any of the above aspects, at least two of the antigenic coronavirus peptides are included in a fusion protein and linked via a linker. In some embodiments, 2 to 10 of the antigenic coronavirus peptides are included in a tandem fusion protein, where the antigenic coronavirus peptides may be linked via a linker. In some embodiments, the antigenic coronavirus peptides are included in a fusion protein that includes a nanoparticle-forming peptide, where the antigenic coronavirus peptide is linked to the nanoparticle-forming peptide via a linker. In any embodiment that includes a linker, the linker can include an amino acid sequence selected from GGGSGGSG (SEQ ID NO:583), GSGGGG (SEQ ID NO:11), GGGG (SEQ ID NO:15), GSGG (SEQ ID NO:5), GGG (SEQ ID NO:16), and SGG (SEQ ID NO:17).

[0014] In some embodiments of any of the above aspects, the first and second antigenic coronavirus peptides are comprised in a mosaic coronavirus spike protein, wherein at least one domain of the mosaic coronavirus spike protein is substituted or added from a heterologous coronavirus strain. In any embodiment in which the antigenic coronavirus peptide is comprised in a fusion protein with a nanoparticle-forming peptide, the fusion protein can comprise a format selected from SpFN, beads on a string, domain fusion, domain swap, loop insertion, and domain insertion. In some such embodiments, the fusion protein comprises a format selected from the formats depicted in Figures 2-7. In any such embodiment, the first and second antigenic coronavirus peptides can be different RBD peptides from different coronavirus strains (e.g., R1, R2) contained in the same fusion protein (e.g., R1R2FN), where the fusion protein may further include a spike protein (e.g., R1R2mosSpFN), and the composition may further include two or more different fusion proteins (e.g., R1R2FN, R2R1FN, or R1R2mosSpFN, R2R1mosSpFN) that include the same two or more different RBD peptides at different positions in the fusion protein, or mRNA molecules encoding the two or more different fusion proteins, or nanoparticles displaying the two or more different fusion proteins. In any such embodiment, the fusion protein can comprise an amino acid sequence selected from the sequences disclosed in Tables 6 and 7 (SEQ ID NOs: 29-551), or a sequence having at least 80% sequence identity thereto (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).

[0015] In some embodiments, the composition comprises nanoparticles comprising at least two antigenic coronavirus peptides. In some embodiments, the composition comprises one or more mRNA molecules encoding at least two antigenic coronavirus peptides, wherein the one or more mRNA molecules may be encapsulated or co-encapsulated in one or more lipid nanoparticles (LNPs). In some embodiments, the composition comprises one mRNA molecule encoding one fusion protein comprising at least two antigenic coronavirus peptides, wherein the fusion protein optionally further comprises a nanoparticle-forming peptide, and wherein the mRNA molecule is encapsulated in a lipid nanoparticle (LNP); in some embodiments, the composition comprises two or more mRNA molecules, optionally in a fusion protein comprising a nanoparticle-forming peptide, wherein the two or more mRNA molecules each encode at least one of the at least two antigenic coronavirus peptides, and wherein each mRNA molecule is encapsulated in a separate lipid nanoparticle (LNP); in some embodiments, the composition comprises two or more mRNA molecules, optionally in a fusion protein comprising a nanoparticle-forming peptide, wherein the two or more mRNA molecules each encode at least one of the at least two antigenic coronavirus peptides, and wherein the two or more mRNA molecules are co-encapsulated in the same lipid nanoparticle (LNP).

[0016] According to any of the above aspects or embodiments, when the composition comprises one or more mRNA molecules, the mRNA molecules can have one or more features selected from a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR), a polyadenylation (poly(A)) sequence, a chemical modification, where optionally, the chemical modification comprises N1-methylpseudouridine, and the mRNA is a self-replicating mRNA or a non-replicating mRNA, and the mRNA molecules can be co-encapsulated in a lipid nanoparticle (LNP). According to any of the above embodiments, the immunogenic composition can further comprise an adjuvant. In some embodiments including an antigen or nanoparticles, the adjuvant comprises one or more selected from ALFQ, Alhydrogel, and combinations thereof.

[0017] In a further aspect, the present disclosure provides nanoparticles comprising a nanoparticle-forming peptide and a fusion protein comprising at least two antigenic coronavirus peptides independently selected from a coronavirus receptor-binding domain (RBD), or a fragment or variant thereof, a coronavirus N-terminal domain (NTD), or a fragment or variant thereof, a coronavirus S1 domain, or a fragment or variant thereof, a coronavirus stabilized extracellular spike S-2P domain, or a fragment or variant thereof, a coronavirus stabilized extracellular spike S domain, or a fragment or variant thereof, a coronavirus stabilized extracellular spike S trimer, or a fragment or variant thereof, and a mosaic coronavirus spike protein, wherein at least one domain of the mosaic coronavirus spike protein has been substituted or added from a heterologous coronavirus strain.

[0018] In a further aspect, the present disclosure provides a DNA molecule comprising a sequence encoding a nanoparticle disclosed herein (e.g., a nanoparticle of the above-described aspect), or a plasmid comprising said DNA molecule, wherein the plasmid may be capable of expressing the DNA molecule in vivo.

[0019] In a further aspect, the present disclosure provides a method of treating or preventing a coronavirus infection in a subject in need thereof, comprising administering to a subject in need thereof an immunogenic composition as disclosed herein (e.g., an immunogenic composition of any of the above aspects or embodiments). Also provided are immunogenic compositions as described herein for use in treating or preventing a coronavirus infection in a subject in need thereof. Also provided is the use of an immunogenic composition as described herein in the preparation of a medicament for treating or preventing a coronavirus infection in a subject in need thereof. In some embodiments, the subject is at risk of contracting a coronavirus infection.

[0020] In some embodiments, the subject has already suffered from a coronavirus infection. In some embodiments, the subject has not previously been administered a vaccine for the prevention of a coronavirus infection. In some embodiments, the subject has previously been administered a vaccine for the prevention of a coronavirus infection. In some embodiments, the method, composition for use, or use elicits an immune response in the subject against a coronavirus, wherein the immune response may comprise neutralizing antibodies, and further wherein the neutralizing antibodies may cross-neutralize two or more coronavirus strains, and further wherein the neutralizing antibodies may cross-neutralize one or more coronavirus strains that are not component strains of the immunogenic composition.

[0021] In another aspect, the present disclosure provides an mRNA molecule comprising or consisting of a sequence selected from any one of SEQ ID NOs: 552-582, or a sequence at least 80% homologous thereto. In some embodiments, the mRNA molecule has a sequence comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity to any one of SEQ ID NOs: 552-582. In some embodiments, the mRNA molecule has a sequence comprising any one of SEQ ID NOs: 552-582. In some embodiments, the mRNA molecule has a sequence consisting of any one of SEQ ID NOs: 552-582. The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the present disclosure as claimed. Other objects, advantages, and novel features will become readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the present disclosure. [Brief explanation of the drawings]

[0022] [Figure 1] 1 shows a schematic diagram of the coronavirus phylogenetic tree. Viral species known to infect humans and cause significant disease, such as HCoV-229E, are named and colored light gray. [Figure 2] Linear schematics of pan-CoV nanoparticle design formats are shown. Ten design formats are shown, including tandemly linked heterologous receptor binding domains (RBDs), tandem RBDs and N-terminal domains (NTDs), an RBD replacing the NTD but retaining the native subdomain (SD) of the S1 polypeptide, and a C-terminal H. pylori ferritin RBD inserted into a loop of the NTD. Three examples are spike ferritin nanoparticles (SpFNs), in which a heterologous RBD molecule is introduced to replace either the native RBD or NTD domain, or as an additional RBD domain linked at the N-terminus. Many of these design formats are orthogonal and can be combined. Specifically, RBD insertion into a loop of the NTD can be combined with NTD-containing constructs. While illustrated using ferritin, ferritin can be replaced by other nanoparticle carrier molecules as discussed herein. [Figure 3-1] Selected "beads-on-a-string" formats are shown in graphical representations, including examples provided for (A) RR-FN, (B) RN-FN, (C) RRN-FN, and (D) RNRN-FN. For example, one or more RBDs of any combination can be linked in tandem to ferritin ("R-FN" or "RR-FN"); one RBD from any coronavirus strain can be linked to an NTD and ferritin from any coronavirus strain ("RN-FN"); two RBDs from different coronavirus strains can be linked in tandem to an NTD and ferritin from different coronavirus strains ("RRN-FN"); and an RBD-NTD-RBD-NTD string comprising an RBD or NTD and ferritin from any coronavirus strain linked in tandem. [Figure 3-2]Selected "beads-on-a-string" formats are shown in graphical representations, including examples provided for (E) R-FN, (F) RR-FN, and (G) RR-FN. For example, one or more RBDs of any combination can be linked in tandem to ferritin ("R-FN" or "RR-FN"); one RBD from any coronavirus strain can be linked to an NTD and ferritin from any coronavirus strain ("RN-FN"); two RBDs from different coronavirus strains can be linked in tandem to an NTD and ferritin from different coronavirus strains ("RRN-FN"); and an RBD-NTD-RBD-NTD string comprising an RBD or NTD and ferritin from any coronavirus strain linked in tandem. [Figure 4-1] Selected "domain fusion" formats are shown in diagrammatic representations, with examples and domain designations provided for (A) R2-SD-FN, (B) R-S1-FN, (C) R2-SD-S2-FN, and (D) (R)-R-SpFN. [Figure 4-2] Selected "domain fusion" formats are shown in graphical representations, with examples and domain designations provided for (E) R-SpFN, (F) RR-SpFN, and (G) RR-SpFN. [Figure 5] Selected "loop insertion" formats are shown in diagrammatic representation, with examples and domain designations provided for a set of chimeric fusion ferritin nanoparticle immunogens where heterologous RBD molecules are inserted into (A) R2N-FN-70, (B) R2N-FN-148, and (C) R2N-FN-164. [Figure 6]Selected "domain swap" formats (also referred to herein as "mosaic" formats) are shown in diagrammatic representations, including examples and domain designations provided for a set of chimeric fusion spike ferritin nanoparticle immunogens where a heterologous RBD molecule is (A) inserted into the CoV SpFN molecule (mosaic SpFN) and (B) an additional heterologous RBD molecule is added to the N-terminus of the SpFN molecule (mosaic R-mosaic SpFN). Chimeric fusion spikes containing the indicated additional heterologous RBD are shown in cartoon representation. [Figures 7A-7B] Selected designs for a set of chimeric fusion spike ferritin nanoparticle (SpFN) immunogens are shown in diagrammatic representations where heterologous RBD and NTD molecules are added to the N-terminus of the mosaic SpFN design (e.g., mosaic format): Figure 7A: various RR-mosaic SpFNs; Figure 7B: RNR-mosaic SpFNs. Additional RBD or NTD molecules can be added to the N-terminus of the mosaic SpFNs. [Figure 8] (A) Negative stain electron microscopy of MERS-CoV RBD-ferritin nanoparticles and (B) 2D classification of M3 RBD-ferritin and M4 RBD-ferritin nanoparticles are shown. Briefly, purified proteins were deposited at 0.02–0.08 mg / mL onto carbon-coated copper grids and stained with 0.75% uranyl formate. Grids were imaged using either an FEI T20 operating at 200 kV with an Eagle 4K CCD using Serial EM or a Thermo Scientific Talos L120C operating at 120 kV with a Thermo Scientific Ceta using EPU. [Figure 9] A cladogram reflecting the phylogenetic grouping of selected sarbecovirus strains based on sequence similarity of the amino acid sequence of the spike protein receptor binding domain (RBD) is shown. [Figure 10]1 shows the results of an octet biolayer interference binding assay of MERS RBD-ferritin nanoparticle immunogens (M.1 to M3.6) evaluated for binding to the MERS-CoV neutralizing human monoclonal antibody CDC-C2 in two formats. [Figure 11] Negative stain electron microscopy and binding studies of the MERS RBD-ferritin nanoparticle immunogen are shown. Panel A shows that the SARS2-RBD-MERS-RBD CoV-ferritin construct forms nanoparticles as shown by negative stain EM, and panel B shows that the SARS2-RBD-MERS-RBD CoV-ferritin construct was evaluated for binding to a MERS-CoV-neutralizing human monoclonal antibody and a SARS-CoV-2 neutralizing mAb. The construct designs for pCoV247 and pCoV248 are shown in Table 3. [Figure 12] In panel A, negative stain electron microscopy of coronavirus spike ferritin nanoparticle constructs shows that they form nanoparticles as shown by negative stain EM; in panel B, 2D classification of SARS-1 (SARS-CoV-1), MERS, HKU-1, and 229E spike ferritin nanoparticles, and in panel C, 3D reconstruction of HKU-1 and 229E spike ferritin nanoparticles. [Figure 13] Graphs reporting the results of the neutralizing antibodies ShAb01 (neutralizing SARS-CoV-1 and SARS-CoV-2), ShAb02 (neutralizing SARS-CoV-2), and R-SpFN designed pCoV316 and pCoV317 evaluated for binding to the human ACE2 receptor by biolayer interferometry. Binding occurred for 180 seconds, followed by 60 seconds of dissociation. [Figure 14]Generation and characterization of constructs pCoV323 (RBD from MZ081380_bat_Yunnan_RsYN04_2020, and RR-SpFN when the SARS-1 RBD is linked to the WA-1 SpFN molecule). Left panel: Size-exclusion chromatography of pCoV323(RR-SpFN) shows that the transiently transfected protein forms large nanoparticles of the expected size. Right panel: SDS-PAGE of pCoV323 after purification by NiNTA affinity purification after size-exclusion chromatography. [Figure 15] A cladogram reflecting the phylogenetic grouping of selected sarbecovirus strains based on sequence similarity of the amino acid sequence of the spike protein receptor binding domain (RBD) is shown. [Figure 16] In panel A, a graphical representation of the antigenic distance set is shown. Sarbecovirus strains that are relatively close to each other have greater immunological similarity than strains that are further apart. Figure 16 also shows, in panel B, a graphical representation of a multivalent RFN containing antigens from three different strains of the antigenic distance set. The different RBDs (i.e., the "R" in "RFN") are depicted with different patterns reflecting the different strains of origin. [Figure 17] A summary of the antigenic distance sets tested in different antigen presentation formats is shown: (+) indicates constructs expressed from co-encapsulated mRNA molecules; (++) indicates constructs expressed from a mixture of separately encapsulated mRNA molecules; + / -FN indicates formats tested with or without empty ferritin particles. [Figure 18] Shown are results from a Western blot (Panel A) following expression of the mRNA constructs in HeLa cells and a blot showing the correct banding (MW) of the monovalent constructs (Panel B). [Figure 19]Results from Western blot and electron microscopy after expression of mRNA constructs in HeLa cells are shown. Panel A shows proteins from cell lysates; Panel B shows proteins from culture supernatants; and Panel C shows negatively stained electron microscopy grids of purified SARS-CoV-2 beta SpFN molecules from the supernatants. Particle assemblies are indicated by black arrows, and spikes are visible on the surface of ferritin nanoparticles. Panel D shows class averages of SpFN particles containing spikes and ferritin particles. [Figure 20] Geometric mean pseudoneutralization titers elicited against coronavirus clade 1b strains WA-1, Delta, Beta, BA5, BQ.1.1, or XBB.1.5, SARS-COV-1 strains from coronavirus clade 1a, and Merbicovirus in sera from mice immunized with monovalent RFN (groups 1-7) (Panel A), SpFN (groups 18-24) (Panel B), or stabilized transmembrane spike (S2P, groups 15-17, 37-40) (Panel C). Further details for each group can be found in Table 8. [Figure 21] Geometric mean pseudoneutralization titers elicited against coronavirus clade 1b strains WA-1, Delta, Beta, BA5, BQ.1.1, or XBB.1.5, SARS-COV-1 strains from coronavirus clade 1a, and Merbecovirus in sera from mice immunized with multivalent mixes A-E RFN (groups 8-12) (Panel A), SpFN (groups 25-29) (Panel B), or stabilized transmembrane spike (S2P, groups 41-44) (Panel C). Further details for each group can be found in Table 8. [Figure 22]Geometric means of pseudoneutralization titers elicited against coronavirus clade 1b strains (A) WA-1, (B) delta, (C) beta, (D) BA5, (E) BQ.1.1, or (F) XBB.1.5 in sera from mice immunized with monovalent RFN or RFN antigenic distance mixes A through E (groups 1 through 12) are shown. Titers and readouts from each group of mice are represented as box plots showing the mean (horizontal line), first standard deviation (box), and second standard deviation (vertical line). Any individual titers more than two standard deviations from the mean (i.e., outliers) are represented as circles. Further details for each group can be found in Table 8. [Figure 23] Figure 1 shows pseudoneutralization titers elicited against coronavirus clade 1b strains (A) WA-1, (B) delta, (C) beta, (D) BA5, (E) BQ.1.1, or (F) XBB.1.5 in sera from mice immunized with monovalent SpFN or SpFN antigenic distance mixes A through E (groups 18 through 29). Titers and readouts from each group of mice are represented as box plots showing the mean (horizontal line), first standard deviation (box), and second standard deviation (vertical line). Any individual titers more than two standard deviations from the mean (i.e., outliers) are represented as circles. Further details for each group can be found in Table 8. [Figure 24] Figure 1 shows pseudoneutralization titers elicited against coronavirus clade 1b strains (A) WA-1, (B) delta, (C) beta, (D) BA5, (E) BQ.1.1, or (F) XBB.1.5 in sera from mice immunized with monovalent S2P or S2P antigenic distance mixes A through E (groups 15-17, 31, 38-44). Titers and readouts from each group of mice are represented as box plots showing the mean (horizontal line), first standard deviation (box), and second standard deviation (vertical line). Any individual titers more than two standard deviations from the mean (i.e., outliers) are represented as circles. Further details for each group can be found in Table 8. [Figure 25]Geometric mean pseudoneutralization titers elicited against coronavirus strains WA-1, Delta, Beta, BA5, BQ.1.1, XBB.1.5, SARS1, or MERS in sera from mice immunized with spike antigens presented as either monovalent or multivalent stabilized transmembrane spikes (S-2P, monovalent groups 15-17, 37-40; multivalent groups 31, 41-44) and monovalent or multivalent spikes conjugated to ferritin (SpFN, monovalent groups 18-24; multivalent groups 25-29). Groups were matched based on component strains and plotted in a scatter plot with the stabilized transmembrane spike group forming the x-axis coordinate and the ferritin-conjugated spike group forming the y-axis coordinate. Monovalent titers (open circles) are distributed around the x = y line (dashed line), suggesting roughly equal titers for each group. See Figures 20 and 21 for individual group titers and titer distributions. [Figure 26] Pseudoneutralization titers elicited against coronavirus strains (A) WA-1, (B) delta, (C) beta, and (D) SARS-1 in sera from mice immunized with Mix C SpFN (Group #), Mix D RFN (Group #), and Mix D RFN with empty ferritin (RFN+FN, Group #). mRNA was administered either as a co-administration of a mixture of LNPs encapsulating mRNA molecules encoding each construct separately (admin) or as LNPs encapsulating mRNA molecules together (encap). [Figure 27] Figure 1 shows the pseudoneutralization titers elicited against coronavirus strain pseudoviruses (A) WA-1, (B) delta, (C) beta, and (D) SARS-1 by sera from mice immunized with RFN: WA-1 (groups 1 and 34), SARS-1 (groups 4 and 36), or BANAL20-247 (groups 7 and 35) with or without empty ferritin. Additionally, multivalent Mix D (WA-1 + SARS-1 + BANAL20-247) RFN was tested in four different formulations: coadministered each monovalent component (without empty FN) (ca-, group 33), coencapsulated each monovalent component with coadministered empty FN (ca ce+, group 14), coencapsulated without empty FN (group 13), and coencapsulated (group 8). [Figure 28] Figure 1 shows that multivalent particles can be generated either by co-encapsulating multiple mRNAs, each expressing a different fusion protein (illustrated as RFNs of different strains), into a single LNP (as shown in the upper panel), or by administering multiple LNPs, each encapsulating a different mRNA. [Figure 29] The various mixes of virus strains used in the experiments (Mixes A to E) and the relative antigenic distances of the virus strains are shown, as well as a schematic diagram of the resulting nanoparticles. [Figure 30] Various examples of multivalent particles are shown. The illustrated examples include particles containing two different RFN fusion proteins (A) and four different RFN fusion proteins (B), although other particles could be formed from three, five, six, or more different RFN fusion proteins. The illustrated examples also include a multivalent particle (C) containing a single RRFN fusion protein in which each RBD (i.e., each "R" in "RRFN") is derived from a different viral strain, and a particle containing two RRFN fusion proteins in which the order of the RBD domains is reversed between the two different RRFN fusion proteins. Other RRFN particles could also be formed containing RRFN fusion proteins containing various RBD domains derived from different viral strains and in different orders. [Figure 31] 1 shows the dosing regimen for the study of multivalent antigens as described herein as booster vaccines. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present disclosure provides immunogenic compositions, including nanoparticle vaccines and mRNA molecules encoding them, for treating or preventing coronavirus infections and diseases, as well as related infections and diseases caused by sarbecoviruses and merbecoviruses. The disclosed immunogenic compositions comprise at least two antigenic coronavirus peptides, including at least a first antigenic coronavirus peptide and a second antigenic coronavirus peptide, or one or more messenger RNA (mRNA) molecules encoding them. According to some embodiments, multivalent immunogenic compositions are described herein that comprise two or more antigenic coronavirus peptides from different virus strains, or mRNA molecules encoding them. Heterologous antigens can focus the immune response to create an additional breadth of recognized antigens. Furthermore, immunization with multiple heterologous virus strains can provide additional breadth of immune response, even across clades.

[0024] The nanoparticles disclosed herein are composed of fusion proteins comprising nanoparticle-forming peptides and antigenic coronavirus peptides (e.g., at least two antigenic coronavirus peptides, which may be derived from different strains of coronavirus), optionally linked together via a linker. The fusion proteins are capable of self-assembly into stable nanoparticles in solution and, when administered to a subject, can generate a protective neutralizing immune response (i.e., production of neutralizing antibodies and / or protective cytokines). Similarly, the disclosed mRNA molecules, when administered and expressed in vivo, result in the production of antigens that generate a protective neutralizing immune response. In some embodiments, immunogenic compositions include one or more mRNA molecules encoding one or more fusion proteins comprising nanoparticle-forming peptides and antigenic coronavirus peptides, which, when administered and expressed in vivo, result in the production of nanoparticles as disclosed herein that generate a protective neutralizing immune response. In any embodiment, the immunogenic compositions as disclosed herein can also include an adjuvant. The disclosed immunogenic compositions (e.g., comprising mRNA molecules or nanoparticles) may provide protection against infection by coronaviruses, such as SARS-CoV-2 and other sarbecoviruses, MERS-CoV and other merbecoviruses, and other coronaviruses. The disclosed immunogenic compositions may also reduce disease caused by coronaviruses. The disclosed immunogenic compositions can induce a protective immune response in individuals who are vaccinated.

[0025] I. Definition It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. Unless otherwise specified, materials and / or methodologies known to those of ordinary skill in the art can be utilized in carrying out the methods described herein, based on the guidance provided herein. As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Reference to a singular object is not intended to mean "one and only," but rather "one or more," unless expressly stated to be so. As used herein, "about," when used in conjunction with a numerical value, means the specified numerical value as well as plus or minus 10% of the numerical value. For example, "about 10" should be understood as both "10" and "9 to 11."

[0026] As used herein, a phrase in the form "A / B" or "A and / or B" means (A), (B), or (A and B); a phrase in the form "at least one of A, B, and C" means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C). As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. As used herein, "variant," when used in the context of referring to a peptide, refers to a peptide sequence derived from a parent sequence by the incorporation of one or more amino acid changes, which may include substitutions, deletions, or insertions. For purposes of this disclosure, a variant can include an amino acid sequence that shares 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 up to about 100% sequence identity or homology with the reference (or "parent") sequence. For purposes of this disclosure, the terms "variant" and "derivative," when used in the context of referring to a peptide, are used interchangeably.

[0027] As used herein, "mutant," when used in the context of referring to a virus (e.g., SARS-CoV-2), refers to a virus that is descended from a reference (or "parent") virus that possesses one or more alterations in its genome (e.g., RNA genome), or a virus that has been genetically engineered to have one or more alterations in its genome compared to the reference (or "parent") virus, which may or may not result in changes to proteins encoded by the RNA sequence (e.g., one or more proteins of the mutant virus may contain substitutions, deletions, or insertions compared to the parent strain). For example, known mutant strains of SARS-CoV-2 include, but are not limited to, Omicron strains, including B.1.1.7 (first identified in the United Kingdom), B.1.351 (first identified in South Africa), and P.1 (first identified in Brazil), as well as XBB.1.5, EG.5.1, BA.1, BA.2, and BA.5. For purposes of this disclosure, a variant strain of a virus can include a genomic sequence that shares 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 up to about 100% sequence identity or homology with a reference (or "parental") genomic sequence.

[0028] As used herein, the phrases "effective amount," "therapeutically effective amount," and "therapeutic level" refer to a dosage or concentration of a disclosed vaccine that provides the specific pharmacological effect for which the vaccine is administered in a subject in need of such treatment, i.e., treatment to treat or prevent a coronavirus infection (e.g., MERS, SARS, or COVID-19). It is emphasized that a therapeutically effective amount or therapeutic level of a vaccine will not always be effective in treating or preventing the infections described herein, even if such dosage would be considered a therapeutically effective amount by one of skill in the art. Solely for convenience, exemplary dosages, drug delivery amounts, therapeutically effective amounts, and therapeutic levels are provided herein. The therapeutically effective amount may vary based on the condition of the subject, including the route of administration and dosage form, the age and weight of the subject, and / or the type and severity of the coronavirus infection.

[0029] The terms "treat," "treatment," or "treating," as used herein in reference to a coronavirus infection, mean reducing or eliminating viral load, or eliminating tissue pathology or viral presence in the airways or lungs. The terms "prevent," "preventing," or "prevention," as used herein with reference to a coronavirus infection, mean making an infection unable or reducing the risk of developing in a subject exposed to the coronavirus, or making an infection unable or reducing the risk of developing a high viral load of the coronavirus, or reducing or eliminating tissue pathology or viral presence in the respiratory tract or lungs. Prevention can also mean preventing a subsequent infection if the initial infection is treated or cured. Prevention can also mean preventing or reducing the risk of transmission of the virus from one subject host to another.

[0030] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any individual mammalian subject, e.g., a bovine, canine, feline, equine, or human. In specific embodiments, the subject, individual, or patient is a human. As used herein, the abbreviation "SD" in the context of the disclosed fusion proteins refers to a subdomain of the coronavirus spike protein. Within the spike protein, there are subdomains 1 and 2 (see, e.g., Wrapp et al., Science 367, 1260-1263 (2020)). Thus, "SD" could refer to either or both of SD1 and SD2 (i.e., subdomains 1 and 2) of the spike protein.

[0031] II. Coronavirus Coronaviruses are a family of viruses (i.e., Coronaviridae) that cause respiratory infections in mammals and contain genomes that are approximately 30 kilobases in length. The Coronaviridae family is divided into four genera, and the genome encodes 28 proteins across multiple open reading frames, including 16 nonstructural proteins (nsp) that are cleaved from the polyprotein after translation. See, e.g., Letko et al., Nature Microbiology, 2020, 5(4):562-569. The Coronaviridae family includes both α-coronaviruses and β-coronaviruses, both of which primarily infect bats but can also infect other mammals, such as humans, camels, and rabbits. β-coronaviruses have been of relatively greater clinical importance to date, causing disease outbreaks with high mortality rates, such as Severe Acute Respiratory Syndrome (SARS-CoV-1), Middle East Respiratory Syndrome (MERS-CoV), and COVID-19 (SARS-CoV-2). Other pathogenic β-coronaviruses include OC43 and HKU1. Non-limiting examples of pathogenic α-coronaviruses include, but are not limited to, 229E and NL63.

[0032] Although SARS-CoV-2 is a newly identified virus, it shares genetic and morphological characteristics with other viruses in the Coronaviridae family, particularly those from the β-coronavirus genus. The genome of the recently isolated SARS-CoV-2 shares 82% nucleotide identity with the human SARS-CoV (SARS-CoV-1) and 89% nucleotide identity with the bat SARS-like CoV ZXC21 (Lu et al., 2020). The spike (S) glycoprotein in particular shares significant structural homology with SARS-CoV-1 compared to other coronaviruses, such as MERS-CoV. Like SARS-CoV-1, the surface spike (S) glycoprotein of SARS-CoV-2 binds to the same host receptor, ACE-2, to mediate cell entry (Letko et al., 2020; Yan et al., 2020a). The class I fusion protein S is also a crucial determinant of viral host range and tissue tropism and a primary target of the host immune response (Li, 2016). Therefore, the majority of coronavirus vaccine candidates developed to date are based on S or one of its subcomponents. The coronavirus S glycoprotein contains three segments: a large ectodomain, a single-pass transmembrane anchor, and a short intracellular tail. The ectodomain consists of the receptor-binding subunit S1, which contains two subdomains: an N-terminal one and a C-terminal one. The latter contains the receptor-binding domain (RBD), which plays a key role in binding the virus to host receptors and inducing a conformational change in the protein that leads to fusion with the host cell membrane via the S2 subunit. Antibodies have been shown to neutralize viral entry by binding to the RBD of the spike protein. This region is also known to be the most variable part of the protein and is likely responsible for immune evasion, which can lead to reinfection or reduced vaccine efficacy.

[0033] As discussed in more detail below, ferritin is a small protein expressed by many organisms that can form homotypic 24mer "nanoparticles." Previous studies have shown that it can function as an antigen-presenting system by modifying the N-terminal region with an antigen of interest. Antigens can be conjugated to the ferritin moiety without impairing nanoparticle formation. Ferritin with antigens conjugated via sufficiently long linkers can form quaternary structures, such as CoV trimers. (8)

[0034] Multiple technology platforms, including nucleic acid vaccines, whole virus vaccines, recombinant protein subunit vaccines, and nanoparticle vaccines, are currently advancing SARS-CoV-2 vaccine development. Among these vaccine platform types, nanoparticle technology has previously been shown to improve antigen structure and stability, as well as vaccine targeted delivery, immunogenicity, and safety. Vaccines containing ferritin nanoparticles conjugated to either the spike protein or the RBD region of the spike protein have been demonstrated to elicit immune responses that are protective against subsequent challenge in animal models. Joyce, et al., Science Translational Med., 14 (632) (DOI: 10.1126scitranslmed.abi5735) (Dec. 16, 2021); Joyce et al., Cell Reports, 37: 110143 (Dec. 21, 2021). Antigen presentation systems using multiple antigens (multivalent) have been demonstrated to increase the breadth of responses to coronaviruses (see, e.g., Cohen at al., Science, 371: 735-741 (Feb. 2021); Cohen at al., Science 377, eabq0839(2022) (DOI:10.1126 / science.abq0839)) and influenza (see, e.g., Kanekiyo et al., Nat. Immunol., 20: 367-72 (Apr. 2019)). Boosting immunizations (or sequential vaccinations) with heterologous virus strains have been shown to improve coverage efficacy against heterologous virus strains from the original virus strain. See, e.g., Tan et al., N. Eng. J. Med., 385: 1401-06 (Aug. 2021).

[0035] In some embodiments of the present disclosure, the coronavirus treated or prevented by the disclosed immunogenic compositions (e.g., vaccines) is a β-coronavirus. In some embodiments, the β-coronavirus is selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (also known by the provisional name 2019 novel coronavirus, or 2019-nCoV, or COVID-19), human coronavirus OC43 (hCoV-OC43), Middle East respiratory syndrome-related coronavirus (MERS-CoV, also known by the provisional name 2012 novel coronavirus, or 2012-nCoV), severe acute respiratory syndrome-related coronavirus (SARS-CoV, also known as SARS-CoV-1), HKU-1, 229E, and NL63. In some embodiments, the β-coronavirus is SARS-CoV-2, the causative agent of COVID-19. In some embodiments, the disclosed vaccines can provide broad-spectrum treatment and / or prevention against multiple different types of coronaviruses, such as MERS-CoV, SARS-CoV-1, and / or SARS-CoV-2, and / or others.

[0036] III. Immunogenic composition Disclosed herein are immunogenic compositions (e.g., vaccines) that can be used to treat or prevent coronavirus infection. In some aspects, the disclosed immunogenic compositions comprise a fusion protein comprising a nanoparticle-forming peptide and an antigenic coronavirus peptide, optionally linked by a linker (i.e., a linker domain). The antigenic coronavirus peptide can comprise one or more fragments or full-length proteins derived from a coronavirus (e.g., SARS-CoV-2, SARS-CoV-1, or MERS-CoV), as described in more detail below.

[0037] A. Nanoparticle-forming Peptides The nanoparticle-forming peptide of the immunogenic compositions as disclosed herein can be any suitable nanoparticle-forming peptide. H. pylori ferritin and fragments and variants thereof are particularly suitable to serve as nanoparticle-forming peptides for the vaccines as disclosed herein. Thus, the nanoparticle-forming peptide of the vaccines as disclosed herein can comprise Helicobacter pylori ferritin protein (HpF) or a fragment or variant thereof. For example, the nanoparticle component can comprise the following amino acid sequence derived from H. pylori ferritin: ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 1).

[0038] Thus, the nanoparticle-forming peptide of the vaccine can comprise the above-described H. pylori ferritin sequence (SEQ ID NO: 1) or a variant thereof that can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutation. For example, the nanoparticle-forming peptide can comprise a variant of SEQ ID NO: 1 that can include a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from the N-terminal domain of SEQ ID NO: 1. In some embodiments, the nanoparticle-forming peptide can include a substitution of a glutamic acid residue (E) at position 13 of SEQ ID NO: 1. In some embodiments, the nanoparticle-forming peptide can include a substitution of a glutamic acid residue (E) at position 13 of SEQ ID NO: 1 and a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from the N-terminal domain of SEQ ID NO: 1, such as in the following sequence: DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 2); or SKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 3).

[0039] In some embodiments, the nanoparticle-forming peptide can comprise a variant of any of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, which can include an amino acid sequence that shares 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 up to 100% sequence identity or homology with any of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. As noted above, in some embodiments, the nanoparticle-forming peptide can be a non-ferritin-based peptide, such as a peptide comprising the following sequence, or a fragment or variant thereof: MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR (SEQ ID NO: 4).

[0040] In some embodiments, the nanoparticle-forming peptide can comprise a variant of SEQ ID NO: 4, which can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutations in SEQ ID NO: 4. In some embodiments, the nanoparticle-forming peptide can comprise a variant of SEQ ID NO: 4, which can include an amino acid sequence that shares 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 up to 100% sequence identity or homology with SEQ ID NO: 4.

[0041] B. Linker Domain The disclosed fusion proteins typically include a flexible amino acid linker; however, the linker domain (i.e., linker) is optional, and in some embodiments, the nanoparticle-forming peptide can be directly linked to the antigenic coronavirus peptide. The linker can be about 3 to about 50 amino acids in length, or more specifically, about 4 to about 42 amino acids in length. In some embodiments, the linker can be 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, or 42 amino acids in length. The linker domain can include glycine (G) repeats or a combination of glycine (G) and serine (S) residues. Several exemplary linker sequences are disclosed in Table 1 below.

[0042] [Table 1] The linker domain can comprise one, two, or three repeats of any one of SEQ ID NOs: 5-17 or 583. In some embodiments, the linker domain comprises a variant of any one of SEQ ID NOs: 5-17 or 583, which can include an amino acid sequence that shares 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 up to 100% sequence identity or homology with any one of SEQ ID NOs: 5-17 or 583. The above linker sequences are not intended to be limiting, and one of skill in the art will understand, based on the guidance provided herein, that other flexible peptide linkers may also be suitable for linking nanoparticle-forming peptides and antigenic coronavirus peptides.

[0043] C. Antigenic Coronavirus Peptides Generally, the antigenic coronavirus peptides of the disclosed immunogenic compositions and fusion proteins comprise the coronavirus spike protein (also known as the "S protein" or "glycoprotein S"), which is generally responsible for viral entry into host cells, or a fragment or variant thereof (such as the RBD domain or a fragment or variant thereof). In some embodiments, the antigenic coronavirus peptide can comprise one, two, or three or more different domains of the coronavirus spike protein that are linked together in sequence; in such embodiments, linkers may separate the different domains.

[0044] The spike protein was selected as the antigenic coronavirus peptide for the vaccine as disclosed herein because antibodies raised against this peptide are likely to be neutralizing. The spike protein contains two functional subunits responsible for binding to host cell receptors (S1 subunit) and fusing viral and cellular membranes (S2 subunit). The fusion proteins of the present disclosure can comprise the entire spike protein, the S1 subunit alone, the S2 subunit alone, or antigenic / immunogenic fragments or variants of any of these. In some embodiments, the fusion protein comprises the full-length coronavirus spike protein sequence. In some embodiments, the fusion protein includes a variant that includes 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the coronavirus spike protein (e.g., SEQ ID NO: 18), so long as the fragment is capable of eliciting an immune response (i.e., is an antigenic fragment).

[0045] Without wishing to be bound by theory, it is understood that the spike protein of SARS-CoV-2 attaches to the human angiotensin-converting enzyme (ACE)-2 cell surface receptor to facilitate human infection. Therefore, antibodies that can bind to the spike glycoprotein and prevent its interaction with the ACE2 receptor can promote protection from infection. The SARS-CoV-2 spike protein (NCBI Reference Sequence: YP_009724390.1) contains 1,273 amino acids and consists of an N-terminally located signal peptide (amino acids 1-13), an S1 subunit (residues 14-685), and an S2 subunit (residues 686-1,273); the last two regions are responsible for receptor binding and membrane fusion, respectively. The amino acid sequence is shown below.

[0046] Specific domains of the coronavirus spike protein that are particularly useful as antigenic coronavirus peptides in the context of the present disclosure include the following domains: the receptor binding domain (RBD) of a coronavirus, or a fragment or variant thereof; the N-terminal domain (NTD) of a coronavirus, or a fragment or variant thereof, - coronavirus receptor binding domain (RBD)-N-terminal domain chimera, or a fragment or variant thereof; the S1 domain of a coronavirus, or a fragment or variant thereof, a stabilized coronavirus spike S-2P domain or a stabilized extracellular spike S-2P domain, or a fragment or variant thereof; a stabilized spike S domain or a stabilized extracellular spike S domain of a coronavirus, or a fragment or variant thereof; or - A stabilized spike S trimer or stabilized extracellular spike S trimer of a coronavirus, or a fragment or variant thereof.

[0047] Thus, the antigenic coronavirus peptides of the present disclosure can comprise an RBD. The RBD can comprise the SARS-CoV-2 RBD amino acid sequence shown below: NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP (SEQ ID NO: 19). In some embodiments, the antigenic coronavirus peptide comprises a variant of SEQ ID NO: 19, which can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutations within SEQ ID NO: 19. In some embodiments, the antigenic coronavirus peptide comprises a variant of SEQ ID NO: 19, which can include an amino acid sequence that shares 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 up to 100% sequence identity or homology with SEQ ID NO: 19. In some embodiments, the antigenic coronavirus peptide comprises a fragment of the RBD, which can be a fragment of SEQ ID NO: 19 comprising 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of SEQ ID NO: 19, so long as the fragment is capable of eliciting an immune response (i.e., is an antigenic fragment).

[0048] Antigenic coronavirus peptides can include one or more specific modifications made to reduce "sticky" hydrophobic regions, which can enhance expression and / or ability to form nanoparticles, for example, variants of the RBD (e.g., SEQ ID NO: 19) with one or more of the following modifications:

[0049] [Table 2] The above-described modifications can increase expression and / or nanoparticle formation of fusion proteins containing RBDs with these modifications.

[0050] Additionally or alternatively, the antigenic coronavirus peptide can be or include an RBD from a coronavirus other than SARS-CoV-2. For example, the RBD domain can be derived from MERS or SARS-CoV-1 (also referred to herein as SARS1 and SARS-1). Exemplary RBD sequences can be found in the full-length constructs provided in the accompanying Tables 6 and 7. Furthermore, in some embodiments, the particles can include multiple RBDs from the same or different coronaviruses, such as those discussed in more detail below.

[0051] Additionally or alternatively, an antigenic coronavirus peptide of the present disclosure can comprise the SARS-CoV-2 NTD amino acid sequence QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTL (SEQ ID NO: 20). In some embodiments, the antigenic coronavirus peptides comprise variants of SEQ ID NO: 20, which can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutations in SEQ ID NO: 20. In some embodiments, the antigenic coronavirus peptides comprise variants of SEQ ID NO: 20, which can include an amino acid sequence that shares 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 up to 100% sequence identity or homology with SEQ ID NO: 20. In some embodiments, the antigenic coronavirus peptide comprises a fragment of the NTD, which can be a fragment of SEQ ID NO:20 comprising 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of SEQ ID NO:20, so long as the fragment is capable of eliciting an immune response (i.e., is an antigenic fragment).

[0052] Additionally or alternatively, the antigenic coronavirus peptides of the present disclosure can be or include an NTD from a coronavirus other than SARS-CoV-2. For example, the NTD domain can be derived from MERS or SARS-CoV-1. Exemplary NTD sequences can be found in the full-length constructs provided in the accompanying Tables 6 and 7. Furthermore, in some embodiments, the particles can include multiple NTDs from the same or different coronaviruses.

[0053] In some embodiments, the particles can include a combination of one or more RBDs and one or more NTDs, where the RBDs and NTDs can be derived from the same or different coronaviruses or virus strains.

[0054]

[0055] Additionally or alternatively, antigenic coronavirus peptides of the present disclosure can comprise the S-2P sequence or a fragment or variant thereof. The S-2P sequence is a stabilized version of the spike ectodomain that contains two proline substitutions and stabilizes the prefusion conformation. Alternatively, the S-2P domain comprises a transmembrane domain. Specifically, S-2P contains proline modifications K986P and V987P and a furin cleavage site (RRAS to GSAS). In some embodiments, antigenic coronavirus peptides can comprise variants of the S-2P sequence, which can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutations in the S-2P sequence. In some embodiments, the antigenic coronavirus peptide can comprise a variant of an S-2P sequence, which can include an amino acid sequence that shares 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 up to 100% sequence identity or homology with stabilized S-2P. In some embodiments, the antigenic coronavirus peptide can comprise a fragment of S-2P, including 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of stabilized S-2P, so long as the fragment is capable of eliciting an immune response (i.e., is an antigenic fragment).

[0056] Additionally or alternatively, antigenic coronavirus peptides of the present disclosure can comprise a spike S domain or extracellular spike S domain (e.g., a stabilized spike S domain or a stabilized extracellular spike S domain), or a fragment or variant thereof. A stabilized extracellular spike S domain can comprise one or more modifications to stabilize the refusion conformation of the domain or extracellular domain. In some embodiments, antigenic coronavirus peptides can comprise a stabilized extracellular spike S domain that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutations in the extracellular spike S domain. In some embodiments, the antigenic coronavirus peptide can comprise a stabilized extracellular spike S domain comprising an amino acid sequence that shares 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 up to 100% sequence identity or homology with the extracellular spike S domain. In some embodiments, the antigenic coronavirus peptide can comprise a fragment of the extracellular spike S domain (e.g., a stabilized extracellular spike S domain) comprising 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the extracellular spike S domain (e.g., a stabilized extracellular spike S domain), so long as the fragment is capable of eliciting an immune response (i.e., is an antigenic fragment). In some embodiments, the antigenic coronavirus peptide can comprise a stabilized spike S domain that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutations in the spike S domain.In some embodiments, the antigenic coronavirus peptide can comprise a stabilized spike S domain comprising an amino acid sequence that shares 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 up to 100% sequence identity or homology with the spike S domain. In some embodiments, the antigenic coronavirus peptide can comprise a fragment of the spike S domain (e.g., a stabilized spike S domain) comprising 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the spike S domain (e.g., a stabilized spike S domain), so long as the fragment is capable of eliciting an immune response (i.e., is an antigenic fragment).

[0057] Additionally or alternatively, antigenic coronavirus peptides as described herein can comprise spike S trimers or extracellular spike S trimers (e.g., stabilized spike S trimers or stabilized extracellular spike S trimers), or fragments or variants thereof. The stabilized extracellular spike S trimers can comprise one or more modifications to stabilize the refusion conformation of the extracellular trimer. In some embodiments, antigenic coronavirus peptides can comprise stabilized extracellular spike S trimers that include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutations in the extracellular spike S trimer. In some embodiments, the antigenic coronavirus peptide can comprise a stabilized extracellular spike S trimer comprising an amino acid sequence that shares 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 up to 100% sequence identity or homology with the extracellular spike S trimer. In some embodiments, the antigenic coronavirus peptide can comprise a fragment of the extracellular spike S trimer (e.g., a stabilized extracellular spike S trimer) that comprises 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the extracellular spike S trimer (e.g., a stabilized extracellular spike S trimer), so long as the fragment is capable of eliciting an immune response (i.e., is an antigenic fragment). The stabilized spike S trimer can include one or more modifications to stabilize the pre-fusion conformation of the trimer (e.g., the trimerization domain). In some embodiments, the antigenic coronavirus peptide can comprise a stabilized spike S trimer that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutations in the spike S trimer.In some embodiments, the antigenic coronavirus peptide can comprise a stabilized spike S trimer comprising an amino acid sequence that shares 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 up to 100% sequence identity or homology with the spike S trimer. In some embodiments, the antigenic coronavirus peptide can comprise a fragment of the spike S trimer (e.g., a fragment of a stabilized extracellular spike S trimer) that comprises 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the spike S trimer (e.g., a stabilized spike S trimer), so long as the fragment is capable of eliciting an immune response (i.e., is an antigenic fragment).

[0058] Additionally or alternatively, antigenic coronavirus peptides as described herein can include a stabilized variant containing six prolines (i.e., "Hexapro"), another variant of the spike protein that contains the two S-2P proline substitutions plus the F817P, A892P, A899P, and A942P substitutions. In some embodiments, antigenic coronavirus peptides can include variants of Hexapro, which can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more substitution, deletion, or insertion mutations in Hexapro. In some embodiments, the antigenic coronavirus peptide can comprise a variant of Hexapro, which can include an amino acid sequence that shares 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 up to 100% sequence identity or homology with Hexapro. In some embodiments, the antigenic coronavirus peptide can comprise a fragment of Hexapro that comprises 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of Hexapro, so long as the fragment is capable of eliciting an immune response (i.e., is an antigenic fragment).

[0059]

[0060] D. Fusion Proteins and Vaccine Nanoparticles The disclosed vaccine nanoparticles are composed of multiple fusion glycoprotein domains that self-assemble into nanoparticles. As described above, the fusion protein includes a nanoparticle-forming peptide, which may be H. pylori ferritin protein or a fragment or variant thereof. Ferritin is a naturally occurring protein that self-assembles into 24-membered spherical particles composed of multiple three-, four-, and / or two-fold axes. Thus, the nanoparticles can include three-, four-, or two-fold axes. With a three-fold axis, eight antigenic trimeric coronavirus peptides can be displayed on the surface of the self-assembling protein nanoparticle. In the case of a monomeric antigen, such as RBD, 24 coronavirus peptides can be displayed on the surface of the self-assembling protein nanoparticle.

[0061] E. Multivalent Immunogenic Compositions As noted above, the evolution of SARS-CoV-2 mutant strains has presented a challenge to vaccine efficacy. As viruses evolve, amino acids within epitopes of protein antigens can mutate. Due to this process, antibodies generated by a host against one protein (e.g., after exposure to the virus or immunization with the protein) may bind strongly, weakly, or not at all to the same epitope in an evolutionarily related protein. When binding is observed at the same position in two or more closely related (but non-identical) virus strains, the binding position may be referred to as a "shared" epitope, which is typically a neutralizing epitope. As a neutralizing epitope, a shared epitope may be subject to evolutionary pressure and may exhibit short-term evolution (e.g., changes in amino acid sequence). By definition, a shared epitope has the potential to mutate but has not previously mutated sufficiently to abolish binding by overlapping populations of antibodies and therefore remains common to virus strains. Due to the variability of shared epitopes, binding is unlikely to be observed in more distantly related virus strains. Generating antibodies that can better tolerate variability in shared epitopes (e.g., exhibit binding across shared epitopes) represents one approach to generating a broad antigenic response that can provide protection against current and future coronavirus strains. Less commonly recognized epitopes (i.e., subdominant epitopes) may not change over small timescales (i.e., from season to season) due to a lack of evolutionary pressure, but may still change over larger timescales (i.e., decades / centuries) due to evolutionary drift. In some cases, mutations may be less tolerated for subdominant epitopes that coincide with functional regions of proteins. When an antibody consistently binds to one such epitope in distantly related antigens, the binding location may be referred to as a "conserved" epitope. The generation of antibodies against less commonly recognized conserved epitopes represents an alternative approach to generating a broad antigenic response that could provide protection against current and future circulating coronavirus strains.The multivalent embodiments described herein can take advantage of either or both of these approaches by inducing antibodies against common and / or conserved epitopes.

[0062] In some aspects, the present disclosure relates to optimized multivalent presentation of antigenic coronavirus peptides on nanoparticles (e.g., on mRNA-encoded ferritin nanoparticles) that, when administered, generate a neutralizing immune response against a broad set of coronaviruses (Figure 15). As described herein, the antigenic breadth of the immune response is enhanced through simultaneous, localized presentation of multiple antigens to the immune system. This multivalent vaccination can be achieved through mosaic antigens, such as those that may be formed in vivo after administration of nanoparticles or mRNA molecules encoding fusion proteins comprising one or more antigenic coronavirus peptides and a nanoparticle-forming protein, such as ferritin. mRNA molecules encoding different fusion proteins (containing different antigenic coronavirus peptides) may be co-encapsulated in a single lipid nanoparticle (LNP) or encapsulated in separate LNPs that are formulated together in an immunogenic composition to achieve simultaneous delivery. Upon administration, in vivo expression of the mRNA molecules results in the formation of multivalent nanoparticles displaying antigenic coronavirus peptides from different viral strains. These antigens may be from closely related, distantly related, or different sarbecovirus strains. (Figure 16, panel A). Co-localized presentation of multiple antigens from virus strains with selected antigenic distances (as illustrated in Figure 16, panel B) is designed to promote the maturation of antibodies capable of binding epitopes from multiple antigens from different coronavirus strains. This will likely increase the breadth of protection against current and future circulating coronavirus strains.

[0063] Thus, some embodiments of the present disclosure relate to the co-localized presentation of multiple antigens from different virus strains with a selected antigenic distance (as illustrated in FIG. 17). In this context, antigenic distance refers to the immunogenic similarity of the antibody binding profiles of sera resulting from exposure to two different antigens, as measured by their similarity in readouts in an assay (e.g., a pseudoneutralization assay). A smaller antigenic distance implies greater immunogenic similarity (e.g., greater similarity in neutralizing titers across multiple virus strains), while a larger distance implies greater immunogenic difference (e.g., less similarity in neutralizing titers across multiple virus strains). Biologically, antigenic similarity refers to sera resulting from two different antigens having antibodies that bind to overlapping and / or identical epitopes. Thus, antigens with small antigenic distances are expected to have common epitopes that share physicochemical properties and promote binding of similar antibodies, while antigens at greater distances may lack common epitopes and promote binding of non-overlapping sets of antibodies. In some cases, antigens can induce responses against distantly related virus strains; this is likely mediated by antibodies that bind to conserved, subdominant epitopes.

[0064] According to some multivalent embodiments, the present disclosure provides immunogenic compositions designed based on antigenic distance sets of antigens that, when presented in close physical proximity, promote the generation of antibodies with broad binding spectrums. By presenting antigens in close physical proximity, such as on the same nanoparticle, the immune system will likely elicit antibodies that simultaneously bind to two distinct but evolutionarily related epitopes. This provides additional breadth of the immune response, as antibodies that bind to two variable shared epitopes will bind to a wider range of antigens. The immune system may also elicit antibodies that bind to two conserved, subdominant epitopes, such as due to tolerance of mutations in those two epitopes and / or relatively strong binding by the antigen-binding site of the antibody, leading to stabilization of the antibody-antigen interaction. Thus, as described above, the multivalent embodiments described herein induce antibodies against shared and / or conserved epitopes. Figure 15 shows how coronaviruses are phylogenetically grouped into clades according to sequence similarity in the RBD protein. Figure 16, panel A provides antigenic distance maps for several virus strains. Figure 17 illustrates various multivalent embodiments (Mix A through Mix F) based on antigenic distance sets.

[0065] The antigenic coronavirus peptide component of the disclosed immunogenic compositions or fusion proteins comprised therein, or the mRNA encoding same, can comprise one, two, three, or more different domains, which can be selected from the exemplary antigenic peptides discussed above. For example, in some embodiments, the antigenic coronavirus peptide can comprise one or more domains selected from the RBD, NTD, full-length spike protein, stabilized extracellular spike S-2P domain, stabilized extracellular spike S domain, stabilized extracellular spike S trimer (optionally including a transmembrane component), and variants or fragments thereof. For example, the antigenic coronavirus peptide of the immunogenic compositions as disclosed herein can comprise a combination of two domains, such as two domains independently selected from the RBD, NTD, full-length spike protein, stabilized extracellular spike S-2P domain, stabilized extracellular spike S domain, stabilized extracellular spike S trimer, HexaPro, and variants or fragments thereof. Alternatively, the antigenic coronavirus peptide can comprise a combination of three domains, such as three domains independently selected from the RBD, NTD, full-length spike protein, S1 subunit, S2 subunit, stabilized extracellular spike S-2P domain, stabilized extracellular spike S domain, stabilized extracellular spike S trimer, HexaPro, and variants or fragments thereof. For non-nanoparticle embodiments, the antigenic coronavirus peptide can additionally or alternatively comprise one or more domains selected from the stabilized extracellular spike S-2P domain, stabilized extracellular spike S domain, stabilized extracellular spike S trimer, and variants or fragments thereof. As described above and exemplified in more detail below, the disclosed immunogenic compositions (e.g., vaccines) may be capable of eliciting immune responses from multiple types of coronaviruses (i.e., SARS, MERS, etc.), and therefore the various domains of the antigenic coronavirus peptide can be derived from different coronaviruses, different strains of the same coronavirus, or combinations thereof.

[0066] Exemplary fusion protein formats include, but are not limited to, (1) RBD and ferritin ("R-FN"), (2) spike protein from a strain of coronavirus and ferritin ("SpFN"), where the spike protein may have RBDs from different viral strains ("mosaic SpFN"), and (3) RBD-RBD and ferritin, where the two RBDs are from two different strains of coronavirus. strain ("RR-FN"), (4) RBD-NTD and ferritin ("RN-FN"), (5) RBD-RBD-NTD and ferritin ("RRN-FN"), (6) RBD-NTD-RBD-NTD and ferritin ("RNRN-FN"), (7) RBD-SD-RBD-SD ("R2-SD-FN") and ferritin, (8) RBD-NTD-SD-RBD-SD and ferritin ("R-S1-FN"), (9) (10) spike protein and ferritin in which the NTD is replaced by a second RBD ("R2-SD-S2-FN"), (11) RBD-spike protein and ferritin in which the RBD is derived from a different strain of coronavirus ("RmosSpFN"), (12) spike protein and ferritin containing an additional RBD ("(R)-SpFN"), (13) spike protein and ferritin containing two additional RBDs, which may be derived from two different strains of coronavirus ("RR-SpFN"), and (14) fusion protein containing RBD-NTD / RBD / NTD in which a second RBD is inserted into the NTD loop, and ferritin ("R2N-FN"). Linear diagrams of many of these fusion protein constructs are shown in Figures 2-7. The various combinations of RBD, NTD, and SD utilized in the disclosed fusion proteins can be derived from the same strain of coronavirus or different strains of coronavirus, but in multivalent embodiments are derived from at least two different strains.

[0067] Generally, the fusion proteins disclosed herein fall into one of four major design formats: beads-on-a-string (e.g., RN-FN, RRN-FN, RNRN-FN, R-FN, and RR-FN; see Figure 3), domain fusion (R-S1-FN, R2-SD-S2-FN, and (R)-R-SpFN or RR-SpFN; see Figure 4), loop insertion (R2N-FN; see Figure 5), and domain swap (mosaic SpFN; see Figure 6). SpFN formats are also included. Exemplary fusion protein sequences are disclosed in Tables 6 and 7. Exemplary mRNA sequences encoding the exemplary fusion protein sequences of Table 7 are also set forth in Table 7 (SEQ ID NOS: 552-566 and 575-582).

[0068] Negative stain electron microscopy 3D reconstructions of selected nanoparticles are shown in FIGS. 8, 11, and 12. Exemplary nanoparticles and their respective designs are disclosed in Table 3 below.

[0069] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0070] Nanoparticles as disclosed herein (either administered directly or formed following administration of mRNA encoding a component protein) are capable of binding to the human ACE-2 receptor. Additionally or alternatively, the nanoparticles as disclosed herein can bind to bat ACE2 proteins, such as proteins from the Chinese horseshoe bat (R. sinicus 3364), the Chinese horseshoe bat (R. sinicus 1434), the middle horseshoe bat (R. affinis 787), the middle horseshoe bat (R. affinis 9479), the Lander's horseshoe bat (R. landeri) of sub-Saharan Africa (including Kenya), the Alcyone's horseshoe bat (R. alcyone) of West and Central Africa, the relatively large greater horseshoe bat (Rhinolophus ferrumequinum) found in Asia, but also in Europe and North Africa, and the least horseshoe bat (Rhinolophus pusillus). Additionally or alternatively, the nanoparticles as disclosed herein can bind to the human DPP4 receptor.

[0071] The disclosed fusion proteins that self-assemble into the disclosed nanoparticles, including those listed in Table 3 above, and those listed in Tables 6 and 7 below, can be expressed alone or co-expressed (e.g., on two different plasmids) in a suitable expression system, which can include a mammalian or eukaryotic expression system. Some of the fusion proteins disclosed in Tables 6 and 7 can include a histidine tag (i.e., His tag) containing a repeat of 5-10 histidine (H) residues or other tag sequences that may be useful in protein processing or purification, but which can ultimately be cleaved from the active protein prior to nanoparticle assembly. Alternatively, as discussed above and in more detail below, the disclosed fusion proteins that self-assemble into the disclosed nanoparticles, including those listed in Table 3 above, and those listed in Tables 6 and 7 below, can be encoded by mRNA molecules that, when administered and expressed in vivo, result in the formation of nanoparticles as disclosed herein.

[0072] All of the proteins disclosed in Table 6 and some of the proteins disclosed in Table 7 are exemplary nanoparticle-forming proteins capable of forming RBD-ferritin or spike-ferritin nanoparticles. These sequences include a set of alternative sequences to improve the stability and immunogenicity of the RBD-ferritin or spike-ferritin constructs. This includes a stabilizing disulfide bond, a D614G mutation, a mutation to remove the glycan in the spike at N165 to allow the RBD greater freedom of movement and to allow the RBD to exist in a more exposed "upper" conformation, and an N234Q mutation to remove the glycan at 234 in the spike to allow the RBD to exist in a closer conformation. Additionally or alternatively, glycans at N146 or N77 of the ferritin sequence can improve and stabilize the ferritin molecule.

[0073] The "beads-on-a-string" fusion protein format can be used to create nanoparticles containing antigenic components from multiple coronaviruses, such as SARS-CoV-2, SARS-CoV-1, Khosta-2, BANAL-20-247, HKU-1, MERS-CoV, 229E, NL63, OC43, or closely related coronaviruses, including those identified from bats, camels, or pangolins. These embodiments can be utilized to create pan-sarbecovirus vaccines, pan-merbecovirus vaccines, pan-sarbecovirus-merbecovirus vaccines, pan-β-coronavirus vaccines, or pan-coronavirus vaccines. For example, multiple RBD, NTD, or combination "beads" composed of different antigenic sequences can be presented together on a single "string" (i.e., in a single construct) to elicit a broad immune response against coronaviruses. For example, an antigen "string" such as SARS-CoV-2-RBD-SARS-CoV-1-RBD-Khosta-2-RBD-BANAL-20-247-RBD or SARS-CoV-2-RBD-SARS-CoV-1-RBD-HKU-1-RBD-MERS-CoV-RBD-229E-RBD-NL63-RBD could be used with an antigen "string" such as SARS-CoV-2-omicron-BQ.1.1-RBD-SARS-CoV-1-RBD or SARS-CoV-2-RBD-pangolinSARS-CoV-1-RBD-OC43-RBD-camelMERS-CoV-RBD-229E-RBD-NL63-RBD to boost or focus the immune response towards specific pan-reactive or pan-protective immunity. A "beads-on-a-string" can include, for example, sequences for 1 to 10 RBDs, NTDs, or both RBD and NTD domains in tandem, or in other words, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RBDs, NTDs, or both. A linker sequence, including but not limited to, the linker sequences disclosed in Table 1, can connect one or more, or each, of the tandem RBD and / or NTD sequences.

[0074] "Beads on a string" can also be added to SpFN or mos-SpFN molecules that contain an additional 1 to 10 RBDs, NTDs, or both in tandem, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RBDs linked to an SpFN or mos-SpFN molecule. A linker sequence, including but not limited to a linker selected from the linker sequences disclosed in Table 1, can link one or more, or each, of the RBD and / or NTD sequences in tandem.

[0075] The "domain fusion" format of fusion proteins can be used to create nanoparticles containing antigenic components from multiple coronaviruses, such as SARS-CoV-2 and its variants and derivatives, including those of interest (e.g., alpha, beta, delta, omicron), SARS-CoV-1, Khosta-2, BANAL-20-247, HKU-1, MERS-CoV, 229E, NL63, OC43, or closely related coronaviruses, including those identified from bats, camels, or pangolins. These embodiments can be utilized to create pan-β-coronavirus vaccines or pan-coronavirus vaccines. In this format, a heterologous RBD can be added to the N-terminus of a previously described construct. Exemplary constructs can include multiple (i.e., at least two) RBD domains from different strains of coronavirus, along with other antigenic domains, such as the NTD, SD1, SD2, S-2P, or Hexapro domains.

[0076] The "loop insertion" format of the fusion protein can be used to create nanoparticles containing antigenic components from multiple coronaviruses, such as SARS-CoV-2, SARS-CoV-1, HKU-1, MERS-CoV, 229E, NL63, OC43, or closely related coronaviruses, including those identified from bats, camels, or pangolins. These embodiments can be utilized to create pan-β-coronavirus vaccines or pan-coronavirus vaccines. In this format, one or more RBDs are linked to or inserted into the loop domain of the NTD via a linker (e.g., a linker from Table 1). In some embodiments, at least two RBDs will be inserted into either the same loop or different loops of the NTD. The RBDs can be derived from different strains or variants of coronavirus. The NTD can be derived from the same virus strain or variant as one or both of the RBDs, or the NTD can also be derived from a different strain or variant of coronavirus for one or both of the RBDs.

[0077] The "domain insertion" or "mosaic" format of fusion proteins can be used to create nanoparticles containing antigenic components from multiple coronaviruses, such as SARS-CoV-2 and its variants and derivatives, including those of interest (e.g., alpha, beta, delta, omicron), SARS-CoV-1, Khosta-2, BANAL-20-247, HKU-1, MERS-CoV, 229E, NL63, OC43, or other coronaviruses, including those identified from bats, camels, or pangolins. Examples of useful coronaviruses include, but are not limited to, sarbecoviruses (e.g., ZXC21, BANAL-20-247, Rf4092, Shaanxi2011, HeB2013, Rp3, Rs_672, HKU3-1, Rs4081, RmYN02, Rf1, Yun11, BM48-31, BB9904, Khosta-1, Khosta-2, RhGB01, BtKY72, RsYN04, RatG 15 (Ra7909), SHC014, WIV1, LyRa3, Rs4084, Rs4231, BANAL-20-103, RaTG13, BANAL-20-52, Pang17 (GX-P5L), or RshSTT182 / 200), Merbecovirus (e.g., MER1 (ErinaceusCoV / 2012-174 / GER / 2012), MER2 (Neoromicia / 5038), MER3 (HKU4 SM3A), MER4 (BatCoV-Ita2 206645-63), MER5 (BatCoV-Ita1 206645-40), or HKU5), or a combination thereof. These embodiments can be utilized to generate a pan-β-coronavirus vaccine or a pan-coronavirus vaccine. In this format, the spike protein or a segment thereof is linked to a ferritin peptide, and one or more heterologous domains (e.g., the RBD, NTD, or any combination thereof) are substituted for the native domains or added as additional domains. For example, a heterologous RBD from one virus strain can substitute for the native RBD of a given spike protein, forming a "mosaic."Additionally or alternatively, the RBD of a heterologous viral species or strain can substitute for the native NTD of the spike protein to form a "mosaic." Additionally or alternatively, one or more RBDs of a heterologous viral strain can be added to one end (i.e., the C-terminus or N-terminus) of the native spike protein to form a mosaic. Multiple constructs can be combined together in a single nanoparticle by coexpression to generate stable protein nanoparticles, where the spike trimer on the surface of the nanoparticle can be a heterologous mixture, e.g., a promoter derived from the WA-1 strain, a promoter derived from BA.4 / 5, and a promoter derived from beta. These heterologous nanoparticles can also be encoded as mRNA constructs, in which case mRNA molecules encoding different spike-ferritin molecules can be encapsulated in a single lipid nanoparticle to facilitate heterologous nanoparticle formation in the vaccinated individual. Heterogeneous nanoparticles could also be encoded within a single construct, with an exemplary cleavage site encoded between a given construct, such as F2A (see, e.g., ncbi.nlm.nih.gov / pmc / articles / PMC4622431 / ).

[0078] In some embodiments, an immunogenic composition as described herein comprises antigenic coronavirus peptides (or mRNA molecules encoding them) from two or more coronavirus strains independently selected from Clade 1a, Clade 1b, Clade 2, Clade 3, and Middle East Respiratory Syndrome-associated coronavirus (MERS-CoV). In some embodiments, an immunogenic composition as described herein comprises antigenic coronavirus peptides (or mRNA molecules encoding them) from different coronavirus strains independently selected from WA-1, Beta, Omicron BQ.1.1, and XBB.1.5; viral strains of SARS-CoV-1, BANAL20-247, Khosta2, and MERS-CoV. In some embodiments, the immunogenic compositions as described herein comprise antigenic coronavirus peptides (or mRNA molecules encoding them) selected from the following combinations of viral strains: (i) two or more selected from WA-1, beta, and Omicron BQ1.1 or XBB.1.5; (ii) one or more selected from WA-1, beta, Omicron XBB.1.5, and Omicron BQ1.1, and a viral strain of SARS-CoV-1; (iii) WA-1, beta, Omicron XBB.1.5, and Omicron BQ1. (iv) one or more selected from WA-1, Beta, Omicron XBB.1.5, and Omicron BQ1.1, and one or more selected from SARS-CoV-1 virus strains, BANAL20-247, and Khosta-2, and MERS-CoV; and (v) two or more selected from SARS-CoV-1 virus strains, BANAL20-247, and Khosta-2.

[0079] Fusion proteins can be designed with different antigen presentation formats. Fusion proteins can alternatively contain a single antigenic coronavirus peptide (e.g., spike protein and / or RBD antigen) conjugated to a ferritin moiety, or can contain two or more antigenic coronavirus peptides in tandem conjugated to a nanoparticle-forming protein (e.g., ferritin moiety) (also referred to herein as "multidomain" fusion proteins). For fusion proteins containing multiple antigenic coronavirus peptides, the fusion proteins used in a single immunogenic composition (e.g., to form a single nanoparticle) can have different configurations of antigenic coronavirus peptides (e.g., AB and BA) to provide antigens peripherally and laterally presented on the nanoparticle. In this context, "peripherally" refers to epitopes on fusion proteins that are in close proximity on the nanoparticle, while "laterally" refers to epitopes within the same fusion protein. For example, multidomain fusion proteins (e.g., RBD-RBD or RBD-RBD-Spike, etc.) enable peripheral and lateral antibody binding. See, e.g., Figure 30. Thus, to allow for both peripheral and lateral antibody binding, fusion proteins can be prepared to "pattern" the nanoparticle surface by altering the order of antigens (e.g., RBDs) of the component strains, so that combinations of epitopes are adjacent both laterally and peripherally. For embodiments involving spike proteins, "mosaic" formats can be designed in which the spike proteins are derived from one viral strain and the RBD domains or additional RBD domains of the spike proteins are derived from different coronaviruses (mosSp, RmosSp).As an example, consider a fusion protein where the series of antigenic peptides are RBD-RBD-spike (where spike includes the RBD domain) obtained from different strains of sarbecovirus (A, B, and C, respectively, with spikes from the C strain); additional fusion proteins could be RBD-RBD-mosspike where the RBDs are (B, C, and A, respectively, with spikes from the C strain) and (C, A, and B, respectively, with spikes from the C strain), thereby allowing an antigenic domain from each virus strain to be presented at each possible location in the fusion protein.

[0080] To pursue this approach, fusion proteins were designed according to the following antigen conjugation framework ("antigen presentation") across a range of antigenic distance sets to provide multivalent nanoparticles that present antigens at selected antigenic distances to allow common and conserved epitopes to be optimally recognized and cross-linked by B cell receptors. Specifically, we generated formulations containing up to five distinct mRNA constructs (Figure 16, panel A and Table 8 below), each encoding a component strain from an antigenic distance set corresponding to the antigen presentation formats (RFN, SpFN, RRFN, RR-SpFN) as shown in Table 8 and Figures 17 and 29.

[0081] Administration of an immunogenic composition as described herein containing several different mRNA molecules (e.g., encoding different fusion proteins), either co-encapsulated in the same LNP or encapsulated separately in separate LNPs and then administered in the same composition, results in co-expression of each encoded fusion protein (e.g., containing a ferritin-conjugated antigen). Ferritin-conjugated antigens produced in the same cell self-assemble into ferritin nanoparticles that display the expressed fusion proteins on the same nanoparticle, resulting in what are referred to herein as "mosaic nanoparticles" or "multivalent nanoparticles."

[0082] For the purposes of the present disclosure, seven virus strains with different levels of antigenic (i.e., sequence) distance were selected to span the antigenic space of sarbecoviruses. The virus strains included three antigenically distinct SARS-CoV-2 (clade 1b) strains: parental WA-1, Beta, Omicron XBB.1.5, and Omicron BQ.1.1; a SARS-CoV-1 (clade 1a) strain (Frankfurt); two increasingly distant bat zoonotic coronaviruses (clades 2 and 3): BANAL20-247 and Khosta2, respectively; and an outlier Merbecovirus (MERS-CoV) strain representing a non-ACE2-binding strain (Figure 16, panel A). From this set of virus strains, we designed the following sets of virus strains based on the antigenic distance paradigm to be selected: low: parental WA-1, Beta, Omicron BQ.1.1 (Mix A); medium: parental WA-1, Omicron BQ.1.1, SARS-CoV-1 (Mix B); medium-high: parental WA-1, SARS-CoV-1, Khosta-2 (Mix C); medium-high: parental WA-1, SARS-CoV-1, BANAL20-247 (Mix D); high: parental WA-1, SARS-CoV-1, MERS-CoV (Mix E); high: SARS-CoV-1, Khosta-2, BANAL20-247 (Mix F).

[0083] As described in the Examples, mRNA molecules encoding antigenic coronavirus peptides (e.g., spike and / or RBD antigens) (with or without ferritin moieties) from virus strains in these antigenic distance sets were constructed to obtain immunogenic compositions that provide multivalent presentation of antigens reflecting various antigenic distance paradigms. Thus, in some embodiments, the immunogenic compositions as described herein comprise antigenic coronavirus peptides (or mRNA molecules encoding them) selected from the following combinations of virus strains: (i) WA-1, beta, and omicron BQ.1.1 (or XBB.1.5), where the antigenic coronavirus peptide is the RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(beta)FN, and R(BQ1.1)FN, or the spike antigen contained in S-2P (e.g., S(WA-1)-2P, S(beta)-2P, and S(BQ1.1)-2P) or the SpFN fusion proteins Sp(WA-1)FN, Sp(beta)FN, and Sp(BQ1.1)FN, or the RmosSpFN or RRmosSpFN fusion proteins (ii) WA-1, Omicron BQ.1.1 (or XBB1.5), and SARS-CoV-1, wherein the antigenic coronavirus peptide is an RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(BQ1.1)FN, and R(SARS-CoV-1)FN, or a spike antigen contained in S-2P (e.g., S(WA-1)-2P, S(BQ1.1)-2P, and S(SARS-CoV-1)-2P) or the SpFN fusion proteins Sp(WA-1)FN, Sp(BQ1.1)FN, and Sp(SARS-CoV-1)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion protein, or any combination thereof;(iii) WA-1, SARS-CoV-1, and Khosta2, where the antigenic coronavirus peptide is an RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(SARS-CoV-1)FN, and R(Khosta2)FN, or an S-2P (e.g., S(WA-1)-2P, S(SARS-CoV-1)-2P, and S(Khosta2)-2P) or an SpFN fusion protein Sp(WA-1)FN, Sp(SARS-CoV-1)FN, and Sp(Khosta2)FN. (iv) WA-1, SARS-CoV-1, and BANAL20-247, where the antigenic coronavirus peptide is the RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(SARS-CoV-1)FN, and R(BANAL20-247)FN, or S-2P (e.g., S(WA-1)-2P, S(SARS-CoV-1)-2P, and (v) WA-1, SARS-CoV-1, and MERS-CoV, wherein the antigenic coronavirus peptide is a spike antigen contained in the RFN fusion proteins R(WA-1)FN, R(SARS-CoV-1)FN, and R(SARS-CoV-2)FN, or a mosaic antigen contained in the RFN fusion proteins R(WA-1)FN, R(SARS-CoV-2)FN, and R(SARS-CoV-2)FN, or a combination of any of them; (vi) WA-1, SARS-CoV-1, and MERS-CoV, wherein the antigenic coronavirus peptide is a spike antigen contained in the RFN fusion proteins R(WA-1)FN, R(SARS-CoV-2)FN, and R(SARS-CoV-2)FN, or a mosaic antigen contained in the RFN fusion proteins R(WA-1)FN, R(SARS-CoV-2)FN, and R(SARS-CoV-2)FN, or a combination of any of them; - the RBD antigen contained in R(MERS-CoV)FN, and R(MERS-CoV)FN, or the spike antigen contained in S-2P (e.g., S(WA-1)-2P, S(SARS-CoV-1)-2P, and S(MERS-CoV)-2P) or the SpFN fusion proteins Sp(WA-1)FN, Sp(SARS-CoV-1)FN, and Sp(MERS-CoV)FN, or the mosaic antigen contained in RmosSpFN or RRmosSpFN fusion proteins, or any combination thereof;and (vi) SARS-CoV-1, Khosta-2, and BANAL20-247, wherein the antigenic coronavirus peptide is the RBD antigen contained in the RFN fusion proteins R(SARS-CoV-1)FN, R(Khosta2)FN, and R(BANAL20-247)FN, or S-2P (e.g., S(SARS-CoV-1)-2P, S(Khosta2)-2P, and S(BANAL20-247)-2P) or the SpFN fusion proteins Sp(SARS-CoV-1)FN, Sp(Khosta2)FN, and (vii) beta, omicron BQ.1.1 (or XBB1.5), and SARS-CoV-1, wherein the antigenic coronavirus peptide is the RBD antigen contained in the RFN fusion proteins R(beta)FN, R(BQ1.1)FN, and R(SARS-CoV-1)FN, or S-2P (e.g., S(beta)FN). (viii) beta, omicron XBB.1.5, and SARS-CoV-1, wherein the antigenic coronavirus peptide is a spike antigen contained in the RFN fusion protein R(beta)FN, Sp(BQ1.1)FN, and Sp(SARS-CoV-1)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion protein, or any combination thereof; ... the RBD antigen contained in S-2P (e.g., S(beta)-2P, S(XBB1.5)-2P, and S(SARS-CoV-1)-2P) or the SpFN fusion proteins Sp(beta)FN, Sp(XBB1.5)FN, and Sp(SARS-CoV-1)FN, or the mosaic antigen contained in RmosSpFN or RRmosSpFN fusion proteins, or any combination thereof;(ix) Omicron BQ.1.1, SARS-CoV-1, and Khosta2, wherein the antigenic coronavirus peptide is an RBD antigen contained in the RFN fusion proteins R(Omicron BQ.1.1)FN, R(SARS-CoV-1)FN, and R(Khosta2)FN, or a spike antigen contained in S-2P (e.g., S(Omicron BQ.1.1)-2P, S(SARS-CoV-1)-2P, and S(Khosta2)-2P) or SpFN fusion proteins Sp(Omicron BQ.1.1)FN, Sp(SARS-CoV-1)FN, and Sp(Khosta2)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion protein, or any combination thereof;

[0084] Any of the fusion proteins, nanoparticles, mRNA molecules, and immunogenic compositions (e.g., vaccines) disclosed herein can be used to treat or prevent coronavirus infection. The optimal dose and route of administration may vary depending on the nature of the immunogenic composition (e.g., mRNA or nanoparticles), the virus being treated, and the subject being treated.

[0085] F. Antigen-Encoding Nucleic Acids and Nanoparticles While the above discussion has focused on antigens, fusion proteins, and nanoparticles, it should be understood that the present disclosure encompasses mRNA molecules encoding antigenic coronavirus antigens as described herein, and immunogenic compositions comprising one or more mRNA molecules encoding antigenic coronavirus antigens as described herein, where the mRNA molecules may be encapsulated or co-encapsulated in lipid nanoparticles (LNPs), as described in more detail below. Accordingly, additionally disclosed herein are nucleic acid-based vaccines (e.g., mRNA vaccines), priming agents (i.e., vaccine primers), and boosters that can be used to treat or prevent coronavirus infections, such as COVID-19 caused by SARS-CoV-2, or to treat or prevent SARS-CoV-1 infections. For example, the disclosed nucleic acids can include DNA or mRNA encoding any antigenic coronavirus peptide or fusion protein (i.e., a fusion protein comprising a nanoparticle-forming peptide and an antigenic coronavirus peptide, optionally linked by a linker), as described herein. The antigenic coronavirus peptides encoded by the nucleic acid can include one or more fragments or full-length proteins derived from a coronavirus (e.g., SARS-CoV-2 or SARS-CoV-1), such as the S protein and, in particular, the RBD of the S protein, or any antigenic coronavirus peptide as described herein.

[0086] Although the discussion herein focuses on mRNA, it should be understood that the nucleic acids of the present disclosure can be RNA (including mRNA) or DNA. The nucleic acids of the present disclosure can be single-stranded or double-stranded. In certain embodiments, the nucleic acid is RNA, e.g., mRNA. According to some aspects, the present disclosure provides an mRNA molecule comprising or consisting of a sequence selected from any one of SEQ ID NOs: 552-582, or a sequence at least 80% homologous thereto. In some embodiments, the mRNA molecule has a sequence comprising or consisting of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity to any one of SEQ ID NOs: 552-582. In some embodiments, the mRNA molecule has a sequence comprising any one of SEQ ID NOs: 552-582. In some embodiments, the mRNA molecule has a sequence consisting of any one of SEQ ID NOs: 552-582.

[0087] i. DNA vaccines, primers, and boosters DNA encoding a fusion protein as disclosed herein or an antigenic coronavirus peptide as disclosed herein (e.g., a coronavirus S protein or a fragment or variant thereof) can be used as a vaccine, either as a primer that can be administered prior to administration of a nanoparticle or mRNA vaccine as disclosed herein, or as a booster after administration of a nanoparticle or mRNA vaccine as disclosed herein. For example, the DNA can encode all, a fragment, or a variant of the RBD (or other antigenic peptide) of a coronavirus S protein (e.g., the S protein of SARS-CoV-2 or SARS-CoV-1). The DNA can be incorporated into a plasmid that can include components (e.g., a promoter) necessary for expression of the DNA in vivo after administration to a subject, and the plasmid can be operably structured for expression in a mammal, such as a human.

[0088] (1) Vector In one aspect, a vector comprising the nucleic acid disclosed herein is disclosed herein. In some embodiments, the mRNA as described herein can be cloned into a vector. Vectors include, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors also include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription (IVT). In certain embodiments, vectors can be used to express mRNA in host cells.In various embodiments, vectors can be used as templates for IVT.The construction of optimally translated IVT mRNA suitable for therapeutic use is disclosed in detail in Sahin, et al. (2014). Nat. Rev. Drug Discov. 13, 759-780;Weissman (2015). Expert Rev. Vaccines 14, 265-281.

[0089] In some embodiments, the vectors disclosed herein can comprise, from 5' to 3', at least the following sequences: an RNA polymerase promoter; a polynucleotide sequence encoding a 5' UTR; a polynucleotide sequence encoding an ORF; a polynucleotide sequence encoding a 3' UTR; and a polynucleotide sequence encoding at least one RNA aptamer. In some embodiments, the vectors disclosed herein can comprise a polynucleotide sequence encoding a poly(A) sequence and / or a polyadenylation signal. Various RNA polymerase promoters are known. In some embodiments, the promoter may be a T7 RNA polymerase promoter. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3, and SP6 promoters are known.

[0090] Also disclosed herein are host cells (e.g., mammalian cells, e.g., human cells) comprising the vectors or nucleic acids disclosed herein. A "host cell" includes an individual cell or cell culture that can be or has been a recipient of exogenous nucleic acid. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and / or variation. A host cell includes cells transfected or infected in vivo or in vitro with a nucleic acid or vector disclosed herein.

[0091] Vectors can be introduced into target cells using any of a number of different methods, including, but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM830(BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, biolistic particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. (2001). Hum Gene Ther. 12(8):861-70, or TransIT-RNA transfection kit (Mirus, Madison, Wis.)).

[0092] Chemical means for introducing vectors into host cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose the cells to the inhibitors of the present disclosure, various assays can be performed to confirm the presence of the mRNA sequences in the host cells.

[0093] ii. mRNA vaccines In some embodiments, the nucleic acid of the present disclosure is messenger RNA (mRNA). The mRNA can be modified or unmodified. The mRNA can include one or more coding and non-coding regions. The coding region is alternatively referred to as an open reading frame (ORF). The non-coding regions in the mRNA include the 5' cap, 5' untranslated region (UTR), 3' UTR, and poly A tail. The mRNA can be purified from natural sources, produced using a recombinant expression system (e.g., in vitro transcription), and optionally purified, or chemically synthesized. In certain embodiments, the mRNA comprises an ORF encoding an antigen of interest. In certain embodiments, the RNA (e.g., mRNA) further comprises at least one of a 5' UTR, a 3' UTR, a poly(A) tail, and / or a 5' cap.

[0094] An mRNA vaccine can be prepared by preparing an mRNA molecule encoding any one or more of the antigenic coronavirus peptides or fusion proteins disclosed herein. As a result of the self-assembling nature of the disclosed nanoparticles, expression of such mRNA after administration to a subject results in the formation of nanoparticles in vivo, which can elicit an immunogenic response from the subject, thereby causing the subject to produce coronavirus-specific antibodies. Thus, the present disclosure provides mRNAs that can be used as vaccines, encoding any one or more of the antigenic coronavirus peptides or fusion proteins disclosed herein. An mRNA as disclosed herein can encode any of the proteins listed in Table 6 or Table 7. The mRNA can be linear or circular.

[0095] In some embodiments, an immunogenic composition as described herein comprises one or more mRNA molecules encoding at least two antigenic coronavirus peptides from different viral strains, wherein the one or more mRNA molecules may be encapsulated or co-encapsulated in one or more lipid nanoparticles (LNPs). In some embodiments, a composition comprises one mRNA molecule encoding a fusion protein comprising at least two antigenic coronavirus peptides from different viral strains, wherein the fusion protein may further comprise a nanoparticle-forming peptide, and wherein the mRNA molecule is encapsulated in a lipid nanoparticle (LNP). In some embodiments, a composition comprises two or more mRNA molecules, each encoding at least one antigenic coronavirus peptide, in a fusion protein optionally comprising a nanoparticle-forming peptide, wherein each mRNA molecule is encapsulated in a separate lipid nanoparticle (LNP). In some embodiments, the composition comprises two or more mRNA molecules, each encoding at least one antigenic coronavirus peptide, in a fusion protein optionally comprising a nanoparticle-forming peptide, wherein the two or more mRNA molecules are co-encapsulated in the same lipid nanoparticle (LNP).

[0096] In any of the mRNA embodiments, the mRNA molecule can have one or more features selected from a 5' untranslated region (5'UTR); a 3' untranslated region (3'UTR); a polyadenylation (poly(A)) sequence; and chemical modifications, which may include N1-methylpseudouridine. Additionally or alternatively, the mRNA can be a self-replicating mRNA or a non-replicating mRNA. In one aspect, the present disclosure provides a composition comprising a nucleic acid as described herein, comprising a nucleotide sequence (e.g., mRNA) encoding an antigen as described herein (e.g., spike protein, RBD domain, and combinations thereof, etc.) or a fusion protein as described herein (e.g., RFN, RRFN, SpFN, mosSpFN, R-SpFN, R-mosSpFN, RR-SpFN, RR-mosSpFN, etc.).

[0097] Accordingly, the present disclosure provides compositions comprising one, two, three, or four or more nucleic acids (e.g., mRNA molecules) comprising nucleotide sequences encoding an antigenic coronavirus peptide as described herein (e.g., a sarbecovirus spike antigen or fragment thereof as described herein), optionally in a fusion protein with a nanoparticle-forming protein such as a ferritin portion. A single mRNA molecule can encode two or more antigenic coronavirus peptides as described herein, optionally in a fusion protein with a nanoparticle-forming protein such as a ferritin portion. Alternatively, a single mRNA molecule can encode only one antigenic coronavirus peptide as described herein, optionally in a fusion protein with a nanoparticle-forming protein such as a ferritin portion. Contemplated herein are embodiments in which all component antigens are encoded by different mRNA molecules, embodiments in which two or more or all of the component antigens are encoded by the same mRNA molecule, and all rearrangements and combinations thereof.

[0098] An immunogenic composition as disclosed herein can include a single type of mRNA molecule (e.g., mRNA molecules encoding the same antigenic coronavirus peptide) or a combination of mRNA molecules (e.g., mRNA molecules encoding different coronavirus peptides) formulated in the same composition. Additionally or alternatively, a composition as described herein can include an mRNA molecule encoding only one antigenic coronavirus peptide, optionally for use in combination with another such monovalent composition or for use in combination with a multivalent composition as described herein (e.g., a combination of compositions for simultaneous, separate, or sequential administration). Embodiments contemplated herein include those in which two or more or all of the mRNA molecules are formulated in the same composition (which may be encapsulated or co-encapsulated in the same or separate LNPs) and those in which two or more or all of the mRNA molecules are formulated in separate compositions. In some embodiments, all of the mRNA molecules are formulated in the same composition (which may be encapsulated in separate LNPs or co-encapsulated in the same LNPs). In other embodiments, each mRNA molecule is formulated in a separate composition. In some embodiments, two or more mRNA molecules are formulated in one composition (which may be encapsulated in separate LNPs or co-encapsulated in the same LNP), and one or more additional mRNA molecules are formulated in a second composition (which may be encapsulated in separate LNPs or co-encapsulated in the same LNP). In some embodiments, one, two, three, four or more mRNA molecules as described herein are formulated in one composition, or in two, three, four or more compositions, each having any combination or subcombination thereof.

[0099] The mRNA compositions as described herein may also include one or more additional components, such as one or more small molecule immunostimulants (eg, TLR agonists). The mRNA compositions as described herein may comprise a delivery system for nucleic acids (e.g., mRNA), such as liposomes, oil-in-water emulsions, or microparticles. In some embodiments, in compositions as described herein comprising mRNA, the mRNA is encapsulated in lipid nanoparticles (LNPs), such as in an LNP formulation.

[0100] (1) 5' cap The mRNA 5' cap provides resistance to nucleases found in most eukaryotic cells and can enhance translation efficiency. Several types of 5' caps are known: 7-methylguanosine cap ("m 7 Cap-0 (also referred to as "Cap-G" or "Cap-0") contains a guanosine linked to the first transcribed nucleotide via a 5'-5'-triphosphate bond.

[0101] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphate groups; subsequently, guanosine triphosphate (GTP) is added to the terminal phosphate group via a guanylyltransferase, resulting in a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp, (5'(A,G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in U.S. Patent Application Publication Nos. 2016 / 0032356 and 2018 / 0125989, which are incorporated herein by reference.

[0102] 5'-capping of polynucleotides can be achieved concomitantly during in vitro transcription reactions using the following chemical RNA cap analogs to generate a 5'-guanosine cap structure according to the manufacturer's protocol: 3'-O-Me-m7G(5')ppp(5')G (ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G; m7G(5')ppp(5')(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; m7G(5')ppp(5')(2'OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies). 5'-capping of modified RNAs can be achieved post-transcriptionally using vaccinia virus capping enzyme to generate the Cap 0 structure: m7G(5')ppp(5')G. Cap 1 structures can be generated using both vaccinia virus capping enzyme and 2'-O-methyltransferase to generate m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structures can be generated from the Cap 1 structure, followed by 2'-O-methylation of the 5'-antepenultimate nucleotide using 2'-O-methyltransferase. Cap 3 structures can be generated from the Cap 2 structure, followed by 2'-O-methylation of the 5'-preantepenultimate nucleotide using 2'-O-methyltransferase. In certain embodiments, an mRNA of the disclosure comprises a 5' cap selected from the group consisting of 3'-O-Me-m7G(5')ppp(5')G (ARCA cap), G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7G(5')ppp(5')(2'OMeG)pG. In certain embodiments, the mRNA of the present disclosure comprises the following 5' cap:

[0103] [ka] (2) Untranslated region (UTR) In some embodiments, mRNAs of the present disclosure include 5' and / or 3' untranslated regions (UTRs). In an mRNA, the 5' UTR begins at the transcription initiation site and continues up to, but not including, the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal.

[0104] In some embodiments, the mRNAs disclosed herein can comprise a 5' UTR that includes one or more elements that affect mRNA stability or translation. In some embodiments, the 5' UTR can be about 10 to 5,000 nucleotides in length. In some embodiments, the 5' UTR can be about 50 to 500 nucleotides in length. In some embodiments, the 5' UTR can be at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, or about 600 nucleotides in length. The length is 50 nucleotides, about 700 nucleotides, about 750 nucleotides, about 800 nucleotides, about 850 nucleotides, about 900 nucleotides, about 950 nucleotides, about 1,000 nucleotides, about 1,500 nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about 3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides, about 4,500 nucleotides, or about 5,000 nucleotides.

[0105] In some embodiments, the mRNAs disclosed herein can include a 3' UTR that includes one or more of a polyadenylation signal, binding sites for proteins that affect the stability of mRNA localization in a cell, or one or more binding sites for an miRNA. In some embodiments, the 3' UTR can be about 50 to 5,000 or more nucleotides in length. In some embodiments, the 3' UTR can be about 50 to 1,000 or more nucleotides in length. In some embodiments, the 3'UTR is at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 nucleotides in length.

[0106] In some embodiments, the mRNAs disclosed herein can include a 5' or 3' UTR that is derived from a gene other than that encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0107] In certain embodiments, the 5' and / or 3' UTR sequences can be derived from stable mRNAs (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to increase mRNA stability. For example, the 5' UTR sequence can include a subsequence of the CMV immediate early 1 (IE1) gene, or a fragment thereof, to improve nuclease resistance and / or improve the half-life of the mRNA. Inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof, in the 3' end or untranslated region of the mRNA is also contemplated. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the mRNA compared to their unmodified counterparts, including modifications made to, for example, improve the resistance of such mRNA to in vivo nuclease digestion. Exemplary 5'UTRs include sequences derived from the CMV immediate early 1 (IE1) gene (U.S. Patent Application Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated by reference herein), or the sequence GGGAUCCUACC (SEQ ID NO: 25) (U.S. Patent Application Publication No. 2016 / 0151409, incorporated by reference herein).

[0108] In various embodiments, the 5'UTR can be derived from the 5'UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5'-terminal oligopyrimidine (TOP) tract. Furthermore, the majority of TOP genes are characterized by growth-associated translational regulation. However, TOP genes with tissue-specific translational regulation are also known. In certain embodiments, the 5'UTR derived from the 5'UTR of a TOP gene lacks the 5'TOP motif (oligopyrimidine tract) (e.g., U.S. Patent Application Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0109] In certain embodiments, the 5'UTR is derived from the ribosomal protein Large 32 (L32) gene (US Patent Application Publication No. 2017 / 0029847, supra). In certain embodiments, the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (U.S. Patent Application Publication No. 2016 / 0166710, supra). In certain embodiments, the 5'UTR is derived from the 5'UTR of the ATP5A1 gene (US Patent Application Publication No. 2016 / 0166710, supra). In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5'UTR.

[0110] In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO:26 and reproduced below: GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO: 26). In some embodiments, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO:27 and reproduced below: CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 27). The 5'UTR and 3'UTR are described in further detail in WO 2012 / 075040, which is incorporated herein by reference.

[0111] (3) Polyadenylation tail As used herein, the terms "poly(A) sequence," "poly(A) tail," and "poly(A) region" refer to a sequence of adenosine nucleotides at the 3' end of an mRNA molecule. A poly(A) tail can confer stability to an mRNA and protect it from exonuclease degradation. A poly(A) tail can enhance translation. In some embodiments, a poly(A) tail is essentially homopolymeric. For example, a poly(A) tail of 100 adenosine nucleotides can have a length of essentially 100 nucleotides. In certain embodiments, a poly(A) tail can be interrupted by at least one nucleotide other than adenosine nucleotides (e.g., a nucleotide that is not adenosine nucleotides). For example, a poly(A) tail of 100 adenosine nucleotides can have a length of 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide, or a stretch of nucleotides other than adenosine nucleotides). In certain embodiments, the poly(A) tail comprises the sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 28).

[0112] As used herein, a "poly(A) tail" typically relates to RNA. However, in the context of the present disclosure, the term also relates to corresponding sequences in DNA molecules (e.g., "poly(T) sequences"). The poly(A) tail can comprise from about 10 to about 500 adenosine nucleotides, from about 10 to about 200 adenosine nucleotides, from about 40 to about 200 adenosine nucleotides, or from about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail can be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides. In some embodiments, where the nucleic acid is RNA, the poly(A) tail of the nucleic acid is obtained from a DNA template during in vitro transcription of the RNA. In certain embodiments, the poly(A) tail is obtained in vitro by common methods of chemical synthesis without being transcribed from a DNA template. In various embodiments, the poly(A) tail is generated by enzymatic polyadenylation of the RNA (after in vitro transcription of the RNA) using a commercially available polyadenylation kit and corresponding protocol, or alternatively, by using an immobilized poly(A) polymerase, for example, using the methods and means described in WO 2016 / 174271.

[0113] The nucleic acid can include a poly(A) tail obtained by enzymatic polyadenylation, wherein the majority of the nucleic acid molecule includes about 100 (±20) to about 500 (±50) or about 250 (±20) adenosine nucleotides. In some embodiments, the nucleic acid can include a poly(A) tail derived from the template DNA and can additionally include at least one additional poly(A) tail generated by enzymatic polyadenylation, e.g., as described in WO 2016 / 091391. In certain embodiments, the nucleic acid comprises at least one polyadenylation signal. In various embodiments, the nucleic acid can include at least one poly(C) sequence. The term "poly(C) sequence," as used herein, is intended to refer to a sequence of cytosine nucleotides of up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence contains about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence contains about 30 cytosine nucleotides.

[0114] (4) Chemical modification The mRNA disclosed herein can be modified or unmodified. Typically, the mRNA contains at least one chemical modification. In some embodiments, the mRNA disclosed herein can contain one or more modifications that typically enhance RNA stability. Exemplary modifications include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including, but not limited to, purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNAs may be selected from a wide variety of nucleotides, including, for example, 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5 -carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, phosphoramidate, phosphorothioate, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and modified nucleotide analogs or derivatives of purines and pyrimidines such as inosine.

[0115] In some embodiments, the disclosed mRNAs can contain at least one chemical modification, including, but not limited to, pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0116] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. In some embodiments, the chemical modification comprises N1-methylpseudouridine. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0117] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified. The preparation of such analogs is described, for example, in U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642.

[0118] (5)mRNA synthesis The mRNA disclosed herein can be synthesized according to any of a variety of methods. For example, mRNA according to the present disclosure can be synthesized via in vitro transcription (IVT). Some methods for in vitro transcription are described, for example, in Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) Methods Enzymol. 530:101-14. Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that can include DTT and magnesium ions, an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions may vary depending on the specific application. The presence of these reagents is generally undesirable in the final mRNA product, and these reagents can be considered impurities or contaminants that can be purified or removed to provide clean and / or homogeneous mRNA suitable for therapeutic use. While mRNA provided from an in vitro transcription reaction may be desirable in some embodiments, other sources of mRNA can be used in accordance with the present disclosure, including wild-type mRNA produced from bacteria, fungi, plants, and / or animals.

[0119] iii. Other RNA vaccines (1) Self-replicating RNA, trans-replicating RNA and non-replicating RNA Typically, the nucleic acid molecules described herein are non-replicating RNA, however, the nucleic acid molecules described herein may alternatively be self-replicating RNA or trans-replicating RNA.

[0120] (2) Self-replicating RNA Self-replicating (or self-amplifying) RNAs can be generated, for example, by using replication elements derived from alphaviruses and replacing structural viral proteins with nucleotide sequences encoding a protein of interest (e.g., a sarbecovirus spike antigen). Self-replicating RNAs are typically positive-strand molecules that can be directly translated after delivery to cells. This translation provides an RNA-dependent RNA polymerase, which subsequently generates both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the generation of multiple daughter RNAs. These daughter RNAs, as well as colinear subgenomic transcripts, can themselves be translated to provide in situ expression of the encoded antigen, or can be transcribed to provide additional transcripts with the same sense as the delivered RNA, which are translated to provide in situ expression of the antigen. The overall result of this round of transcription is a large amplification in the number of introduced replicon RNAs, such that the encoded antigen becomes the major polypeptide product of the cell.

[0121] One suitable system for achieving self-replication in this manner is to use alphavirus-based replicons. These replicons are positive-strand (positive-sense) RNAs that, after delivery to cells, result in the translation of a replicase (or replicase-transcriptase). The replicase is translated into a polyprotein that self-cleaves to provide a replication complex, producing genomic copies of the positive-strand delivered RNA. These negative-strand transcripts can themselves be transcribed to provide additional copies of the positive-strand parent RNA and also to provide subgenomic transcripts encoding antigens. Translation of the subgenomic transcripts thus results in in situ expression of the antigen by the infected cell. Suitable alphavirus replicons can use replicases from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc. Mutant or wild-type viral sequences can be used, for example, the attenuated TC83 mutant of VEEV has been used in replicons, see the following reference: WO 2005 / 113782, which is incorporated herein by reference.

[0122] In one embodiment, each self-replicating RNA described herein encodes (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule and (ii) a spike polypeptide antigen as disclosed herein. The polymerase may be, for example, an alphavirus replicase, including one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4. While naturally occurring alphavirus genomes encode structural virion proteins in addition to nonstructural replicase polyproteins, in certain embodiments, the self-replicating RNA molecule does not encode alphavirus structural proteins. Thus, while a self-replicating RNA may result in the production of its own genomic RNA copies in a cell, it does not result in the production of RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecule is unable to persist itself in an infectious form. Alphavirus structural proteins necessary for wild-type virus persistence are absent from the self-replicating RNAs of the present disclosure, and their place is occupied by a gene encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins. Self-replicating RNAs are described in further detail in WO2011005799, which is incorporated herein by reference.

[0123] (3) trans-replicating RNA Trans-replicating (or trans-amplifying) RNAs possess elements similar to the self-replicating RNAs described above. However, with trans-replicating RNAs, two separate RNA molecules are used. A first RNA molecule encodes the RNA replicase described above (e.g., an alphavirus replicase), and a second RNA molecule encodes a protein of interest (e.g., a spike protein described herein). The RNA replicase replicates one or both of the first and second RNA molecules, thereby greatly increasing the copy number of the RNA molecule encoding the protein of interest. Trans-replicating RNAs are described in further detail in International Publication No. WO2017162265, which is incorporated herein by reference.

[0124] (4) Non-replicating RNA Non-replicating (or non-amplifying) RNA is RNA that does not have the ability to replicate itself.

[0125] G.LNP In certain embodiments, the immunogenic composition as described herein comprises lipid nanoparticles (LNPs) encapsulating one or more mRNA molecules as described herein. The mRNA can be encapsulated in the lipid nanoparticles (LNPs) through methodologies known in the art, such as a modified ethanol drop nanoprecipitation process. Briefly, ionizable, structural, helper, and polyethylene glycol lipids can be mixed with the mRNA in a given lipid:mRNA ratio in acetate buffer, pH 5.0. The mixture can be neutralized with Tris-Cl, pH 7.5, sucrose added as a cryoprotectant, sterile filtered, and stored frozen at -70°C until further use. The mRNA and LNP can be as follows: lipid nanoparticles containing RNA, an ionizable lipid, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), a PEGylated lipid, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, and two structural lipids, 1,2-disteroyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol. Those skilled in the art will understand that this is only one exemplary method of formulating mRNA, and that other methods and formulation agents (e.g., other lipids) used in the art may be similarly suitable. Parallel methodologies can be used to implement other embodiments of the mRNA vaccines contemplated herein. In certain embodiments, a composition of the present disclosure (e.g., a composition comprising a nucleic acid of the present disclosure) further comprises a lipid nanoparticle (LNP). In certain embodiments, the nucleic acid of the present disclosure is encapsulated in the LNP. The LNPs of the present disclosure can include four classes of lipids: (i) ionizable lipids (e.g., cationic lipids); (ii) PEGylated lipids; (iii) cholesterol-based lipids, and (iv) helper lipids.

[0126] i. Ionizable lipids The ionizable lipid facilitates mRNA encapsulation and may be a cationic lipid, which provides a positively charged environment at low pH to facilitate efficient encapsulation of negatively charged mRNA drug substances. In some embodiments, the cationic lipid is OF-02:

[0127] [ka] Formula (I) OF-02 is a non-degradable structural analog of OF-Deg-Lin. While OF-Deg-Lin contains a degradable ester linkage connecting the diketopiperazine core and the doubly unsaturated tail, OF-02 contains a non-degradable 1,2-amino alcohol linking the same diketopiperazine core and the doubly unsaturated tail (Fenton et al., Adv Mater. (2016) 28:2939; U.S. Patent No. 10,201,618). The exemplary LNP formulation herein, lipid A, contains OF-2. In some embodiments, the cationic lipid is cKK-E10 (Dong et al., PNAS (2014) 111(11):3955-60; U.S. Patent No. 9,512,073):

[0128] [ka] cKK-E10 Formula (II) An exemplary LNP formulation herein, lipid B, contains cKK-E10. In some embodiments, the cationic lipid is GL-HEPES-E3-E10-DS-3-E18-1(2-(4-(2-((3-(bis((Z)-2-hydroxyoctadec-9-en-1-yl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate), a HEPES-based disulfide cationic lipid comprising a piperazine core having formula III (WO 2022 / 221688):

[0129] [ka] Formula (III) An exemplary LNP formulation herein, Lipid C, contains GL-HEPES-E3-E10-DS-3-E18-1. Lipid C is of the same composition as Lipid A or Lipid B, except for the difference in the cationic lipid. In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-4-E10 (2-(4-(2-((3-(bis(2-hydroxydecyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate), a HEPES-based disulfide cationic lipid comprising a piperazine core having formula IV (WO 2022 / 221688):

[0130] [ka] Formula (IV) An exemplary LNP formulation herein, Lipid D, contains GL-HEPES-E3-E12-DS-4-E10. Lipid D is of the same composition as Lipid A or Lipid B, except for the difference in the cationic lipid. In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-3-E14 (2-(4-(2-((3-(bis(2-hydroxytetradecyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate), a HEPES-based disulfide cationic lipid comprising a piperazine core having formula V (WO 2022 / 221688):

[0131] [ka] Formula (V) An exemplary LNP formulation herein, Lipid E, contains GL-HEPES-E3-E12-DS-3-E14. Lipid E ​​is of the same composition as Lipid A or Lipid B, except for the difference in the cationic lipid. The cationic lipids GL-HEPES-E3-E10-DS-3-E18-1 (III), GL-HEPES-E3-E12-DS-4-E10 (IV), and GL-HEPES-E3-E12-DS-3-E14 (V) can be synthesized according to the general procedure presented in Scheme 1: Scheme 1: General synthetic scheme for lipids of formulae (III), (IV), and (V)

[0132] [ka] In some embodiments, the cationic lipid is MC3, having formula VI:

[0133] [ka] Formula (VI) In some embodiments, the cationic lipid is SM-102 (9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate), having formula VII:

[0134] [ka] Formula (VII) In some embodiments, the cationic lipid is ALC-0315 [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate), having formula VIII:

[0135] [ka] Formula (VIII) In some embodiments, the cationic lipid is cOrn-EE1 having formula IX:

[0136]

change

[0137] [ka] Formula (X) In some embodiments, the cationic lipid is IS-001 having formula XI (European Patent Application No. 23306049.0):

[0138] [ka] Formula (XI)

[0139] In some embodiments, the cationic lipid is biodegradable. In some embodiments, the cationic lipid is non-biodegradable. In some embodiments, the cationic lipid is cleavable. In some embodiments, the cationic lipid is non-cleavable. Cationic lipids are described in further detail in Dong et al. (PNAS. 111(11):3955-60. 2014); Fenton et al. (Adv Mater. 28:2939. 2016); U.S. Patent Nos. 9,512,073; and 10,201,618, each of which is incorporated herein by reference.

[0140] ii. PEGylated lipids PEGylated lipid components provide control over nanoparticle size and stability. The addition of such components can prevent complex aggregation and provide a means for increasing circulation life and delivery of lipid-nucleic acid pharmaceutical compositions to target tissues (Klibanov et al. FEBS Letters 268(1):235-7. 1990). These components can be selected to rapidly exchange from the pharmaceutical composition in vivo (see, for example, U.S. Patent No. 5,885,613).

[0141] Contemplated PEGylated lipids include, but are not limited to, C6-C derivatized ceramides such as N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). 20 (e.g., C8, C 10 , C 12 , C 14 , C 16 , or C 18 PEGylated lipids include polyethylene glycol (PEG) chains up to 5 kDa long covalently attached to lipids having alkyl chains up to 5 kDa long. In some embodiments, the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); or 1,2-distearoyl-rac-glycero-polyethylene glycol (DSG-PEG), PEG-DAG; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; PEG-dialkyoxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

[0142] In certain embodiments, the PEG has a high molecular weight, e.g., 2000-2400 g / mol. In certain embodiments, the PEG is PEG2000 (or PEG-2K). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8 PEG2000, or ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000.

[0143] iii. Cholesterol-based lipids The cholesterol component provides stability to the lipid bilayer structure within the nanoparticle. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S. Patent No. 5,744,335), imidazole cholesterol ester ("ICE"; WO 2011 / 068810), sitosterol (22,23-dihydrostigmasterol), β-sitosterol, sitostanol, fucosterol, stigmasterol (stigmasta-5,22-dien-3-ol), ergosterol; desmosterol (3β-hydroxy-5,24-cholestadiene); lanosterol (8,24-lanostadien-3b-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); dihydrolanosterol (24,25-dihydrolanosterol); Examples of cholesterol-based lipids include immosterol (5α-cholesta-8,24-dien-3β-ol); lathosterol (5α-cholest-7-en-3β-ol); diosgenin ((3β,25R)-spirost-5-en-3-ol); campesterol (campest-5-en-3β-ol); campestanol (5a-campestan-3b-ol); 24-methylenecholesterol (5,24(28)-cholestadien-24-methylene-3β-ol); cholesteryl margarate (cholest-5-en-3β-ylheptadecanoate); cholesteryl oleate; cholesteryl stearate and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP is cholesterol.

[0144] iv. Helper lipids The helper lipid enhances the structural stability of the LNP and aids the LNP in endosomal escape. The helper lipid improves the uptake and release of the mRNA drug payload. In some embodiments, the helper lipid is a zwitterionic lipid with fusogenic properties to enhance the uptake and release of the drug payload. Examples of helper lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (DEPE); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), DMPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-distearoylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0145] Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelin, ceramide, cerebroside, ganglioside, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-steroyl-2-oleoyl-phosphatidylethanolamine (SOPE), or combinations thereof. In certain embodiments, the helper lipid is DOPE. In certain embodiments, the helper lipid is DSPC.

[0146] In various embodiments, the LNPs of the present disclosure comprise (i) a cationic lipid selected from OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE. In other embodiments, the LNPs of the present disclosure comprise (i) SM-102; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DSPC. In yet other embodiments, the LNPs of the disclosure comprise (i) ALC-0315; (ii) ALC-0159; (iii) cholesterol; and (iv) DSPC.

[0147] v. Molar ratio of lipid components The molar ratios of the above components are important for the effectiveness of LNPs in delivering mRNA. The molar ratio of cationic lipid, PEGylated lipid, cholesterol-based lipid, and helper lipid is A:B:C:D, where A+B+C+D=100%. In some embodiments, the molar ratio of cationic lipid to total lipid in the LNP (i.e., A) is 35-55%, e.g., 35-50% (e.g., 38-42%, e.g., 40%, or 45-50%). In some embodiments, the molar ratio of PEGylated lipid component to total lipid in the LNP (i.e., B) is 0.25-2.75% (e.g., 1-2%, e.g., 1.5%). In some embodiments, the molar ratio of cholesterol-based lipid to total lipid (i.e., C) is 20-50% (e.g., 27-30%, e.g., 28.5%, or 38-43%). In some embodiments, the molar ratio of helper lipid to total lipid (i.e., D) is 5-35% (e.g., 28-32%, e.g., 30%, or 8-12%, e.g., 10%). In some embodiments, the (PEGylated lipid + cholesterol) components have the same molar amount as the helper lipid. In some embodiments, the LNPs comprise a molar ratio of cationic lipid to helper lipid that is greater than 1.

[0148] In certain embodiments, the LNPs of the present disclosure comprise cationic lipids at a molar ratio of 35% to 55% or 40% to 50% (e.g., 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55% cationic lipids); 0.25% to 2.75% or 1.00% to 2.00% Polyethylene glycol (PEG)-conjugated (PEGylated) lipids in molar ratios (e.g., PEGylated lipids in molar ratios of 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75%); cholesterol-based lipids in molar ratios of 20% to 50%, 25% to 45%, or 28.5% to 43% (e.g., 20%, cholesterol-based lipids in a molar ratio of 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%, or 50%; and helper lipids in a molar ratio of 5% to 35%, 8% to 30%, or 10% to 30%. helper lipids at a molar ratio of 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%, or 35%, where all of the molar ratios are relative to the total lipid content of the LNP.

[0149] In certain embodiments, the LNP comprises a cationic lipid at a molar ratio of 40%; a PEGylated lipid at a molar ratio of 1.5%; a cholesterol-based lipid at a molar ratio of 28.5%; and a helper lipid at a molar ratio of 30%. In certain embodiments, the LNPs of the present disclosure comprise a cationic lipid at a molar ratio of 45-50%; a PEGylated lipid at a molar ratio of 1.5-1.7%; a cholesterol-based lipid at a molar ratio of 38-43%; and a helper lipid at a molar ratio of 9-10%. In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000). In various embodiments, the cholesterol-based lipid is cholesterol. In some embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE).

[0150] In certain embodiments, the LNPs comprise OF-02 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%. In certain embodiments, the LNP comprises cKK-E10 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%. In certain embodiments, the LNPs comprise GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0151] In certain embodiments, the LNPs comprise GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%. In certain embodiments, the LNPs comprise GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%. In certain embodiments, the LNPs comprise SM-102 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%. In certain embodiments, the LNPs comprise ALC-0315 at a molar ratio of 35% to 55%; ALC-0159 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0152] In certain embodiments, the LNPs comprise OF-02 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is designated herein as "Lipid A." In certain embodiments, the LNP comprises cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is designated herein as "Lipid B." In certain embodiments, the LNPs comprise GL-HEPES-E3-E10-DS-3-E18-1 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is designated herein as "Lipid C." In certain embodiments, the LNPs comprise GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is designated herein as "Lipid D." In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is designated herein as "Lipid E."

[0153] In certain embodiments, the LNPs comprise DLin-MC3-DMA (MC3) at a molar ratio of 50%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 38.5%, and DSPC at a molar ratio of 10%. This LNP formulation is designated herein as "Lipid F." In certain embodiments, the LNPs comprise 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102) at a molar ratio of 50%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 10%; cholesterol at a molar ratio of 38.5%; and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) at a molar ratio of 1.5%.

[0154] In certain embodiments, the LNPs comprise (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 46.3%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 9.4%; cholesterol at a molar ratio of 42.7%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a molar ratio of 1.6%. In certain embodiments, the LNPs comprise (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 47.4%; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 10%; cholesterol at a molar ratio of 40.9%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a molar ratio of 1.7%.

[0155] In certain embodiments, the LNP comprises IM-001 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%. In certain embodiments, the LNP comprises IS-001 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%. In some embodiments, the LNP formulation is as defined in terms of "Lipid A," "Lipid B," or "Lipid D." To calculate the actual amount of each lipid to be incorporated into the LNP formulation, the molar amount of cationic lipid is first determined based on the desired N / P ratio, where N is the number of nitrogen atoms in the cationic lipid, and P is the number of phosphate groups in the target mRNA to be transported by LNP.Then, the molar amount of each of the other lipids is calculated based on the molar amount of cationic lipid and the selected molar ratio.Then, these molar amounts are converted into mass using the molecular weight of each lipid.

[0156] vi. Nucleic acids in LNPs The LNP compositions described herein can include nucleic acids (eg, mRNA) of the present disclosure. LNPs can be multivalent, if desired. In some embodiments, LNPs can carry nucleic acids, such as mRNAs, encoding two or more polypeptides of the present disclosure, such as two, three, four, five, six, seven, or eight polypeptides. For example, LNPs can carry multiple nucleic acids (e.g., mRNAs) of the present disclosure, each encoding a different polypeptide of the present disclosure, or can carry a polycistronic mRNA (e.g., each antigen-encoding sequence is separated by a nucleotide linker encoding a self-cleaving peptide, such as a 2A peptide) that can be translated into two or more polypeptides of the present disclosure. LNPs carrying different nucleic acids (e.g., mRNAs) typically contain (encapsulate) multiple copies of each nucleic acid. For example, LNPs carrying or encapsulating two different nucleic acids typically carry multiple copies of each of the two different nucleic acids. In some embodiments, a single LNP formulation can include multiple types (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of LNPs, each type carrying a different nucleic acid (e.g., mRNA).

[0157] When the nucleic acid is mRNA, the mRNA can be unmodified (i.e., containing only natural ribonucleotides A, U, C, and / or G linked by phosphodiester bonds) or chemically modified (e.g., containing nucleotide analogs such as pseudouridine (e.g., N-1-methylpseudouridine), 2'-fluororibonucleotides, and 2'-methoxyribonucleotides, and / or phosphorothioate linkages). The mRNA molecule can include a 5' cap and a poly-A tail.

[0158] vii. Buffers and Other Components To stabilize the nucleic acid and / or LNP (e.g., to extend the shelf life of a vaccine product), to facilitate administration of the LNP pharmaceutical composition, and / or to enhance in vivo expression of the nucleic acid, the nucleic acid and / or LNP can be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and / or other pharmaceutically acceptable excipients. Examples of such excipients are parabens, thimerosal, thiomersal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA). The LNP compositions of the present disclosure can be provided in a frozen liquid or lyophilized form. A variety of cryoprotectants can be used, including, but not limited to, sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. The cryoprotectant can comprise 5-30% (w / v) of the LNP composition. In some embodiments, the LNP composition contains, for example, 5-30% (e.g., 10%) (w / v) trehalose. Once formulated with a cryoprotectant, the LNP composition can be frozen (or lyophilized and cryopreserved) at -20°C to -80°C. The LNP compositions can be provided to patients in an aqueous buffer solution that is thawed if previously frozen, or reconstituted in an aqueous buffer solution at the clinic if previously lyophilized. The buffer solution is preferably isotonic and suitable for, for example, intramuscular or intradermal injection. In some embodiments, the buffer solution is phosphate buffered saline (PBS).

[0159] viii. Processes for Making the LNP Compositions of the Present Disclosure The LNPs of the present disclosure can be prepared by various techniques currently known in the art. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, such as by dissolving the lipids in a suitable solvent to deposit selected lipids on the inner wall of a suitable container or vessel, and then evaporating the solvent to leave a thin layer inside the vessel, or by spray drying. An aqueous phase can then be added to the vessel while vortexing, resulting in the formation of MLVs. Unilamellar vesicles (ULVs) can then be formed by homogenization, sonication, or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0160] Various methods are described in U.S. Patent Application Publication Nos. 2011 / 0244026, 2016 / 0038432, 2018 / 0153822, 2018 / 0125989, and International Application No. PCT / US2020 / 043223 (filed July 23, 2020) and can be used to practice the present disclosure. One exemplary process involves encapsulating mRNA by mixing with a mixture of lipids without first preforming the lipids into lipid nanoparticles, as described in U.S. Patent Application Publication No. 2016 / 0038432. Another exemplary process involves encapsulating mRNA by mixing preformed LNPs with the mRNA, as described in U.S. Patent Application Publication No. 2018 / 0153822.

[0161] In some embodiments, the process for preparing mRNA-loaded LNPs comprises heating one or more of the solutions to a temperature above ambient temperature, the one or more solutions being a solution containing preformed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing mRNA encapsulated in LNPs. In some embodiments, the process comprises heating one or both of the mRNA solution and the preformed LNP solution prior to the mixing step. In some embodiments, the process comprises heating one or more of the solution containing preformed LNPs, the solution containing mRNA, and the solution containing mRNA encapsulated in LNPs during the mixing step. In some embodiments, the process comprises heating the mRNA encapsulated in LNPs after the mixing step. In some embodiments, the temperature to which one or more of the solutions is heated is about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C or higher. In some embodiments, the temperature to which one or more of the solutions are heated ranges from about 25-70° C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about 50-70° C., or about 60-70° C. In some embodiments, the temperature is about 65° C.

[0162] Various methods can be used to prepare mRNA solutions suitable for the present disclosure. In some embodiments, mRNA can be dissolved directly in a buffer solution as described herein. In some embodiments, mRNA solutions can be prepared by mixing an mRNA stock solution with a buffer solution prior to mixing with a lipid solution for encapsulation. In some embodiments, mRNA solutions can be prepared by mixing an mRNA stock solution with a buffer solution immediately prior to mixing with a lipid solution for encapsulation. In some embodiments, suitable mRNA stock solutions can contain mRNA in water or buffer at a concentration of about 0.2 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, or 5.0 mg / mL or greater.

[0163] In some embodiments, the mRNA stock solution is mixed with the buffer solution using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps. Typically, the buffer solution is mixed at a speed greater than that of the mRNA stock solution. For example, the buffer solution can be mixed at a speed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times greater than that of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate in the range of about 100 to 6000 mL / min (e.g., about 100 to 300 mL / min, 300 to 600 mL / min, 600 to 1200 mL / min, 1200 to 2400 mL / min, 2400 to 3600 mL / min, 3600 to 4800 mL / min, 4800 to 6000 mL / min, or 60 to 420 mL / min). In some embodiments, the buffer solution is mixed at a flow rate of about 60 mL / min, 100 mL / min, 140 mL / min, 180 mL / min, 220 mL / min, 260 mL / min, 300 mL / min, 340 mL / min, 380 mL / min, 420 mL / min, 480 mL / min, 540 mL / min, 600 mL / min, 1200 mL / min, 2400 mL / min, 3600 mL / min, 4800 mL / min, or 6000 mL / min or greater.

[0164] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging from about 10 to 600 mL / min (e.g., about 5 to 50 mL / min, about 10 to 30 mL / min, about 30 to 60 mL / min, about 60 to 120 mL / min, about 120 to 240 mL / min, about 240 to 360 mL / min, about 360 to 480 mL / min, or about 480 to 600 mL / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of about 5 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 25 mL / min, 30 mL / min, 35 mL / min, 40 mL / min, 45 mL / min, 50 mL / min, 60 mL / min, 80 mL / min, 100 mL / min, 200 mL / min, 300 mL / min, 400 mL / min, 500 mL / min, or 600 mL / min or greater.

[0165] The process of incorporating desired mRNA into lipid nanoparticles is referred to as "loading". Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255-8. The nucleic acid incorporated into LNP can be completely or partially located in the lumen of the lipid nanoparticle, be within the bilayer membrane of the lipid nanoparticle, or be associated with the outer surface of the lipid nanoparticle membrane. The incorporation of desired mRNA into lipid nanoparticles is also referred to herein as "encapsulation", where the nucleic acid is completely or substantially contained within the lumen of the lipid nanoparticle.

[0166] Suitable LNP can be produced in various sizes.In some embodiments, the size reduction of lipid nanoparticles is associated with more efficient delivery of mRNA.Selection of suitable LNP size can take into account the location of target cell or tissue and to some extent the application that lipid nanoparticles are produced for. Various methods known in the art are available for sizing lipid nanoparticle populations. A preferred method herein utilizes a Zetasizer NanoZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 μL of LNP sample is mixed with 990 μL of 10% trehalose. This solution is loaded into a cuvette and then placed in the Zetasizer instrument. The z-average diameter (nm), or cumulant average, is considered the average size for the LNPs in the sample. The Zetasizer instrument can also be used to measure the polydispersity index (PDI) by using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. The average LNP diameter can be reduced by sonication of the formed LNPs. Intermittent sonication cycles can be alternated with quasi-elastic light scattering (QELS) evaluation to guide efficient lipid nanoparticle synthesis.

[0167] In some embodiments, the majority of the purified LNPs, i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs, have a size of about 70 to 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all (e.g., greater than 80 or 90%) of the purified lipid nanoparticles have a size of about 70 to 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).

[0168] In some embodiments, the LNPs in the compositions of the present disclosure have an average size of less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm. In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the LNPs in a composition of the present disclosure have a size in the range of about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm), or about 50-70 nm (e.g., 55-65 nm), which are particularly suitable for pulmonary delivery via nebulization.

[0169] In some embodiments, the dispersity, or particle size heterogeneity measure (PDI), of the LNPs in the pharmaceutical compositions provided herein is less than about 0.5. In some embodiments, the LNPs have a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08. The PDI can be measured using a Zetasizer instrument, as described above. In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified LNPs in the pharmaceutical compositions provided herein encapsulate mRNA within each individual particle. In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles in the pharmaceutical composition encapsulate mRNA within each individual particle. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of 50% to 99%; or greater than about 60, 65, 70, 75, 80, 85, 90, 92, 95, 98, or 99%. Typically, lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91, 92, 93, 94, or 95%). In some embodiments, the LNPs have an N / P ratio of 1 to 10. In some embodiments, the lipid nanoparticles have an N / P ratio of 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or greater than about 8. In further embodiments, exemplary LNPs herein have an N / P ratio of 4.

[0170] In some embodiments, pharmaceutical compositions according to the present disclosure contain at least about 0.5 μg, 1 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg of encapsulated mRNA. In some embodiments, pharmaceutical compositions contain between about 0.1 μg and 1000 μg, at least about 0.5 μg, at least about 0.8 μg, at least about 1 μg, at least about 5 μg, at least about 8 μg, at least about 10 μg, at least about 50 μg, at least about 100 μg, at least about 500 μg, or at least about 1000 μg of encapsulated mRNA.

[0171] In some embodiments, mRNA can be produced by chemical synthesis or in vitro transcription (IVT) of a DNA template. An exemplary process for producing and purifying mRNA is described in Example 1. In this process, a cDNA template is used to generate mRNA transcripts during the IVT process, and the DNA template is degraded by DNase. The transcripts are purified by depth filtration and tangential flow filtration (TFF). The purified transcripts are further modified by adding caps and tails, and the modified RNA is again purified by depth filtration and TFF. The mRNA is then prepared in an aqueous buffer and mixed with an amphipathic solution containing the lipid components of the LNP. The amphipathic solution for dissolving the four lipid components of the LNP can be an alcohol solution. In some embodiments, the alcohol is ethanol. The aqueous buffer can be, for example, a citrate, phosphate, acetate, or succinate buffer and can have a pH of about 3.0 to 7.0, e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5. The buffer can contain other components, such as salts (e.g., sodium, potassium, and / or calcium salts). In certain embodiments, the aqueous buffer has 1 mM citrate, 150 mM NaCl, and a pH of 3.5 or 4.5.

[0172] An exemplary, non-limiting process for producing mRNA-LNP compositions is described in Example 1. The process involves mixing a buffered mRNA solution with a solution of lipids in ethanol in a controlled, uniform manner, where the lipid:mRNA ratio is maintained throughout the mixing process. In this illustrative example, the mRNA is presented in an aqueous buffer containing citric acid monohydrate, trisodium citrate, and sodium chloride. The mRNA solution is added to a solution (1 mM citrate buffer, 150 mM NaCl, pH 4.5). A lipid mixture of four types of lipids (e.g., cationic lipids, PEGylated lipids, cholesterol-based lipids, and helper lipids) is dissolved in ethanol. The aqueous mRNA solution and the ethanolic lipid solution are mixed in a 4:1 volumetric ratio in a "T" mixer with a nearly pulseless pumping system. The resulting mixture is then subjected to downstream purification and buffer exchange. Buffer exchange can be achieved using a dialysis cassette or a TFF system. TFF can be used to concentrate and buffer exchange the resulting nascent LNPs immediately after their formation via the T-mix process. The diafiltration process is a continuous operation in which the volume is kept constant by adding an appropriate buffer at the same rate as the filtered product stream.

[0173] H. Nucleic Acid Formulations and Adjuvants The nucleic acid vaccines, primers, and boosters disclosed herein can be formulated for systemic administration via parenteral delivery. Parenteral administration includes intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, or intramuscular injection or infusion. Formulations for parenteral administration include sterile aqueous solutions, which may also contain buffers, diluents, and other pharmaceutically acceptable additives known to those skilled in the art. For intravenous use, the total concentration of solutes can be controlled to make the preparation isotonic. Intravenous, intraarterial, subcutaneous, or intramuscular injection is a preferred route of administration. Additionally or alternatively, the disclosed vaccines can be formulated for intranasal administration or contact with other mucous membranes. The nucleic acid formulation for injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers, with or without added preservatives. The formulation can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. The formulation can contain any suitable pharmaceutically acceptable excipient. Generally, the nucleic acid administered to a subject is formulated in a lipid composition, such as a lipid nanoparticle, as discussed above.

[0174] I. Vaccine Adjuvants The disclosed immunogenic compositions (e.g., comprising a fusion protein, nanoparticle, or mRNA molecule as described herein) can include an adjuvant to improve the immune response and promote a protective response. Adjuvants are components used in some vaccines that help generate a stronger immune response in the person receiving the vaccine. Adjuvants help the body generate a strong enough immune response to protect the person from the disease they are vaccinated against. The present disclosure provides vaccine formulations containing any of the disclosed antigens and / or nanoparticles (or combinations thereof) and at least one adjuvant selected from the group consisting of ALFQ, Alhydrogel, and combinations thereof.

[0175] The adjuvant ALFQ is a U.S. Army liposomal formulation (ALF) developed by the U.S. Army that contains a high amount of cholesterol along with QS21 saponin (ALFQ). ALFQ has been used with various immunogens in numerous animal studies and has shown effectiveness in eliciting robust immune responses. In contrast to some adjuvants, ALFQ tends to elicit a balanced Th1 / Th2 immune response, avoiding the skewed immune response that has been associated with vaccine-associated enhanced respiratory disease (VAERD). In some embodiments, the ALFQ adjuvant is a liposomal formulation containing monophosphoryl lipid A (MPLA) and QS-21 saponin. In some embodiments, the ALFQ liposomes can contain approximately 600 μg / mL monophosphoryl 3-deacyl lipid A (3D-PHAD) and approximately 300 μg / mL QS-21. To make ALFQ, in one exemplary embodiment, 14.7 mL of ALF55 (containing 1.236 mg / mL 3D-PHAD) can be diluted with 6.5 mL of isotonic Sorensen's PBS pH 6.15 in a sterile glass vial and, with slow stirring, 9.08 mL of QS-21 (1 mg / mL) can be added to make diluted ALF55.

[0176] Alhydrogel refers to a range of aluminum hydroxide gel formulations specifically developed for use as adjuvants in human and veterinary vaccines. The gel is a suspension of boehmite-like (aluminum oxyhydroxide) hydrated nano- / micron-sized crystals in loose aggregates. The product has very low conductivity due to the absence of buffering ions. The product is positively charged at neutral pH and effectively adsorbs negatively charged antigens. The primary purpose of adjuvants in vaccines is to enhance antibody-mediated (Th2) immune responses to antigens. Alhydrogel products can be combined with other adjuvant types (e.g., monophosphoryl lipids) to achieve a balanced Th1 / Th2 immune response. For purposes of formulating the disclosed vaccines, the alhydrogel stock can be diluted prior to combination with the disclosed nanoparticles to provide an aluminum concentration of about 500 μg / mL, about 550 μg / mL, about 600 μg / mL, about 650 μg / mL, about 700 μg / mL, about 750 μg / mL, about 800 μg / mL, about 850 μg / mL, about 900 μg / mL, about 950 μg / mL, about 1000 μg / mL, about 1050 μg / mL, about 1100 μg / mL, about 1150 μg / mL, about 1200 μg / mL, about 1250 μg / mL, about 1300 μg / mL, about 1350 μg / mL, about 1400 μg / mL, about 1450 μg / mL, or about 1500 μg / mL, or more.

[0177] Other vaccine adjuvants are known in the art, and based on the results reported herein for ALFQ and Alhydrogel, one skilled in the art will understand that other adjuvants can also be used with the disclosed antigens and nanoparticles to complement their function. Other adjuvants suitable for use with the disclosed antigens and nanoparticles include, but are not limited to, monophosphoryl lipid A (MPLA), oil-in-water emulsions, ADJUPLEX™ (lecithin and carbomer homopolymer), ADDAVAX™ (squalene-based oil-in-water nanoemulsion), CARBOPOL® polymer (a cross-linked polyacrylic acid polymer), Poly IC:LC (carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA synthetic complex), poly I:C (polyinosinic acid:polycytidylic acid), CpG oligonucleotides, flagellin, Iscomatrix (composed of saponin, cholesterol, and dipalmitoylphosphatidylcholine), virosomes, MF59 (a squalene-based oil-in-water emulsion), AS03 (a squalene-based oil-in-water emulsion), and AS04 (alum-absorbed 3-O-desacyl-4'-monophosphoryl lipid A).

[0178] J. Pharmaceutical Compositions Pharmaceutical compositions of the present disclosure include immunogenic compositions (eg, vaccines) comprising nanoparticles or mRNA molecules as disclosed herein. In some embodiments, the pharmaceutical composition will also include an adjuvant (e.g., ALFQ, Alhydrogel, or a combination thereof, or an adjuvant suitable for use with an mRNA vaccine). The nanoparticles or mRNA molecules, alone or in combination with one or more adjuvants, can be formulated into a suitable carrier to form a pharmaceutical composition suitable for the intended route of administration. It should also be understood that an immunogenic composition as described herein can itself be a pharmaceutical composition and can include, for example, an adjuvant and / or a suitable carrier for the intended route of administration. Accordingly, in the following discussion, references to a "pharmaceutical composition" should be understood to encompass embodiments of the immunogenic composition as described herein.

[0179] In some embodiments, the pharmaceutical composition is formulated for systemic administration via parenteral delivery. Parenteral administration includes intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, or intramuscular injection or infusion. Formulations for parenteral administration include sterile aqueous solutions, which may also contain buffers, diluents, and other pharmaceutically acceptable additives known to those skilled in the art. For intravenous use, the total concentration of solutes can be controlled to make the preparation isotonic. Intravenous, intraarterial, subcutaneous, or intramuscular injection is a preferred route of administration. Additionally or alternatively, the disclosed vaccine can be formulated for administration via intranasal administration or contact with another mucous membrane.

[0180] Pharmaceutical compositions for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with or without added preservatives. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. The disclosed vaccines can be formulated using any suitable pharmaceutically acceptable excipient. Pharmaceutical compositions for intranasal administration can take the form of dispersions, suspensions, solutions, or emulsions and can be incorporated into nasal aerosols or nasal sprays. Such compositions can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents, and can be formulated using any suitable pharmaceutically acceptable excipient. Intranasal administration includes administration via the nose, with or without simultaneous inhalation during administration. Such administration typically involves contact of the disclosed vaccines with the nasal mucosa, nasal turbinates, or paranasal sinuses. Administration by inhalation can include intranasal administration or oral inhalation. Such administration can also include contact with the oral mucosa, bronchial mucosa, and other epithelia.

[0181] The disclosed immunogenic compositions can be formulated for administration simultaneously with another therapeutic agent. The immunogenic compositions can be formulated for administration sequentially with another therapeutic agent. For example, the immunogenic compositions can be administered either before or after a subject undergoes an antiviral therapy regimen. Any of the immunogenic and pharmaceutical compositions disclosed herein can be used to treat or prevent a coronavirus infection, such as a SARS-CoV-2 infection (e.g., COVID-19) or a SARS-CoV-1 infection. A pharmaceutical or immunogenic composition for use against a particular coronavirus infection (e.g., SARS-CoV-2) will typically contain an antigenic peptide (or mRNA encoding it) of the target coronavirus (e.g., SARS-CoV-2) but may additionally or alternatively contain an antigenic peptide (or mRNA encoding it) of a closely related coronavirus (e.g., SARS-CoV-1). The optimal dosage and route of administration may vary depending on the nature of the immunogenic composition (e.g., mRNA or nanoparticles), the virus being treated, and the subject being treated.

[0182] IV. Treatment and prevention of coronavirus infections The present disclosure provides methods for treating and preventing coronaviruses, including but not limited to, sarbecovirus and merbecovirus infections, by administering an immunogenic composition (e.g., a vaccine) as described herein, comprising one or more of the nanoparticles or mRNA molecules disclosed herein. The present disclosure also provides uses of the disclosed immunogenic and pharmaceutical compositions for treating or preventing coronavirus infections, such as SARS-CoV-2 infection (e.g., COVID-19), SARS infection, and MERS infection. Following any of the methods and uses disclosed herein, the subject may be at risk of coronavirus infection or may already be infected with a coronavirus. Additionally or alternatively, the subject may not have previously received a vaccine for the prevention of coronavirus infection, or may have previously received a vaccine for the prevention of coronavirus infection. Methods for targeting a specific coronavirus infection (such as SARS-CoV-2) will typically employ immunogenic or pharmaceutical compositions that include antigenic peptides (or mRNA encoding same) of the target coronavirus (e.g., SARS-CoV-2), but may additionally or alternatively include antigenic peptides (or mRNA encoding same) of a closely related coronavirus (such as SARS-CoV-1).

[0183] The disclosed methods include administering to a subject an effective amount of one or more of the immunogenic compositions (e.g., vaccines) or pharmaceutical compositions disclosed herein. Administration can be via intravenous, intraarterial, intramuscular, subcutaneous, or intradermal injection. In some embodiments, the subject may be at risk of exposure to a coronavirus, such as SARS-CoV-2, MERS, or SARS-CoV-1. In some embodiments, the subject may have previously been exposed to a coronavirus, such as SARS-CoV-2, MERS, or SARS-CoV-1. In some embodiments, the subject has not previously been administered a vaccine for the prevention of coronavirus infection. In some embodiments, the subject has previously been administered a vaccine for the prevention of coronavirus infection. In some embodiments, the subject may have an active infection that can be treated as a result of administration. In some embodiments, administration of the vaccine prevents the subject from developing a coronavirus infection. In some embodiments, the methods elicit an immune response in a subject against a coronavirus, wherein the immune response may comprise neutralizing antibodies, and further wherein the neutralizing antibodies may cross-neutralize two or more coronavirus strains, and further wherein the neutralizing antibodies may cross-neutralize one or more coronavirus strains that are not component strains of the immunogenic composition.

[0184] The method can further include administering a priming agent (i.e., a "primer") to the nanoparticle vaccine or immunogenic composition as described herein. The primer can be administered prior to administration of the nanoparticle vaccine (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks or more prior). The primer can include a nucleic acid (i.e., DNA or mRNA) encoding all, a fragment, or a variant of the RBD of a fusion protein or a coronavirus S protein (e.g., the S protein of SARS-CoV-2 or SARS-CoV-1).

[0185] For purposes of the disclosed methods and uses, the treatment and / or prevention of infection by all coronaviruses is specifically contemplated, including the treatment and / or prevention of various strains of SARS-CoV-2. Methods and uses for the treatment and / or prevention of infection by all strains and variants of SARS-CoV-1, SARS-CoV-2, and MERS-CoV, as well as all strains and variants of other coronaviruses disclosed herein, are also contemplated.

[0186] Dosage regimens can be adjusted to provide the optimal desired response (e.g., production of antibodies and / or cytokines against coronavirus). For example, in some embodiments, a single bolus of vaccine (e.g., an immunogenic composition as described herein) can be administered, while in some embodiments, several doses can be administered over time, or the dose can be relatively reduced or increased as indicated by the circumstances. For example, in some embodiments, the disclosed vaccines can be administered once or twice weekly, once or twice monthly, once weekly, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven weeks, once every eight weeks, once every three months, once every four months, once every five months, once every six months, or once yearly. In some embodiments, a subject may receive an initial dose followed by one or more booster doses with a predetermined period of time (e.g., 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, 6, 9, or 12 months) between each dose. In some embodiments, a subject may receive only a single dose. In some embodiments, a subject may receive an initial dose followed by one or more subsequent doses of equal or lower concentration at a period of time after the initial dose, such as 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, or 20 weeks or more, e.g., 24 weeks, 52 weeks, 104 weeks, 260 weeks, or 520 weeks.

[0187] Doses may likewise be adjusted to provide the optimal desired response. For example, in some embodiments, a dose of the disclosed vaccines can include 1 μg to 50 mg of vaccine. Single doses can include about 1 μg, about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, Approximately 70μg, approximately 75μg, approximately 80μg, approximately 85μg, approximately 90μg, approximately 95μg, approximately 100μg, approximately 125μg, approximately 150μg, approximately 175μg, about 200μg, about 225μg, about 250μg, about 275μg, about 300μg, about 325μg, about 350μg, about 37 5μg, about 400μg, about 425μg, about 450μg, about 475μg, about 500μg, about 525μg, about 550μg, about 575μ g, about 600μg, about 625μg, about 650μg, about 675μg, about 700μg, about 725μg, about 750μg, about 775μg, The vaccine or nanoparticles may comprise about 100 μg, about 825 μg, about 850 μg, about 875 μg, about 900 μg, about 925 μg, about 950 μg, about 975 μg, about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.25 mg, about 3.5 mg, about 3.75 mg, about 4 mg, about 4.25 mg, about 4.5 mg, about 5.75 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 45 mg, or about 50 mg. In some embodiments, a single dose can comprise 4 mg or less of the vaccine or nanoparticles.

[0188] Alternatively, dosing can be based on the number of nanoparticles administered to a subject. For example, in some embodiments, the dose of the disclosed vaccine is 1.0 x 10 8 ~1.0×10 12 For example, a single dose may contain 1.0 x 10 nanoparticles. 8 , 1.5×10 8 , 2.0x10 8 , 2.5x10 8 , 3.0x10 8 , 3.5x108 、4.0x10 8 、4.5x10 8 、5.0x10 8 、5.5x10 8 、6.0x10 8 、6.5x10 8 、7.0x10 8 、7.5x10 8 、8.0x10 8 、8.5x10 8 、9.0x10 8 、9.5x10 8 、1.0x10 9 、1.5x10 9 、2.0x10 9 、2.5x10 9 、3.0x10 9 、3.5x10 9 、4.0x10 9 、4.5x10 9 、5.0x10 9 、5.5x10 9 、6.0x10 9 、6.5x10 9 、7.0x10 9 、7.5x10 9 、8.0x10 9 、8.5x10 9 、9.0x10 9 、9.5x10 9 、1.0x10 10 、1.5x10 10 、2.0x10 10 、2.5x10 10 、3.0x10 10 、3.5x10 10 、4.0x10 10 、4.5x10 10 、5.0x10 10 、5.5x10 10 、6.0x10 10 、6.5x10 10 、7.0x10 10 、7.5x10 10 、8.0x10 10 、8.5x10 10 、9.0x10 10 、9.5x10 10 、1.0x10 11 、1.5x1011 , 2.0x10 11 , 2.5x10 11 , 3.0x10 11 , 3.5x10 11 , 4.0x10 11 , 4.5x10 11 , 5.0x10 11 , 5.5x10 11 , 6.0x10 11 , 6.5x10 11 , 7.0x10 11 , 7.5x10 11 , 8.0x10 11 , 8.5x10 11 , 9.0x10 11 , 9.5×10 11 , or 1.0 × 10 12 In some embodiments, the dose may comprise about 9.5 x 10 nanoparticles. 8 , about 9.75×10 8 , about 9.85×10 8 , about 9.95×10 8 , about 1.0×10 9 , about 1.1×10 9 , about 1.15×10 9 , about 1.2×10 9 , about 1.25×10 9 , about 1.3×10 9 , about 1.35×10 9 , about 1.4×10 9 , about 1.45×10 9 , or approximately 1.5 × 10 9 The nanoparticles may include:

[0189] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In embodiments where the subject is a human, the subject may be at least 18, 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, or at least 80 years old or older. In some embodiments, the subject is a pediatric subject (i.e., under 18 years old).

[0190] V. Screening for binding molecules In addition to being used for therapy, the disclosed nanoparticles and fusion proteins can be used to screen binding molecules, such as antibodies, for their ability to bind to and neutralize coronaviruses (e.g., SARS-CoV-1 or SARS-CoV-2). Any of the fusion proteins disclosed herein can be contacted with a putative coronavirus-binding molecule, such as a putative anti-coronavirus antibody, to assess binding to the fusion protein or nanoparticle. Antibodies (or other binding molecules) that bind to the fusion proteins disclosed herein are expected to be neutralizing.

[0191] VI. Passive Immunotherapy and Treatment Using Binding Molecules Binding molecules (e.g., antibodies that bind to SARS-CoV-2 or another coronavirus as disclosed herein) can be used for passive immunotherapy to prevent the onset of coronavirus infection or for treating subjects who already have a coronavirus infection. Generally, coronavirus-specific antibodies can be obtained from subjects administered an immunogenic composition as disclosed herein, or coronavirus-specific antibodies can be identified from subjects who have recovered from a coronavirus infection (e.g., COVID-19) using the disclosed fusion proteins and nanoparticles as bait for screening assays. These antibodies can be administered to subjects who have been exposed to or are at risk of exposure to a coronavirus to prevent the onset of a coronavirus infection, such as a COVID-19 or SARS-CoV-1 infection (i.e., the antibodies can function as "passive immunotherapy"). Additionally or alternatively, these antibodies can be administered to subjects who have been infected with a coronavirus, such as SARS-CoV-1 or SARS-CoV-2, to treat the infection, e.g., by reducing or eliminating viral load.

[0192] The disclosed binding proteins can be or be derived from human IgG1, IgG2, IgG3, or IgG4 antibodies. In some embodiments, the binding proteins can be or be derived from a class of antibody selected from IgG, IgM, IgA, IgE, and IgD. That is, the disclosed binding proteins can comprise all or a portion of the constant region, framework region, or combinations thereof of an IgG, IgM, IgA, IgE, or IgD antibody. For example, a disclosed binding protein comprising an IgG1 immunoglobulin structure can be modified to replace (or "switch") the IgG1 structure with the corresponding structure of another IgG class immunoglobulin or an IgM, IgA, IgE, or IgD immunoglobulin. This type of modification or switch can be performed to enhance the neutralizing functions of the peptide, such as antibody-dependent cellular cytotoxicity (ADCC) and complement fixation (CDC). Those skilled in the art will understand that, for example, recombinant IgG1 immunoglobulin structures can be "switched" for corresponding regions of immunoglobulin structures from other immunoglobulin classes, such as recombinant secretory IgA1 or recombinant secretory IgA2, which may be useful for topical application to mucosal surfaces. For example, immunoglobulin IgA structures are known to have applications in protective immune surveillance targeting the invasion of infectious diseases, making such structures suitable for methods using the disclosed binding proteins in such contexts, e.g., treating or preventing coronavirus infections (e.g., COVID-19 or SARS-CoV-1 infections) or the spread of coronavirus from one individual to another.

[0193] Any of the coronavirus-specific binding proteins or antibodies obtained from subjects vaccinated with the disclosed immunogenic compositions or screened / selected using the disclosed fusion proteins can be used to treat and / or prevent coronavirus infections, such as COVID-19 or SARS-CoV-1 infections. The optimal dosage and route of administration may vary, for example, based on the subject's condition, including the route of administration and dosage form, the subject's age and weight, and / or the type and severity of the coronavirus infection, and can be determined by a skilled practitioner. The binding proteins can be formulated into pharmaceutical compositions suitable for administration to a subject by any intended route of administration.

[0194] VII. Illustrative Embodiments The present disclosure provides the following exemplary embodiments, which are non-limiting with respect to the present disclosure. Embodiment 1: A nanoparticle-forming peptide and: the receptor binding domain (RBD) of a coronavirus, or a fragment or variant thereof; the N-terminal domain (NTD) of a coronavirus, or a fragment or variant thereof; the S1 domain of a coronavirus, or a fragment or variant thereof; a stabilized extracellular spike S-2P domain of a coronavirus, or a fragment or variant thereof; a stabilized extracellular spike S domain of a coronavirus, or a fragment or variant thereof; A stabilized extracellular spike S trimer of a coronavirus, or a fragment or variant thereof, and A mosaic coronavirus spike protein, wherein at least one domain of the mosaic coronavirus spike protein is heterologously substituted or added. Nanoparticles comprising a fusion protein comprising at least two antigenic coronavirus peptides selected from:

[0195] Embodiment 2: The nanoparticle of embodiment 1, wherein the nanoparticle-forming peptide comprises or is a ferritin protein or a fragment or variant thereof. Embodiment 3: Nanoparticles according to embodiment 1 or 2, wherein the nanoparticle-forming peptide comprises or is Helicobacter pyloriferritin (Hpf) or a fragment or variant thereof.

[0196] Embodiment 4: The nanoparticle-forming peptide is a. ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 1) or a fragment or variant thereof; b. DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 2) or a fragment or variant thereof, and c. The nanoparticles of any one of embodiments 1 to 3, comprising an amino acid sequence selected from SKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 3), or a fragment or variant thereof.

[0197] Embodiment 5: The nanoparticle of any one of embodiments 1 to 4, wherein at least two antigenic coronavirus peptides are linked together via a linker. Embodiment 6: The nanoparticle of any one of embodiments 1 to 5, wherein at least two antigenic coronavirus peptides are linked to the nanoparticle-forming peptide via a linker. Embodiment 7: The nanoparticle of embodiment 5 or 6, wherein the linker comprises an amino acid sequence selected from GSGGGG (SEQ ID NO: 11), GGGG (SEQ ID NO: 15), GSGG (SEQ ID NO: 5), GGG (SEQ ID NO: 16), and SGG (SEQ ID NO: 17). Embodiment 8: The nanoparticle of any one of embodiments 1 to 7, wherein the fusion protein comprises 3 to 10 antigenic coronavirus peptides linked in tandem, and the antigenic coronavirus peptides may be linked to each other via a peptide linker. Embodiment 9: The nanoparticle of any one of embodiments 1 to 8, wherein the at least two antigenic coronavirus peptides are isolated or derived from one or more coronaviruses selected from SARS-CoV-2, human coronavirus OC43 (hCoV-OC43), Middle East respiratory syndrome-associated coronavirus (MERS-CoV), severe respiratory syndrome-associated coronavirus (SARS-CoV-1), HKU-1, 229E, or NL63.

[0198] Embodiment 10: The nanoparticle of any one of embodiments 1 to 9, wherein the fusion protein comprises a format selected from beads-on-a-string, domain fusion, loop insertion, or domain insertion. Embodiment 11: Nanoparticles of any one of embodiments 1 to 10, wherein the fusion protein comprises the format shown in Figure 2. Embodiment 12: The nanoparticle of any one of embodiments 1 to 11, wherein the fusion protein comprises an amino acid sequence disclosed in Table 6. Embodiment 13: A vaccine comprising the nanoparticles of any one of embodiments 1 to 12. Embodiment 14: The vaccine of embodiment 13, further comprising one or more adjuvants selected from ALFQ, Alhydrogel, and combinations thereof.

[0199] Embodiment 15: Messenger RNA (mRNA) encoding the nanoparticles according to any one of embodiments 1 to 12. Embodiment 16: A method for treating or preventing a coronavirus infection in a subject in need thereof, comprising administering to a subject in need thereof nanoparticles according to any one of embodiments 1 to 12, a vaccine according to any one of embodiments 13 to 14, or an mRNA according to embodiment 15. Embodiment 17: The method of embodiment 16, wherein the subject is at risk of contracting a coronavirus infection. Embodiment 18: The method of embodiment 16, wherein the subject is already suffering from a coronavirus infection. Embodiment 19: Nanoparticles according to any one of embodiments 1 to 12, a vaccine according to any one of embodiments 13 to 14, or an mRNA according to embodiment 15 for use in the treatment or prevention of a coronavirus infection in a subject in need thereof.

[0200] Embodiment 20: The nanoparticles, vaccine or mRNA for use according to embodiment 19, wherein the subject is at risk of suffering from a coronavirus infection. Embodiment 21: The nanoparticles, vaccine or mRNA for use according to embodiment 19, wherein the subject is already suffering from a coronavirus infection. Embodiment 22: Use of nanoparticles according to any one of embodiments 1 to 12, a vaccine according to any of embodiments 13-14, or an mRNA according to embodiment 16 in the preparation of a medicament for treating or preventing a coronavirus infection in a subject in need thereof. Embodiment 23: A DNA molecule comprising a sequence encoding a nanoparticle according to any one of embodiments 1 to 12. Embodiment 24: A plasmid comprising the DNA molecule of embodiment 23. Embodiment 25: A plasmid of embodiment 24 capable of expressing the DNA molecule in vivo. The following examples are given to illustrate the present disclosure, it being understood that the invention should not be limited to the specific conditions or details set forth in these examples. [Example]

[0201] Example 1 - Screening of the disclosed fusion proteins Binding studies of MERS-CoV RBD-ferritin nanoparticle immunogens were conducted in two formats, evaluating the MERS-CoV RBD-ferritin construct for binding to the MERS-CoV-neutralizing human monoclonal antibody CDC-C2. Briefly, biosensors were hydrated in PBS prior to use. The assay step was performed at 30°C with agitation set at 1,000 crpm on an Octet RED96 instrument (ForteBio). The biosensor was equilibrated in assay buffer (PBS) for 15 seconds, followed by loading with IgG antibody (30 μg / mL diluted in PBS). MERS-CoV-neutralizing antibodies targeting the spike RBD used included D12, F11, CDC2-C2, and JC57-11, while SARS-CoV-2 neutralizing antibodies included WRAIR-2125, WRAIR-5001, and ShAb02. MERS-CoV and SARS-CoV-2 antibodies were immobilized on an AHC biosensor (ForteBio) for 100 seconds, followed by a brief baseline acquisition in assay buffer for 15 seconds. The immobilized antibodies were then immersed in various antigens for 180 seconds. Response values ​​were measured at the end of the binding step. Results for constructs M.1 to M3.6 are shown in Figure 10.

[0202] Similar antigenic characterization of the MERS RBD-containing immunogens M3.7, M3.12, and M3.13, and the dual RBD constructs SARS-CoV-2-MERS-CoV RR-FN immunogens 248-250 and 267-270, was performed with a set of neutralizing antibodies by octet biolayer interferometry. The results (binding responses after 180 seconds of binding) are shown in Table 4 below.

[0203] [Table 4] Antigenic characterization of the SARS-CoV-1 RBD-containing immunogens CoV263, CoV277, CoV278, CoV316, and CoV317 immunogens was assessed using a set of neutralizing antibodies by octet biolayer interferometry. The results are shown in Table 5 below.

[0204] [Table 5]

[0205] Example 2 - Fusion protein generation and size estimation An RR-SpFN construct was designed and tested for expression, yield, and nanoparticle formation. The construct pCoV323 (RR-SpFN MR14-SARS1-SpFN) was expressed in Expi293F cells for 5 days at 37°C and purified by Galanthus nivalis lectin (GNA) affinity chromatography. This construct showed a reasonable expression level of 0.4 mg / L of culture supernatant. The purified protein was evaluated by SDS-PAGE and size-exclusion chromatography to assess expression and estimate size. The results are shown in Figure 14, where the RR-SpFN construct exhibits appropriate size by SDS-PAGE and nanoparticle formation by size-exclusion chromatography.

[0206] Example 3 mRNA construct expression An mRNA construct encoding the spike antigen was provided and expressed in HeLa cells. Additionally, the expressed fusion protein was evaluated using a set of neutralizing antibodies by octet biolayer interferometry.

[0207] method HeLa cells were plated in 24-well plates at 75,000 cells / well in 0.5 mL EMEM + 10% FBS. The following day, cells were transfected with 1 μg / million cells of mRNA constructs using lipofectamine 2000. Different mRNA constructs were tested for in vitro expression and secretion in transfected HeLa cells. 24 hours after transfection, cell lysates and supernatants (to assess secretion) were analyzed in dot blots by probing the spike or RBD with an anti-RBD monoclonal antibody (mAb) targeting a conformational neutralizing epitope (e.g., SA55) and / or an anti-ferritin mAb. For octet binding studies, a total of approximately 12 μg of mRNA was used to transfect 25 mL of Expi 293F cells. Supernatants were harvested after 2–4 days and evaluated for binding to a set of CoV-specific antibodies, human ACE2, and a negative control influenza antibody by biolayer interferometry.

[0208] Preparation of lysates Cells were harvested 22-24 hours later and lysed in 225 μL per well of CelLyticM+1×HALT. Lysates were incubated on ice for 10 minutes and then clarified in a microcentrifuge at maximum speed for 10 minutes at 4°C. Lysates were diluted 2-fold and 4-fold with PBS for downstream analysis. Preparation of supernatant samples 22-24 hours after transfection, cell culture supernatants were collected and the supernatant samples were diluted 2-fold and 4-fold with PBS for downstream analysis. Expression analysis Next, 2x and 4x diluted lysate or supernatant samples were spotted onto dried nitrocellulose membranes with 1µ dots. The membranes were then allowed to dry completely before further manipulation. Blots were blocked with Intercept blocking buffer for 1 hour at room temperature. Blots were stained with human anti-spike antibody or human anti-ferritin antibody. Antibody staining was performed overnight at 4°C in Intercept blocking buffer + 0.2% Tween 20. Blots were washed 3x5 minutes with TBST. Blots were then stained with donkey anti-human IR800 secondary antibody in Intercept blocking buffer + 0.2% Tween 20 for 1 hour at RT. Blots were washed 4x5 minutes with TBST and scanned on a Licorice odyssey.

[0209] Expression results As shown in Table 10, the following constructs were expressed and detected in the lysates and / or supernatants. Expression in the supernatants indicated that the proteins were secreted. All mRNAs expressed the expected antigens in cell lysates; S-2P antigen was not detected in the supernatants, as expected for a transmembrane protein; and some of the more complex multi-virus strain constructs (R-SpFN and RR-SpFN) were not detected in the supernatants, indicating either low or defective secretion. The results are summarized in Table 10 below.

[0210] [Table 6-1] [Table 6-2] Particular constructs for which no sequence number is provided were not carried forward for in vivo studies at this time. Octet connectivity results The results of the octet binding studies are summarized in Table 11 below.

[0211] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0212] Example 4: Mosaic Nanoparticle Assembly Based on the observed differences in the apparent sizes of RFNs or SpFNs from different virus strains using native gel electrophoresis (PAGE), we evaluated the assembly of RBD or spike trimers from different virus strains on a single ferritin nanoparticle by comparing the apparent sizes of monovalent and multivalent RFNs (or SpFNs). We also evaluated whether coexpression of empty FN with RFNs could potentially benefit assembly and secretion efficiency, for example, by reducing the risk of steric hindrance between the RBD and FN. method HeLa cells were plated in 24-well plates at 75,000 cells / well in 0.5 mL EMEM + 10% FBS. The following day, cells were transfected with 1 μg / million cells of mRNA constructs using lipofectamine 2000. 24 hours after transfection, cell lysates and supernatants (to assess secretion) were analyzed by native PAGE Western blot probing with anti-ferritin mAb.

[0213] Preparation of lysates Cells were harvested 22-24 hours later and lysed in 125 μL per well of CelLyticM+1×HALT. Lysates were incubated on ice for 10 minutes and then clarified in a microcentrifuge at maximum speed for 10 minutes at 4°C. 15 μL of lysate was combined with 5 μL of native Tris-glycine sample buffer without reducing agents. Preparation of supernatant samples 22-24 hours after transfection, cell culture supernatants were harvested, and 15 μL of the supernatant was combined with 5 μL of native Tris-glycine sample buffer without reducing agents.

[0214] analysis The resulting lysate or supernatant samples were subjected to 4-12% gradient native PAGE at 185V for 75 minutes. Proteins were transferred to nitrocellulose membranes. Blots were blocked with Intercept blocking buffer for 1 hour at room temperature. Blots were stained with human anti-ferritin antibody. Antibody staining was performed overnight at 4°C in Intercept blocking buffer + 0.2% Tween 20. Blots were washed 3 x 5 minutes with TBST. Subsequently, blots were stained with goat anti-human IR800 secondary antibody in Intercept blocking buffer + 0.2% Tween 20 for 1 hour at RT. Blots were washed 4 x 5 minutes with TBST and scanned on a Licorice odyssey.

[0215] result The results of these studies are shown in Figures 18 and 19. The data show that when SARS-1 RFN (relatively high migration) and BANAL-20-247 RFN (relatively low migration) are coexpressed, the banding pattern shifts to an intermediate pattern compared to that observed with a single RFN. This indicates the presence of multiple molecular species, most likely composed of different ratios of each construct. This same phenomenon is observed when each RFN is combined with empty FN (ferritin alone). Because the difference in migration is much greater, this effect is more dramatic and clearly indicates multiple molecular species in the complex. Additionally, the lowest-migrating multivalent complex is higher than empty FN, indicating that all detectable empty FN is in complex with at least one RFN component. The combination of all three components produces a distinct banding pattern, supporting the formation of a complex containing all three components. Supernatant samples show similar banding patterns, indicating that multimember complexes can be secreted. Interestingly, the lower migrating complexes appear to be secreted much more efficiently compared to the higher migrating complexes, indicating that smaller complexes containing a higher proportion of empty FN may be secreted more efficiently.

[0216] Example 5 - Immunogenicity studies in naive mice This mouse study focused on mRNA designs spanning RFN and SpFN antigen presentation paradigms at different antigenic distances (Table 8 below). Vaccine compositions were formulated in lipid D LNPs and evaluated for their immunogenicity in naive male and female C57BL / 6 mice using a two-dose primary immunization schedule administered intramuscularly, four weeks apart. To compare the contribution of individual virus strains and assess the impact of multivalent presentation, comparative formulations were prepared using monovalent versions of antigens from each component strain of the multivalent mixture. Additionally, stabilized transmembrane spike proteins (S-2P) were generated for all virus strains in monovalent and multivalent formulations to assess the contribution of antigen presentation by ferritin nanoparticles. In one set of formulations, the RFN formulation also contained a construct encoding a ferritin monomer (without a conjugated antigen), designated "empty FN." For one subset of formulations, individual mRNA constructs were encapsulated separately in separate LNPs administered in the same composition and compared to the results achieved with formulations prepared with the corresponding mRNAs co-encapsulated in the same LNPs.

[0217] formulation For each immunization group (see Table 8), the composition to be administered was formulated and diluted to a concentration appropriate for a 1 μg dose per construct per 50 μL. When mRNA encoding empty ferritin alone was included, the mRNA was added at 0.3 μL per construct per 50 μL. Mice receiving the control formulation listed as "co-administration" were inoculated with three separate injections, each containing one listed construct encapsulated in LNP at a 1 μg dose (total dose of 3 μg).

[0218] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] Sera collected two weeks after the second immunization were tested for neutralizing capacity against a panel of virus strains selected for their clinical relevance. This panel included SARS-CoV-2 (clade 1b) variants (WA-1, Delta, Beta, BA.5, BQ.1.1, XBB.1.5) and representative virus strains derived from SARS-CoV-1. Merbicovikovirus readouts were also included.

[0219] Pseudovirus neutralization assay The spike protein (S) expression plasmid sequences for SARS-CoV-2 and SARS-CoV-1 were codon-optimized and modified to remove an 18-amino acid endoplasmic reticulum retention signal in the cytoplasmic tail in the case of SARS-CoV-2 and a 28-amino acid deletion in the cytoplasmic tail in the case of SARS-CoV. This allowed for increased S incorporation into pseudovirions (PSV), thereby improving infectivity. Virions pseudomorphized with vesicular stomatitis virus (VSV) G protein were used as a nonspecific control. SARS-CoV-2 pseudovirions (PSVs) were generated by cotransfection of HEK293T / 17 cells with the SARS-CoV-2 S plasmid (pcDNA3.4) and the HIV-1 NL4-3 luciferase reporter plasmid (reagents were obtained through the NIH HIV Reagent Program, AIDS Division, NIAID, NIH, provided by Drs. Nathaniel Landau and Aaron Diamond: human immunodeficiency virus 1 (HIV-1) NL4-3ΔEnv Vpr luciferase reporter vector (pNL4-3.Luc.RE-), ARP-3418).

[0220] Infectivity and neutralization titers were determined using ACE2-expressing HEK293 target cells (Integral Molecular) in a semi-automated assay format (Biomek NXp Beckman Coulter) with robotic liquid handling. Test sera were diluted 1:40 and serially diluted in growth medium, followed by addition of 25 μL / well to a white 96-well plate. An equal volume of diluted SARS-CoV-2 PSV was added to each well, and the plate was incubated at 37°C for 1 hour. Target cells were added to each well (40,000 cells / well), and the plate was further incubated for 48 hours. RLU was measured using the Bright-Glo luciferase assay system (Promega Corporation, Madison, WI) on an EnVision multimode plate reader (Perkin Elmer, Waltham, MA). Neutralization dose-response curves were fitted by nonlinear regression using 5-parameter curve fitting using LabKey Server®, and final titers were reported as the reciprocal of the dilution of serum required to achieve 50% neutralization (ID50, 50% inhibitory dilution) and 80% neutralization (ID80, 80% inhibitory dilution).

[0221] Results for monovalent constructs and multivalent Mix A-E formulations in either RFN, SpFN, or S2P antigen presentation formats are summarized in Figures 20 and 21. Statistical representations of mouse group titers for mice vaccinated with monovalent or multivalent Mix A-E in either RFN, SpFN, or S2P antigen presentation formats are provided in Figures 22, 23, and 24. In general, across antigen presentation formats, monovalent virus strains provided comparable neutralization titers against similar virus strains (Figure 20). WA-1 (groups 1, 15, and 19) neutralized WA-1, delta, and beta. For the monovalent controls, the greatest spread across clades 1a and 1b was seen for the beta immunizations (groups 2, 22, and 37), with sera from the beta immunizations observed to neutralize WA-1, delta, and beta. In addition, some mice had substantial titers against two consecutively distant Omicron strains, BA.5 and BQ.1.1, as well as clade 1a SARS-CoV-1 pseudoviruses. Sera from BQ.1.1-immunized mice (groups 3, 16, and 21) produced high titers against the tested Omicron strains, BA.5 and BQ.1.1, with reduced titers against beta, delta, and WA-1. These three monovalent controls establish that the selected clade 1b strains span the antigenic landscape of the SARS-CoV-2 pandemic, from the original virus strain to recent Omicron strains. With respect to other antigens, including SARS-CoV-1, Khosta-2, BANAL20-247, and MERS, immunized mouse sera did not produce any neutralizing titers against the tested clade 1b pseudoviruses. SARS-CoV-1 (groups 4, 17, 20) and MERS (6, 24, 39) strains produced high titers only against their cognate pseudoviruses (Figure 20).

[0222] Mix A (WA-1, Beta, BQ.1.1) polyvalent formulations containing three monovalent virus strains (groups 10, 27, and 41) produced broad coverage across clade 1b, as demonstrated by the high titers observed against all clade 1b sarbecoviruses tested, particularly for SpFN and S-2P antigen presentation (groups 27 and 41, respectively). Mix B (WA-1, BQ.1.1, SARS-CoV-1) (groups 11, 25, and 31) similarly produced high titers across all clade 1b sarbecoviruses but also provided at least homogenous coverage within clade 1a. Mixes containing WA-1, SARS-CoV-1, and one of Khosta-2 (Mix C; groups 9, 26, 44), BANAL20-247 (Mix D; groups 8, 28, 42), or MERS (Mix E; groups 12, 29, 43) showed similar titers to WA-1 for clade 1b and SARS-CoV-1 for clade 1a, with little interference from additional virus strains (Figures 21-24).

[0223] Enhanced clade 1b neutralization titers following mix B immunization Sera generated using the antigen design and immunization strategies disclosed herein are considered to have an enhanced breadth of immune response if they have increased neutralizing titers (as measured by pseudoneutralization assays) against one or more sarbecovirus strains not included as vaccine components. The enhanced effect of multivalent antigen presentation as described herein can be assessed by comparing pseudoneutralization titers between groups of mice vaccinated with multivalent formulations and their matched monovalent formulations at the same antigen presentation.

[0224] Neutralization titers against beta strains elicited by the intermediate antigenic distance set (Mix B) containing WA-1, BQ.1.1, and SARS-CoV-1 in a co-encapsulated SpFN antigen presentation format (Group 25) were enhanced compared to those elicited by WA-1, BQ.1.1, or SARS-CoV-1 monovalent SpFN (Groups 19-21) (Figure 23, Panel C). Titers against beta pseudoviruses for Mix B SpFN were also improved across all antigen presentations compared to any other multivalent or monovalent that did not contain beta strains (GMT titers and minimum titers per mouse group). Without being bound by theory, this enhanced titer against beta pseudoviruses may be mediated by the generation of antibodies that bind to a broader range of common epitopes (e.g., antibodies that are tolerant to accumulated mutations between viral strains), allowing simultaneous binding to two of the WA-1, BQ.1.1, and SARS-CoV-1 SpFN constructs displayed on the same nanoparticles. Because the component viral strains in Mix B do not contain beta strain antigens but still elicited neutralization titers greater than 1:2560, this result is evidence of an enhanced breadth of immune response. Similarly, immunization with either co-encapsulated SpFN or co-administered S-2P Mix B also trended toward enhanced cross-neutralization of the XBB.1.5 strain (Figures 23F and 24F). Immunization with co-encapsulated SpFN Mix B also trended toward enhanced homologous titers against BQ.1.1 (Figure 23, panel E).

[0225] Other studies have reported increased titers against several SARS-CoV-1 strains through vaccination with a multivalent presentation system displaying eight SARS-CoV-1 and SARS-CoV-2 strains, but all observed titers in those reports were consistent with vaccination with component antigens. In other words, any increased spread is simply mediated by each individual antigen present, rather than cross-linkage between antigens. In contrast, the results herein demonstrate increased spread that is necessarily mediated by peripheral connectivity between one or more multivalent antigens.

[0226] Multivalent SpFN antigen presentation formats generate higher neutralization titers than multivalent S-2P formats The benefit of the ferritin antigen presentation system is demonstrated by comparing pseudoneutralization titers between groups of mice vaccinated with the mixed B antigenic range set presented as stabilized transmembrane protein (S-2P) and SpFN. Figure 25 compares pseudoneutralization titers between the monovalent and multivalent SpFN and S-2P groups. For the monovalent groups (open circles; S-2P, monovalent groups 15-17, 37-40; SpFN, monovalent groups 18-24), neutralization titers were observed that trended along the x = y line (dashed line), suggesting that titers were roughly equivalent across antigen presentations. However, the multivalent groups (solid circles; S-2P, groups 31, 41-44; SpFN, groups 25-29) showed improved pseudo-neutralization titers against SpFN antigen presentation, as evidenced by titers above the x=y line (dashed line) for four of the six virus strains tested (excluding SARS-CoV-1 and XBB.1.5, not shown in Figure 25).

[0227] Co-encapsulated and co-administered multivalent antigens Comparison of results obtained in groups of mice inoculated with LNP formulations of co-encapsulated mRNA and LNP formulations of mixtures of separately encapsulated mRNA (e.g., with each mRNA molecule separately encapsulated and co-administered in LNPs) revealed no significant differences between co-encapsulation and separate encapsulation. Pseudo-neutralization titers were very similar between all three co-administered mixtures and their co-encapsulated counterparts, suggesting that each type of individual mRNA / LNP formulation was taken up by the respective cells, resulting in similar expression patterns and nanoparticle formation (Figures 26A-D).

[0228] Neutralizing titers elicited by RFN, SpFN, and S2P antigen presentation formats Comparing matched sets of virus strains across these RFN, SpFN, and S-2P antigen presentation formats, the SpFN formulations demonstrated improved titers across all clade 1a and 1b readouts (Figures 20-23), with the exception of the MERS readout, which demonstrated strong immunogenicity for both the RFN and SpFN constructs and the formulation containing the MERS construct.

[0229] Example 6: Multivalent antigens as booster vaccines The ability of the multivalent composition to elicit cross-neutralizing antibodies upon booster vaccination will be evaluated in BALB / C female mice (n=8) primed with mRNA encoding WA-1 on days 0 and 21 and boosted 3 months later (first boost) using the formulations and dose levels as shown in Table 9. Mice will be bled 1 day before the boost to assess pre-boost titers in all groups. Animals will then be bled 2 weeks after the boost to determine and compare post-boost titers between groups. The commercially available S2-P XBB.1.5 SARS CoV-2 (SoC) vaccine will be included in this study as a comparative standard for the proposed new booster formulation.

[0230] [Table 9-1] [Table 9-2]

[0231] The effect of a second boost on potential further increases in the spread of neutralizing antibodies will be tested for selected S-2P and SpFN multivalent formulations (beta, BQ.1.1, SARS-CoV-1) and the monovalent control WA-1 S-2P and SpFN. The second boost will be administered one month after the first boost. To assess pre- and post-second boost titers, mice will be bled one day before the second boost is administered and then again two weeks after the second boost. All bleeds from this study will be tested for their pseudoneutralization titers against a defined panel of virus strains spanning clades 1a, 1b, 3, and 4, selected for their clinical relevance.

[0232] The specific dosing regimen will be as follows: priming: day 0 and D21; bleed: day 35; pre-boost bleed: approximately 1 day before the first boost; first boost: 3 months after day 21 (D119); bleed: 2 weeks after the first boost (D133); pre-2 boost bleed: approximately 1 day before the second boost (D150); second boost: 1 month after the first boost (D151). There will be 8 mice in each group. The dosing regimen is illustrated in Figure 31. For the present study, the multivalent composition comprises a mixture of LNPs that separately encapsulate mRNA molecules encoding a given antigen, although other studies may utilize multivalent compositions that contain multiple mRNA molecules co-encapsulated in a single LNP.

[0233] [Table 10-1]

[0234] [Table 10-2]

[0235] [Table 10-3]

[0236]

Table 10-4

[0237]

Table 10-5

[0238]

Table 10-6

[0239]

Table 10-7

[0240]

Table 10-8

[0241]

Table 10-9

[0242]

Table 10-10

[0243]

Table 10-11

[0244]

Table 10-12

[0245]

Table 10-13

[0246]

Table 10-14

[0247]

Table 10-15

[0248]

Table 10-16

[0249]

Table 10-17

[0250]

Table 10-18

[0251]

Table 10-19

[0252]

Table 10-20

[0253]

Table 10-21

[0254]

Table 10-22

[0255]

Table 10-23

[0256]

Table 10-24

[0257]

Table 10-25

[0258]

Table 10-26

[0259]

Table 10-27

[0260]

Table 10-28

[0261]

Table 10-29

[0262]

Table 10-30

[0263]

Table 10-31

[0264]

Table 10-32

[0265]

Table 10-33

[0266]

Table 10-34

[0267]

Table 10-35

[0268]

Table 10-36

[0269]

Table 10-37

[0270]

Table 10-38

[0271]

Table 10-39

[0272]

Table 10-40

[0273]

Table 10-41

[0274]

Table 10-42

[0275]

Table 10-43

[0276]

Table 10-44

[0277]

Table 10-45

[0278]

Table 10-46

[0279]

Table 10-47

[0280]

Table 10-48

[0281]

Table 10-49

[0282]

Table 10-50

[0283]

Table 10-51

[0284]

Table 10-52

[0285]

Table 10-53

[0286]

Table 10-54

[0287]

Table 10-55

[0288]

Table 10-56

[0289]

Table 10-57

[0290]

Table 10-58

[0291]

Table 10-59

[0292]

Table 10-60

[0293]

Table 10-61

[0294]

Table 10-62

[0295]

Table 10-63

[0296]

Table 10-64

[0297]

Table 10-65

[0298]

Table 10-66

[0299]

Table 10-67

[0300]

Table 10-68

[0301]

Table 10-69

[0302]

Table 10-70

[0303]

Table 10-71

[0304]

Table 10-72

[0305]

Table 10-73

[0306]

Table 10-74

[0307]

Table 10-75

[0308]

Table 10-76

[0309]

Table 10-77

[0310]

Table 10-78

[0311]

Table 10-79

[0312]

Table 10-80

[0313]

Table 10-81

[0314]

Table 10-82

[0315]

Table 10-83

[0316]

Table 10-84

[0317]

Table 10-85

[0318]

Table 10-86

[0319]

Table 11-1

[0320]

Table 11-2

[0321]

Table 11-3

[0322]

Table 11-4

[0323]

Table 11-5

[0324]

Table 11-6

[0325]

Table 11-7

[0326]

Table 11-8

Claims

1. 1. An immunogenic composition comprising at least two antigenic coronavirus peptides, including at least a first antigenic coronavirus peptide and a second antigenic coronavirus peptide, or one or more messenger RNA (mRNA) molecules encoding them, Each antigenic coronavirus peptide is a. the receptor binding domain (RBD or R) of a coronavirus, or a fragment or variant thereof; b. The N-terminal domain (NTD) of a coronavirus, or a fragment or variant thereof; c. The S1 domain of a coronavirus, or a fragment or variant thereof; d. A stabilized coronavirus spike S-2P domain, or a fragment or variant thereof; e. A stabilized spike S domain of a coronavirus, or a fragment or variant thereof, and f. Stabilized spike S trimer of coronavirus, or a fragment or variant thereof are independently selected from g. The antigenic coronavirus peptides are from the following combinations of virus strains: (i) two or more species selected from clade 1b; (ii) one or more selected from Clade 1b and one or more selected from Clade 1a SARS-CoV-1; (iii) one or more selected from Clade 1b, and one or more selected from Clade 1a, and one or more selected from Clade 2; (iv) one or more selected from Clade 1b, and one or more selected from Clade 1a, and one or more selected from Clade 3; (v) one or more selected from Clade 1b, one or more selected from Clade 2, and one or more selected from Clade 3; and (vi) one or more selected from Clade 1a, one or more selected from Clade 2, and one or more selected from Clade 3 an antigenic coronavirus peptide selected from the group consisting of:

2. 2. The composition of claim 1, wherein at least one of the antigenic coronavirus peptides is an S-2P peptide, and the S-2P peptide optionally comprises the amino acid sequence of any one of SEQ ID NOs: 536-543, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity thereto.

3. 3. The composition of claim 1 or 2, wherein at least one of the antigenic coronavirus peptides is comprised in a fusion protein comprising the antigenic coronavirus peptide and a nanoparticle-forming protein, and two or more of the antigenic coronavirus peptides may be comprised in the same or different fusion proteins comprising the nanoparticle-forming protein.

4. 1. An immunogenic composition comprising nanoparticles comprising at least two antigenic coronavirus peptides, comprising at least a first antigenic coronavirus peptide and a second antigenic coronavirus peptide, or one or more messenger RNA (mRNA) molecules encoding the at least two antigenic coronavirus peptides, Each antigenic coronavirus peptide is a. the receptor binding domain (RBD or R) of a coronavirus, or a fragment or variant thereof; b. The N-terminal domain (NTD) of a coronavirus, or a fragment or variant thereof; c. The S1 domain of a coronavirus, or a fragment or variant thereof; d. A stabilized extracellular spike S-2P domain of a coronavirus, or a fragment or variant thereof; e. A stabilized extracellular spike S domain of a coronavirus, or a fragment or variant thereof, and f. A stabilized extracellular spike S trimer of a coronavirus, or a fragment or variant thereof; are independently selected from each antigenic coronavirus peptide is contained in a fusion protein comprising the antigenic coronavirus peptide and the nanoparticle-forming protein, and the antigenic coronavirus peptides may be contained in the same or different fusion proteins; A composition comprising antigenic coronavirus peptides from at least two different coronavirus strains, or one or more mRNA molecules encoding them.

5. 5. The composition of claim 4, wherein each antigenic coronavirus peptide is derived from a coronavirus strain independently selected from Clade 1a, Clade 1b, Clade 2, Clade 3, and Middle East Respiratory Syndrome-related coronavirus (MERS-CoV), and wherein at least the first and second antigenic coronavirus peptides may be derived from coronavirus strains of different clades.

6. 6. The composition of claim 4 or 5, wherein at least the first and second antigenic coronavirus peptides are derived from different coronavirus strains independently selected from WA-1, Beta, Omicron BQ.1.1, Omicron XBB.1.5, SARS-CoV-1 viral strains, BANAL20-247, Khosta2, and MERS-CoV.

7. Antigenic coronavirus peptides are derived from the following combinations of virus strains: (i) two or more selected from WA-1, Beta, Omicron XBB.1.5, and Omicron BQ1.1; (ii) one or more selected from WA-1, Beta, Omicron XBB.1.5, and Omicron BQ1.1, and SARS-CoV-1 virus strains; (iii) one or more selected from WA-1, Beta, Omicron XBB.1.5, and Omicron BQ1.1, and one or more selected from SARS-CoV-1 virus strains, BANAL20-247, and Khosta-2; (iv) one or more selected from WA-1, Beta, Omicron XBB.1.5, and Omicron BQ1.1, and one or more selected from viral strains of SARS-CoV-1, BANAL20-247, and Khosta-2, and MERS-CoV; and (v) two or more selected from SARS-CoV-1 virus strains, BANAL20-247, and Khosta-2 The composition of any one of claims 1 to 6, comprising an antigenic coronavirus peptide selected from:

8. Antigenic coronavirus peptides are derived from the following combinations of virus strains: a. WA-1, Beta, and Omicron BQ1.1; b. WA-1, Omicron BQ.1.1, and SARS-CoV-1; c. WA-1, SARS-CoV-1, and Khosta2; d. WA-1, SARS-CoV-1, and BANAL20-247; e. WA-1, SARS-CoV-1, and MERS-CoV; and f. SAR-CoV-1, Khosta-2, and BANAL20-247 The composition of any one of claims 1 to 7, comprising an antigenic coronavirus peptide selected from:

9. Antigenic coronavirus peptides are derived from the following combinations of virus strains: (i) WA-1, beta, and Omicron BQ.1.1 (or XBB.1.5), in which the antigenic coronavirus peptide may be an RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(beta)FN, and R(BQ1.1)FN, or a spike antigen contained in S-2P (e.g., S(WA-1)-2P, S(beta)-2P, and S(BQ1.1)-2P) or SpFN fusion proteins Sp(WA-1)FN, Sp(beta)FN, and Sp(BQ1.1)FN, or a mosaic antigen contained in an RmosSpFN or RRmosSpFN fusion protein, or any combination thereof; (ii) WA-1, Omicron BQ.1.1 (or XBB1.5), and SARS-CoV-1, wherein the antigenic coronavirus peptide may be an RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(BQ1.1)FN, and R(SARS-CoV-1)FN, or a spike antigen contained in S-2P (e.g., S(WA-1)-2P, S(BQ1.1)-2P, and S(SARS-CoV-1)-2P) or SpFN fusion proteins Sp(WA-1)FN, Sp(BQ1.1)FN, and Sp(SARS-CoV-1)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion protein, or any combination thereof, WA-1, Omicron BQ. 1.1 (or XBB1.5), and SARS-CoV-1; (iii) WA-1, SARS-CoV-1, and Khosta2, where the antigenic coronavirus peptide is the RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(SARS-CoV-1)FN, and R(Khosta2)FN, or S-2P (e.g., S(WA-1)-2P, S(SARS-CoV-1)-2P, and S(Khosta2)-2P). or WA-1, SARS-CoV-1, and Khosta2, which may be spike antigens contained in SpFN fusion proteins Sp(WA-1)FN, Sp(SARS-CoV-1)FN, and Sp(Khosta2)FN, or mosaic antigens contained in RmosSpFN or RRmosSpFN fusion proteins, or any combination thereof; (iv) WA-1, SARS-CoV-1, and BANAL20-247, where the antigenic coronavirus peptide is the RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(SARS-CoV-1)FN, and R(BANAL20-247)FN, or S-2P (e.g., S(WA-1)-2P, S(SARS-CoV-1)-2P, and S(BANAL20-247)-2 WA-1, SARS-CoV-1, and BANAL20-247, which may be spike antigens contained in the SpFN fusion proteins Sp(WA-1)FN, Sp(SARS-CoV-1)FN, and Sp(BANAL20-247)FN, or mosaic antigens contained in the RmosSpFN or RRmosSpFN fusion proteins, or any combination thereof; (v) WA-1, SARS-CoV-1, and MERS-CoV, where the antigenic coronavirus peptide is the RBD antigen contained in the RFN fusion proteins R(WA-1)FN, R(SARS-CoV-1)FN, and R(MERS-CoV)FN, or S-2P (e.g., S(WA-1)-2P, S(SARS-CoV-1)-2P, and S(MERS-CoV)-2P). or spike antigens contained in SpFN fusion proteins Sp(WA-1)FN, Sp(SARS-CoV-1)FN, and Sp(MERS-CoV)FN, or mosaic antigens contained in RmosSpFN or RRmosSpFN fusion proteins, or any combination thereof, for WA-1, SARS-CoV-1, and MERS-CoV; (vi) SARS-CoV-1, Khosta-2, and BANAL20-247, wherein the antigenic coronavirus peptide is an RBD antigen contained in the RFN fusion proteins R(SARS-CoV-1)FN, R(Khosta2)FN, and R(BANAL20-247)FN, or S-2P (e.g., S(SARS-CoV-1)-2P, S(Khosta2)-2P, and S(BANAL20-247)- 2P) or the spike antigen contained in the SpFN fusion proteins Sp(SARS-CoV-1)FN, Sp(Khosta2)FN, and Sp(BANAL20-247)FN, or the mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion proteins, SAR-CoV-1, Khosta-2, and BANAL20-247, which may be any combination thereof; (vii) beta, omicron BQ.1.1 (or XBB1.5), and SARS-CoV-1, wherein the antigenic coronavirus peptide may be an RBD antigen contained in the RFN fusion proteins R(beta)FN, R(BQ1.1)FN, and R(SARS-CoV-1)FN, or a spike antigen contained in S-2P (e.g., S(beta)-2P, S(BQ1.1)-2P, and S(SARS-CoV-1)-2P) or SpFN fusion proteins Sp(beta)FN, Sp(BQ1.1)FN, and Sp(SARS-CoV-1)FN, or a mosaic antigen contained in an RmosSpFN or RRmosSpFN fusion protein, or any combination thereof; 1.1 (or XBB1.5), and SARS-CoV-1; (viii) beta, omicron XBB.1.5, and SARS-CoV-1, wherein the antigenic coronavirus peptide can be an RBD antigen contained in the RFN fusion proteins R(beta)FN, R(XBB1.5)FN, and R(SARS-CoV-1)FN, or a spike antigen contained in S-2P (e.g., S(beta)-2P, S(XBB1.5)-2P, and S(SARS-CoV-1)-2P) or the SpFN fusion proteins Sp(beta)FN, Sp(XBB1.5)FN, and Sp(SARS-CoV-1)FN, or a mosaic antigen contained in an RmosSpFN or RRmosSpFN fusion protein, or any combination thereof; (ix) Omicron BQ.1.1, SARS-CoV-1, and Khosta2, wherein the antigenic coronavirus peptide may be an RBD antigen contained in the RFN fusion proteins R(Omicron BQ.1.1)FN, R(SARS-CoV-1)FN, and R(Khosta2)FN, or a spike antigen contained in S-2P (e.g., S(Omicron BQ.1.1)-2P, S(SARS-CoV-1)-2P, and S(Khosta2)-2P) or SpFN fusion proteins Sp(Omicron BQ.1.1)FN, Sp(SARS-CoV-1)FN, and Sp(Khosta2)FN, or a mosaic antigen contained in the RmosSpFN or RRmosSpFN fusion protein, or any combination thereof; 1.1, SARS-CoV-1, and Khosta2 The composition of any one of claims 1 to 8, comprising an antigenic coronavirus peptide selected from:

10. The composition of any one of claims 3 to 9, wherein the nanoparticle-forming peptide comprises or is a ferritin protein or a fragment or variant thereof.

11. 10. The composition of any one of claims 3 to 9, wherein the nanoparticle-forming peptide comprises or is Helicobacter pylori ferritin or a fragment or variant thereof.

12. The nanoparticle-forming peptide ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 1) or a fragment or variant thereof; DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 2) or a fragment or variant thereof, and SKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS (SEQ ID NO: 3) or a fragment or variant thereof; The composition of any one of claims 3 to 9, comprising an amino acid sequence selected from:

13. The composition of any one of claims 1 to 12, wherein at least two antigenic coronavirus peptides are comprised in a fusion protein, and the antigenic coronavirus peptides are linked via a linker.

14. 14. The composition of any one of claims 1 to 13, wherein 2 to 10 antigenic coronavirus peptides are comprised in a tandem fusion protein, and the antigenic coronavirus peptides may be linked via a linker.

15. 15. The composition of any one of claims 3 to 14, wherein the antigenic coronavirus peptide is comprised in a fusion protein with the nanoparticle-forming peptide, and the antigenic coronavirus peptide is linked to the nanoparticle-forming peptide via a linker.

16. 16. The composition of any one of claims 13 to 15, wherein the linker comprises an amino acid sequence selected from GGGSGGSG (SEQ ID NO:583), GSGGGG (SEQ ID NO:11), GGGG (SEQ ID NO:15), GSGG (SEQ ID NO:5), GGG (SEQ ID NO:16), and SGG (SEQ ID NO:17).

17. 17. The composition of any one of claims 1 to 16, wherein the first and second antigenic coronavirus peptides are comprised in a mosaic coronavirus spike protein, and at least one domain of the mosaic coronavirus spike protein is substituted or added from a heterologous coronavirus strain.

18. 18. The composition of any one of claims 3 to 17, wherein the antigenic coronavirus peptide is comprised in a fusion protein with a nanoparticle-forming peptide, the fusion protein comprising one or more formats selected from SpFn, daisy-chain, domain fusion, domain swap, loop insertion, and domain insertion.

19. 19. The composition of any one of claims 3 to 18, wherein the antigenic coronavirus peptide is comprised in a fusion protein with a nanoparticle-forming peptide, the fusion protein having a format selected from the formats shown in any one of Figures 2 to 7.

20. The antigenic coronavirus peptides are contained in a fusion protein with the nanoparticle-forming peptide, and at least the first and second antigenic coronavirus peptides are contained in the same fusion protein (e.g., R 1 R 2 FN) 1 , R 2 ), and the fusion protein may further comprise a spike protein (e.g., R 1 R 2 mosSpFN), and two or more different fusion proteins (e.g., RBD peptides) containing the same two or more different RBD peptides at different positions in the fusion protein. 1 R 2 F.N., R. 2 R 1 FN or R 1 R 2 mosSpFN, R 2 R 1 20. The composition of claim 3, optionally comprising mRNA molecules encoding the two or more different fusion proteins, or nanoparticles displaying the two or more different fusion proteins.

21. 21. The composition of any one of claims 1 to 20, wherein the antigenic coronavirus peptide is comprised in one or more fusion proteins comprising an amino acid sequence selected from those disclosed in Table 6 or Table 7 (SEQ ID NOS: 29-551), or a sequence having at least 80% sequence identity thereto.

22. 22. The composition of any one of claims 3 to 21, comprising nanoparticles comprising at least two antigenic coronavirus peptides.

23. 22. The composition of any one of claims 1 to 21, comprising one or more mRNA molecules encoding at least two antigenic coronavirus peptides, wherein the one or more mRNA molecules are optionally encapsulated or co-encapsulated in one or more lipid nanoparticles (LNPs).

24. a. a composition comprising an mRNA molecule encoding a fusion protein comprising at least two antigenic coronavirus peptides, said fusion protein optionally further comprising a nanoparticle-forming peptide, wherein said mRNA molecule is encapsulated in a lipid nanoparticle (LNP); b. A composition comprising two or more mRNA molecules, optionally in a fusion protein comprising a nanoparticle-forming peptide, each of the two or more mRNA molecules encoding at least one of at least two antigenic coronavirus peptides, and each mRNA molecule encapsulated in a separate lipid nanoparticle (LNP); and c. A composition comprising two or more mRNA molecules, optionally in a fusion protein containing a nanoparticle-forming peptide, each of the two or more mRNA molecules encoding at least one of at least two antigenic coronavirus peptides, and the two or more mRNA molecules are co-encapsulated in the same lipid nanoparticle (LNP); 24. The composition of claim 23, wherein the composition is selected from:

25. The composition comprises an mRNA molecule, the mRNA molecule comprising: a. 5' untranslated region (5'UTR); b. the 3' untranslated region (3'UTR); c. a polyadenylation (poly(A)) sequence; d. Chemical modifications, which may include N1-methylpseudouridine and e. The mRNA is a self-replicating mRNA or a non-replicating mRNA, and the mRNA molecule may be encapsulated in a lipid nanoparticle (LNP); The composition according to any one of claims 1 to 24.

26. The composition of any one of claims 1 to 25, further comprising an adjuvant.

27. 27. The composition of claim 26, wherein the composition comprises an antigen or nanoparticles and the adjuvant comprises one or more selected from ALFQ, alhydrogel, and combinations thereof.

28. Nanoparticle-forming peptides and: a. the receptor binding domain (RBD) of a coronavirus, or a fragment or variant thereof; b. The N-terminal domain (NTD) of a coronavirus, or a fragment or variant thereof; c. The S1 domain of a coronavirus, or a fragment or variant thereof; d. A stabilized extracellular spike S-2P domain of a coronavirus, or a fragment or variant thereof; e. A stabilized extracellular spike S domain of a coronavirus, or a fragment or variant thereof; f. A stabilized extracellular spike S trimer of a coronavirus, or a fragment or variant thereof, and g. A mosaic coronavirus spike protein, wherein at least one domain of the mosaic coronavirus spike protein has been replaced or added from a heterologous coronavirus strain; at least two antigenic coronavirus peptides independently selected from A nanoparticle comprising a fusion protein comprising:

29. 29. A DNA molecule comprising a sequence encoding the nanoparticle of claim 28, or a plasmid comprising said DNA molecule, optionally capable of expressing the DNA molecule in vivo.

30. 30. A method of treating or preventing a coronavirus infection in a subject in need thereof, comprising the step of administering to the subject in need thereof the immunogenic composition of any one of claims 1 to 29.

31. 30. The immunogenic composition of any one of claims 1 to 29 for use in the treatment or prevention of a coronavirus infection in a subject in need thereof.

32. 30. Use of the immunogenic composition of any one of claims 1 to 29 in the preparation of a medicament for treating or preventing a coronavirus infection in a subject in need thereof.

33. The method, composition for use, or use according to any one of claims 30 to 32, wherein the subject is at risk of contracting a coronavirus infection.

34. 33. The method, composition for use, or use of any one of claims 30 to 32, wherein the subject is already suffering from a coronavirus infection.

35. 35. The method, composition for use, or use of any one of claims 30 to 34, wherein the subject has not previously been administered a vaccine for the prevention of coronavirus infection.

36. 35. The method, composition for use, or use of any one of claims 30 to 34, wherein the subject has previously been administered one or more vaccines for the prevention of coronavirus infection.

37. 37. The method, composition for use, or use of any one of claims 30 to 36, wherein the method elicits an immune response in the subject against coronavirus, wherein the immune response may comprise neutralizing antibodies, and further wherein the neutralizing antibodies may cross-neutralize two or more coronavirus strains, and further wherein the neutralizing antibodies may cross-neutralize one or more coronavirus strains that are not component strains of the immunogenic composition.

38. An mRNA molecule comprising or consisting of a sequence selected from any one of SEQ ID NOs: 552-582, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity thereto.

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